Activin receptor type ii inhibitors for preventing or treating obesity and related disorders

Abstract

Provided herein, in part, is a method of treating or preventing obesity or an obesity-related disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a small molecule inhibitor of an Activin receptor Type II.

Description

Attorney Docket No.: SUTH-002WOMETHODS OF USE OF ACTIVIN RECEPTOR TYPE II INHIBITORS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 730,416, filed December 10, 2024; U.S. Provisional Patent Application No.63 / 758,752, filed February 14, 2025; and U.S. Provisional Patent Application No. 63 / 779,776, filed March 28, 2025; the disclosure of each of which is hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The invention relates generally to methods of using compounds and compositions for treating or preventing obesity or an obesity-related disease or disorder and / or for reducing body weight, fat mass, liver weight, or liver mass.BACKGROUND

[0003] Significant body weight loss in both animals and human subjects has been achieved with both oral and injectable Glucagon-like peptide- 1 (GLP-1) agonists (incretins), primarily acting through suppression of appetite and food intake. Although this weight loss comprises a combination of both fat mass loss and lean mass loss, the clinical consequences of lean mass loss are not yet clearly understood. Nevertheless, enhancement of fat loss selectively either as monotherapy or in combination with incretin therapy is expected to provide additional clinical benefit and / or dosing flexibility. As a result, despite the progress in managing weight loss in subjects, there is an ongoing need for agents that promote fat mass loss over lean mass loss.SUMMARY

[0004] Described herein are methods of treating or preventing obesity or an obesity-related disease or disorder and / or for reducing body weight, fat mass, liver weight, or liver mass using a compound or composition described herein. The present disclosure is based, in part, on the discovery that oral, small molecule inhibitors of the ACTRIIB kinase domain can reduce fat mass and overall body weight in a subject, as well as reduce liver weight and liver fat, without a substantial increase in muscle mass. These oral small molecule inhibitors, when combined with the incretin semaglutide, synergistically enhance fat loss and body weight loss, and this enhancement is observed even after discontinuation of semaglutide treatment and concomitant increases in food intake. As generally described herein, theIPTS / 2002322363present disclosure provides methods of treating or preventing obesity or an obesity-related disease or disorder and / or for reducing body weight, fat mass, liver weight, or liver mass.

[0005] Provided herein, in part, is a method of treating or preventing obesity or an obesity-related disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a small molecule inhibitor of an Activin receptor Type II.

[0006] Also provided herein is a method of reducing body weight, fat mass, liver weight, or liver fat in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a small molecule inhibitor of an Activin receptor Type II.

[0007] Also provided herein is a method of increasing oxygen consumption or energy expenditure or decreasing respiratory exchange ratio in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a small molecule inhibitor of an Activin receptor Type II.

[0008] In some embodiments, an Activin receptor Type II is the Activin receptor Type II A isoform or B isoform. In certain embodiments, the small molecule inhibitor is administered to the subject orally. In certain embodiments, the method further comprises administering to the subject a second therapeutic agent. In other embodiments, the second therapeutic agent is a GLP-1 agonist. In some embodiments, the method results in synergistic effect with the second therapeutic agent.

[0009] Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing Detailed Description, Examples, and Claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1A-1L are graphs showing KINOMEscan™ results demonstrating the potency of ACTRIIA and ACTRIIB kinase inhibition by Compounds 1, 2, and 3.

[0011] FIG. 2A-2F are graphs showing PanQinase activity dose response curves of Compounds 1, 2, and 3 against ACVR2A and ACVR2B.

[0012] FIG. 3A-3C are graphs showing SBE reporter cell activity of Compounds 1, 2, and 3.

[0013] FIG. 4A-4C are Western blots of P-SMAD2 / 3 levels in mice tissue (adipose, Figure 4A; muscle, Figure 4B; liver, Figure 4C) upon exposure to Compounds 1 and 2.IPTS / 200232236.3 2

[0014] FIG. 5A-5B are bar charts showing reductions in fat mass and body weight in DIO mice after administration of Compound 1, 2, or 3.

[0015] FIG. 6 is a bar chart showing reductions in liver size and liver fat in DIO mice after administration of Compound 1, 2, or 3.

[0016] FIG. 7 is a bar chart showing that small molecule ACTRIIB inhibitors do not cause lean mass hypertrophy.

[0017] FIG. 8 is a bar chart showing reductions in fat mass and overall body weight in DIO mice using combination of Compound 2 and a GLP-1 agonist.

[0018] FIG. 9 is a bar chart showing reductions in fat mass and overall body weight in DIO mice following discontinuation of a GLP-1 agonist.

[0019] FIG. 10A and FIG. 10B are graphs showing daily average food intake following discontinuation of a GLP-1 agonist.

[0020] FIG. 11 provides representative images of 2 mice from each treatment group in a 3-week diet-induced obesity mouse model experiment. The Day 0 images are from a separate group of mice sacrificed at the beginning of the experiment, while all other images are after 3 weeks of the indicated treatment.

[0021] FIG. 12A and FIG. 12B are bar charts showing reductions in fat mass, body weight, liver mass, and liver fat in DIO mice.

[0022] FIG. 13A-13F are graphs showing changes in certain metabolic parameters in DIO mice after Compound 3 administration.

[0023] FIG. 14 is a bar chart showing reductions in fat mass and body weight in DIO mice with Compound 3 either as a monotherapy or as a combination therapy with a GLP-1 agonist.DETAILED DESCRIPTION

[0024] As generally described herein, the present disclosure provides methods of using compounds and compositions for treating or preventing obesity or an obesity -related disease or disorder and / or for reducing body weight, fat mass, liver weight, or liver mass.I. DEFINITIONS

[0025] The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the present invention. When describing the invention, which may include methods of using compounds and IPTS / 200232236.3 3compositions, the following terms, if present, have the following meanings unless otherwise indicated. It should also be understood that when described herein any of the moieties defined forth below may be substituted with a variety of substituents, and that the respective definitions are intended to include such substituted moieties within their scope as set out below.

[0026] When a range of values is listed, it is intended to encompass each value and subrange within the range. For example, “Ci-6 alkyl” is intended to encompass, Ci, C2, C3, C4, C5, Ce, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6. C4-5, and C5-6 alkyl.

[0027] In general, the term “substituted,” whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.

[0028] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. Also contemplated are embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. Also contemplated are embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0029] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0030] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.IPTS / 200232236.3 4

[0031] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0032] The terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable excipient” are used interchangeably herein and refer to buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see e.g., Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23d ed. 2020).

[0033] The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describes pharmaceutically acceptable salts in detail in J. PHARMACEUTICAL SCIENCES (1977) 66: 1-19, and Gould, Salt selection for basic drugs, INTERNATIONAL JOURNAL OF PHARMACEUTICS, 33 (1986) 201-217. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, IPTS / 200232236.3 5oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(Ci-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0034] As used herein, a “subject” to which administration is contemplated includes, but is not limited to, humans (e.g., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and / or a non-human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. The terms “human,” “patient,” and “subject” are used interchangeably herein.

[0035] Disease, disorder, and condition are used interchangeably herein.

[0036] As used herein, “treat,” “treating,” and “treatment” refer to the treatment of a disease, disorder, or symptom or manifestation of such in a subject, e.g., in a human. This includes: (a) preventing a disease or disorder, (b) inhibiting the disease, disorder, etc., i.e., slowing or arresting its progress or development; and (b) relieving the disease, disorder, etc., i.e., causing regression of the disease state. As used herein, “prevent”, “preventing” and “prevention” refer to causing a disease, disorder, or symptom or manifestation of such not to occur for at least a period of time in at least some subjects.

[0037] As used herein, “obese” or “obesity” means a condition in which an individual has a body mass index (BMI) that is >30.0 kg / m2, or in which an individual has a BMI that is > 27.0 kg / m2and additionally has some co-morbid disorder (i.e. such as Type II diabetes (T2D) or heart failure). See generally, “Overweight & Obesity” by the Center for Disease Control and Prevention available at cdc.gov / obesity / adult / defining.html; and “Definitions & Facts for Adult Overweight & Obesity” by the National Institutes of Health at iddk.nih.gov / health-information / weight-management / adult-overweight-obesity / definition -facts.

[0038] As used herein, “obesity-related disease or disorder” means any diseases or disorders that are induced / exacerbated by obesity including, but not limited to, angina IPTS / 200232236.3 6pectoris, cardiovascular disease, cholecystitis, cholelithiasis, congestive heart failure, dyslipidemia, fatty liver disease, fertility complications, glucose intolerance, gout, hypertension, hypothyroidism, hyperinsulinemia, insulin resistance, osteoarthritis, polycystic ovary syndrome (PCOS), pregnancy complications, psychological disorders, sleep apnea and other respiratory problems, stress urinary incontinence stroke, type 2 diabetes mellitus (T2DM), uric acid nephrolithiasis (kidney stones), and cancer of the breast, colon, endometrium, esophagus, gall bladder, kidney, prostate and rectum.

[0039] As used herein, “overweight” means a condition in which an individual has a BMI that is about 25.0 kg / m2to <30 kg / m2. See, id.

[0040] The term “effective amount” as used herein refers to the amount of a compound, e.g., a compound or composition described herein, sufficient to elicit a beneficial or desired result or biological effect. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound or composition described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route. For example, an effective amount may be an amount of a compound or composition sufficient to achieve one or more of the following: (i) inhibit an activity of Activin receptor Type 11 (ACTRI1A / ACTRIIB); (ii) treat or prevent a disease or condition associated with an activity of Activin receptor Type II (ACTRIIA / ACTRIIB), e.g., obesity or an obesity-related disease, or (iii) reduce body weight, fat mass, liver weight, or liver mass, increase oxygen consumption or energy expenditure, or decrease respiratory exchange ratio.

[0041] As used herein, and unless otherwise specified, a “therapeutically effective amount” of a compound or composition is an amount sufficient to provide a therapeutic benefit in the treatment of a disease or condition, or to delay or minimize one or more symptoms associated with the disease, disorder or condition. As will be appreciated by those of ordinary skill in this art, a therapeutically effective amount of a compound or composition described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject. A therapeutically effective amount of a compound or composition can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route. A therapeutically effective amount of a compound or composition also IPTS / 200232236.3 7includes an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disease, disorder or condition. In addition, a therapeutically effective amount can encompass an amount of a compound or composition that improves overall therapy, reduces or avoids symptoms or causes of disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0042] At various places in the present specification, variables or parameters are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges. For example, an integer in the range of 0 to 20 is specifically intended to individually disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and an integer in the range of 1 to 10 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0043] Throughout the description, where compounds or compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compounds or compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps. Similarly, throughout the description, where compounds or compositions are described as consisting essentially of specific components, or where processes and methods are described as consisting essentially of specific steps, it is contemplated that, additionally, there are compounds or compositions of the present invention that consist of the recited components, and that there are processes and methods according to the present invention that consist of the recited processing steps.

[0044] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0045] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and / or in methods of the presentIPTS / 200232236.3 8invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0046] The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0047] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0048] The term “substantially increase” in the context of a given value described herein refers to value (e.g., a value of lean mass or muscle mass in a subject) that increases by at least 10% as compared to a baseline value.

[0049] The term “does not substantially increase” in the context of a given value described herein refers to value (e.g., a value of lean mass or muscle mass in a subject) in which an increase, if present, is less than 10% as compared to a baseline value.

[0050] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0051] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0052] The claims encompass all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claimIPTS / 200232236.3 9that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.II. METHODS OF USE

[0053] As generally described herein, the present disclosure provides methods of treating or preventing obesity or an obesity -related disease or disorder and / or for reducing body weight, fat mass, liver weight, or liver mass.

[0054] In one aspect, provided herein is a method of treating or preventing obesity or an obesity -related disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a small molecule inhibitor of an Activin receptor Type II.

[0055] In another aspect, the present disclosure provides a method of reducing body weight, fat mass, liver weight, or liver fat in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a small molecule inhibitor of an Activin receptor Type II.

[0056] Also provided herein, in part is a method of increasing oxygen consumption or energy expenditure or decreasing respiratory exchange ratio in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a small molecule inhibitor of an Activin receptor Type II.

[0057] In each of the foregoing, one or more of the following embodiments may apply.

[0058] In some embodiments, the Activin receptor Type II is the Activin receptor Type II A isoform or B isoform.

[0059] In some embodiments, the subject is obese or overweight.

[0060] In other embodiments, the small molecule inhibitor is administered to the subject orally.

[0061] In certain embodiments, the method does not substantially increase muscle mass.

[0062] In some embodiments, the method does not substantially increase lean mass.

[0063] In other embodiments, the method does not result in lean mass hypertrophy.

[0064] In some embodiments, the method further comprises administering to the subject a second therapeutic agent. In other embodiments, the second therapeutic agent is a GLP-1 agonist, for example, the GLP-1 agonist may be selected from the group consisting of IPTS / 200232236.3 10dulaglutide, liraglutide, lixisenatide, semaglutide, exenatide, tirzepatide, albiglutide, retatrutide, orforglipron, and danuglipron. In some embodiments, the GLP-1 agonist is semaglutide. In some embodiments, the GLP-1 agonist may be co-formulated with, or be administered as part of a molecule with, an agent having another activity such as GIP agonism, GIP antagonism, or Glucagon receptor modulation.

[0065] In some embodiments, the method results in synergistic effect with the second therapeutic agent. In certain embodiments, the synergistic effect continues after discontinuing the administration of the second therapeutic agent. In other embodiments, the synergistic effect continues after increasing food intake by the subject.Small molecule inhibitors of an Activin receptor Type II

[0066] In some embodiments, the small molecule inhibitor of an Activin receptor Type II is a compound described in WO2024 / 114661, the entire disclosure of which is hereby incorporated by reference. The compound described in the present disclosure, e.g., the compound of formula (I), (II), (III), (IV), or (V), or a pharmaceutically acceptable salt thereof, as described below, may be prepared according to the procedures described in WO2024 / 114661.

[0067] In certain embodiments, the small molecule inhibitor of an Activin receptor Type II is a compound of Formula (I):wherein:Ar is pyrazole, indole or pyrazole containing substituents; in the pyrazole containing substituents, the substituent is selected from C1-4 hydrocarbon groups, and the number of substituents is 1 or 2; andR is 4-methylpiperazine, piperazine, morpholine,IPTS / 200232236.3 11

[0068] In some embodiments of Formular I, (a) when R is morpholine, Ar is not(b) when R is(c) when Ar is indole, R is notor

[0069] In certain embodiments, the small molecule inhibitor of an Activin receptor Type II is a compound of Formula (II):or a pharmaceutically acceptable salt thereof, wherein:Ar is selected from pyrazole, indole, pyrazole containing substituents, isoxazole containing substituents, pyrimidine or 3-pyridyl; in the pyrazole containing substituents and the isoxazole containing substituents, the substituent is selected from C1-4 hydrocarbon groups, and the number of substituents is 1 or 2; andL, N v R is selected from 4-methylpiperazine, piperazine, morpholine,0,o

[0070] In some embodiments of Formula II, (a) when R is morpholine, Ar is not \IPTS / 200232236.3 12(b) when Ris, Ar is notoro(c) when Ar is indole, R is notor

[0071] In other embodiments, the small molecule inhibitor of an Activin receptor Type II is a compound of Formula (III):(III)or a pharmaceutically acceptable salt thereof, wherein:Ar is selected from pyrazole, indole, pyrazole containing substituents, isoxazole containing substituents, pyrimidine or 3-pyridine; in the pyrazole containing substituents and the isoxazole containing substituents, the substituent is selected from C1-4 hydrocarbon groups, and the number of substituents is 1 or 2; andR is selected from 4-methylpiperazine, piperazine,°o O, o or

[0072] In some embodiments, the small molecule inhibitor of an Activin receptor Type II is a compound of Formula (IV):T ilArNH(IV)or a pharmaceutically acceptable salt thereof, wherein:Ar is selected from pyrazole, indole, pyrazole containing substituents, isoxazole IPTS / 200232236.3 13containing substituents, pyrimidine or 3-pyridine; in the pyrazole containing substituents and the isoxazole containing substituents, the substituent is selected from C1-4 hydrocarbon groups, and the number of substituents is 1 or 2; andR is selected from 4-methylpiperazine, piperazine, morpholine,°,

[0073] In some embodiments of Formula (IV), (a) when R is morpholine, Ar is notN(b) when R is / N, Ar is notO(c) when Ar is indole, R is notor

[0074] In certain embodiments, the small molecule inhibitor of an Activin receptor Type II is a compound of Formula (V):(V)or a pharmaceutically acceptable salt thereof, wherein:Ar is selected from pyrazole, indole, pyrazole containing substituents, isoxazole containing substituents, pyrimidine, pyridine or phenyl; in the pyrazole containing substituents and the isoxazole containing substituents, the substituent is selected from C1-4 hydrocarbon groups, and the number of substituents is 1 or 2; ando'^R is selected from 4-methylpiperazine, piperazine, morpholine,IPTS / 200232236.3 14o

[0075] In some embodiments of Formula (V),HN—,a) when R is morpholine, Ar is not;\N-,Oj1b) when Ris, Ar is not; orOc) when Ar is indole, R is notor

[0076] In certain embodiments, in Formulae (I), (II), (III), (IV), (V) or pharmaceutically Hacceptable salts thereof, the Ar is indole. In certain embodiments, the indole isx

[0077] In certain embodiments, in Formulas (I), (II), (III), (IV), (V) or pharmaceutically acceptable salts thereof, the Ar is pyrazole or pyrazole containing substituents.

[0078] In certain embodiments, in Formulas (I), (II), (III), (IV), (V) or pharmaceutically acceptable salts thereof,^ 'NH N-NH < JN, ii -•< „.. J 7" r' the Ar is selected from,S''In some embodiments, the Ar is

[0079] In certain embodiments, in Formulas (I), (II), (III), (IV), (V) or pharmaceutically acceptable salts thereof,the R is 4-methylpiperazine. In some embodiments, the R is”.

[0080] In certain embodiments, the small molecule inhibitor of an Activin receptor Type II is selected from the group consisting of:IPTS / 200232236.3 15IPTS / 200232236.3 16, or a pharmaceutically acceptable salt thereof.EXAMPLES

[0081] In order that the invention described herein may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions and methods provided herein and are not to be construed in any way as limiting their scope.

[0082] In the Examples section, Compound 1 refers to the compound of the structure:. Compound 2 refers to the compound of the structure:Compound 3 refers to the compound of the structure:. The synthesis of each of Compounds 1, 2, and 3 is described in WO2024 / 114661.Example 1: Compounds 1, 2, and 3 inhibit ACTRIIB in vitro and in vivo

[0083] Activins (A-E) signal through a complex of a Type I receptor (ALK1-7) and a Type II receptor, in particular ACTRII (A / B), in various tissues including muscle andIPTS / 200232236.3 17adipose, with a range of downstream gene transcription effects that depend on cell type. This example describes the inhibition of the Activin receptor Type II, isoform B (ACTRIIB) kinase activity by small molecule compounds used to demonstrate the biological consequences of such inhibition (Compounds 1, 2, and 3 in this application).Biochemical inhibition

[0050] Two different biochemical assays were used to demonstrate the potency of ACTRIIA and ACTRIIB kinase inhibition by Compounds 1, 2, and 3. Each of these assays are described separately below.

[0051] KINOMEscan™ (Eurofins Discovery) is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active-site directed ligand. The assay is performed by combining three components: DNA-tagged kinase; immobilized ligand; and a test compound. The ability of the test compound to compete with the immobilized ligand is measured via quantitative PCR of the DNA tag.

[0052] Kinase-tagged T7 phage strains were prepared in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage and incubated with shaking at 32°C until lysis. The lysates were centrifuged and filtered to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% bovine serum albumin (BSA), 0.05% Tween 20, 1 mM dithiothreitol (DTT)) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in lx binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 111X stocks in 100% dimethylsulfoxide (DMSO). Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements were distributed by acoustic transfer (non-contact dispensing) in 100% DMSO. The compounds were then diluted directly into the assays such that the final concentration of DMSO was 0.9%. All reactions were performed in polypropylene 384-well plate. Each was a final volume of 0.02 mL. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (lx phosphate-buffered saline (PBS), 0.05% Tween 20). The beads were then re-suspended in elution buffer (lx PBS, 0.05% Tween 20, 0.5 uM nonbiotinylatedIPTS / 200232236.3 18affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.

[0053] Binding constants (Kds) were calculated with a standard dose-response curve using the Hill equation: Response = Background + ((Signal - Background) / (1 + (Kd Hill slope / DoseHill slope))). The Hill Slope was set to -1. Curves were fitted using a non-linear least square fit with the Levenberg-Marquardt algorithm.

[0054] KinomeScan results are shown in Table 1 and Figures 1A-1L. The calculated Kd for ACTRIIB inhibition was 100 nM for Compound 1, 1400 nM for Compound 2, and 260 nM for Compound 3. The calculated Kd for ACTRIIA inhibition was 230 nM for Compound 1, 1700 nM for Compound 2, and 900 nM for Compound 3.Table 1: KinomeScan activity dose response data and Kd calculationCmpd DiscoveRx Kd (nM) DiscoveRx Kd (nM)Gene GeneSymbol Symbol1 ACVR2B 100 ACVR2A 2302 ACVR2B 1400 ACVR2A 17003 ACVR2B 260 ACVR2A 900Raw KinomeScan dataCmpd DiscoveRx Compound Signal Replicate DiscoveRx Compound Signal Replicate Gene Cone. ID Gene Cone. (nM) ID Symbol (nM) Symbol1 ACVR2B 0 1.29E-06 1 ACVR2A 0 1.51E-06 1 1 ACVR2B 0 2.20E-06 1 ACVR2A 0 1.71E-06 1 1 ACVR2B 0 1.68E-06 1 ACVR2A 0 1.37E-06 1 1 ACVR2B 0.169 2.18E-06 1 ACVR2A 0.169 1.52E-06 1 1 ACVR2B 0.508 2.02E-06 1 ACVR2A 0.508 1.48E-06 1 1 ACVR2B 1.52 2.11E-06 1 ACVR2A 1.52 1.31E-06 1 1 ACVR2B 4.57 1.66E-06 1 ACVR2A 4.57 1.84E-06 1 1 ACVR2B 13.7 1.86E-06 1 ACVR2A 13.7 1.19E-06 1 1 ACVR2B 41.2 1.19E-06 1 ACVR2A 41.2 9.50E-07 1IPTS / 200232236.3 191 ACVR2B 123 7.50E-07 1 ACVR2A 123 9.40E-07 1 1 ACVR2B 370 2.79E-07 1 ACVR2A 370 5.35E-07 1 1 ACVR2B 1110 1.44E-07 1 ACVR2A 1110 3.25E-07 1 1 ACVR2B 3330 7.77E-08 1 ACVR2A 3330 1.58E-07 1 1 ACVR2B 10000 4.58E-08 1 ACVR2A 10000 1.24E-07 1 1 ACVR2B 0 2.03E-06 2 ACVR2A 0 2.32E-06 2 1 ACVR2B 0 2.21E-06 2 ACVR2A 0 2.28E-06 2 1 ACVR2B 0 1.82E-06 2 ACVR2A 0 2.01E-06 2 1 ACVR2B 0.169 2.12E-06 2 ACVR2A 0.169 1.62E-06 2 1 ACVR2B 0.508 2.09E-06 2 ACVR2A 0.508 1.27E-06 2 1 ACVR2B 1.52 2.05E-06 2 ACVR2A 1.52 1.22E-06 2 1 ACVR2B 4.57 1.70E-06 2 ACVR2A 4.57 1.85E-06 2 1 ACVR2B 13.7 1.56E-06 2 ACVR2A 13.7 1.40E-06 2 1 ACVR2B 41.2 1.86E-06 2 ACVR2A 41.2 1.69E-06 2 1 ACVR2B 123 9.42E-07 2 ACVR2A 123 1.40E-06 2 1 ACVR2B 370 4.29E-07 2 ACVR2A 370 6.28E-07 2 1 ACVR2B 1110 1.31E-07 2 ACVR2A 1110 3.54E-07 2 1 ACVR2B 3330 6.04E-08 2 ACVR2A 3330 1.97E-07 2 1 ACVR2B 10000 4.25E-08 2 ACVR2A 10000 1.49E-07 2 2 ACVR2B 0 2.63E-06 1 ACVR2A 0 1.22E-06 1 2 ACVR2B 0 2.59E-06 1 ACVR2A 0 1.62E-06 1 2 ACVR2B 0 1.75E-06 1 ACVR2A 0 1.69E-06 1 2 ACVR2B 0.169 1.76E-06 1 ACVR2A 0.169 1.41E-06 1 2 ACVR2B 0.508 1.82E-06 1 ACVR2A 0.508 1.54E-06 1 2 ACVR2B 1.52 1.66E-06 1 ACVR2A 1.52 1.44E-06 1 2 ACVR2B 4.57 1.66E-06 1 ACVR2A 4.57 1.70E-06 1 2 ACVR2B 13.7 1.64E-06 1 ACVR2A 13.7 1.30E-06 1 2 ACVR2B 41.2 2.07E-06 1 ACVR2A 41.2 1.17E-06 1IPTS / 200232236.3 202 ACVR2B 123 2.47E-06 1 ACVR2A 123 1.44E-06 1 2 ACVR2B 370 1.44E-06 1 ACVR2A 370 1.05E-06 1 2 ACVR2B 1110 1.06E-06 1 ACVR2A 1110 7.16E-07 1 2 ACVR2B 3330 6.53E-07 1 ACVR2A 3330 2.71E-07 1 2 ACVR2B 10000 1.97E-07 1 ACVR2A 10000 2.52E-07 1 2 ACVR2B 0 2.00E-06 2 ACVR2A 0 1.56E-06 2 2 ACVR2B 0 2.63E-06 2 ACVR2A 0 1.52E-06 2 2 ACVR2B 0 2.50E-06 2 ACVR2A 0 1.38E-06 2 2 ACVR2B 0.169 1.62E-06 2 ACVR2A 0.169 1.41E-06 2 2 ACVR2B 0.508 2.48E-06 2 ACVR2A 0.508 1.34E-06 2 2 ACVR2B 1.52 2.32E-06 2 ACVR2A 1.52 1.37E-06 2 2 ACVR2B 4.57 1.92E-06 2 ACVR2A 4.57 1.29E-06 2 2 ACVR2B 13.7 2.07E-06 2 ACVR2A 13.7 1.19E-06 2 2 ACVR2B 41.2 1.79E-06 2 ACVR2A 41.2 1.70E-06 2 2 ACVR2B 123 1.94E-06 2 ACVR2A 123 1.29E-06 2 2 ACVR2B 370 1.52E-06 2 ACVR2A 370 1.27E-06 2 2 ACVR2B 1110 1.40E-06 2 ACVR2A 1110 1.04E-06 2 2 ACVR2B 3330 5.03E-07 2 ACVR2A 3330 5.61E-07 2 2 ACVR2B 10000 1.59E-07 2 ACVR2A 10000 1.67E-07 2 3 ACVR2B 0 1.49E-06 1 ACVR2A 0 2.12E-06 1 3 ACVR2B 0 1.43E-06 1 ACVR2A 0 1.68E-06 1 3 ACVR2B 0 1.55E-06 1 ACVR2A 0 1.46E-06 1 3 ACVR2B 0.169 1.41E-06 1 ACVR2A 0.169 1.34E-06 1 3 ACVR2B 0.508 2.00E-06 1 ACVR2A 0.508 1.62E-06 1 3 ACVR2B 1.52 1.41E-06 1 ACVR2A 1.52 1.69E-06 1 3 ACVR2B 4.57 1.39E-06 1 ACVR2A 4.57 1.40E-06 1 3 ACVR2B 13.7 1.51E-06 1 ACVR2A 13.7 1.47E-06 1 3 ACVR2B 41.2 1.11E-06 1 ACVR2A 41.2 1.33E-06 1IPTS / 200232236.3 213 ACVR2B 123 9.43E-07 1 ACVR2A 123 1.48E-06 1 3 ACVR2B 370 6.15E-07 1 ACVR2A 370 8.98E-07 1 3 ACVR2B 1110 2.93E-07 1 ACVR2A 1110 7.67E-07 1 3 ACVR2B 3330 1.38E-07 1 ACVR2A 3330 5.02E-07 1 3 ACVR2B 10000 7.56E-08 1 ACVR2A 10000 2.66E-07 1 3 ACVR2B 0 1.44E-06 2 ACVR2A 0 1.91E-06 2 3 ACVR2B 0 1.39E-06 2 ACVR2A 0 1.50E-06 2 3 ACVR2B 0 1.49E-06 2 ACVR2A 0 2.00E-06 2 3 ACVR2B 0.169 1.11E-06 2 ACVR2A 0.169 1.56E-06 2 3 ACVR2B 0.508 1.78E-06 2 ACVR2A 0.508 1.78E-06 2 3 ACVR2B 1.52 1.25E-06 2 ACVR2A 1.52 1.50E-06 2 3 ACVR2B 4.57 1.53E-06 2 ACVR2A 4.57 1.61E-06 2 3 ACVR2B 13.7 1.39E-06 2 ACVR2A 13.7 1.65E-06 2 3 ACVR2B 41.2 1.07E-06 2 ACVR2A 41.2 1.15E-06 2 3 ACVR2B 123 1.11E-06 2 ACVR2A 123 1.28E-06 2 3 ACVR2B 370 6.29E-07 2 ACVR2A 370 1.12E-06 2 3 ACVR2B 1110 3.15E-07 2 ACVR2A 1110 7.29E-07 2 3 ACVR2B 3330 1.47E-07 2 ACVR2A 3330 4.78E-07 2 3 ACVR2B 10000 1.38E-07 2 ACVR2A 10000 3.03E-07 2

[0055] The IC50 profile of Compounds 1, 2, and 3 against ACTRIIA and ACTRIIB was determined using the 33PanQinaseTM Activity Assay (Reaction Biology Europe). IC50 values were measured by testing 10 concentrations (IE-05 M to 3E-10 M) of each compound in duplicate.

[0056] The compounds were dissolved to IE-03 M in volumes of 100% DMSO, and 100 pL of each of the stock solutions were transferred into column 2 of a 96 well master plate. Prior to testing, the IE-03 M stock solutions in column 2 of the master plate were subjected to a serial, semi -logarithmic dilution using 100 % DMSO as a solvent. This resulted in 10 distinct concentrations, with a dilution endpoint of 3E-08 M / 100 % DMSO in column 12. Columns 1 and 7 were filled with 100 % DMSO as controls. Subsequently, 4 x 5 pL from IPTS / 200232236.3 22each well of the serial diluted copy plate were aliquoted with a 96 channel pipettor into four identical compound dilution plates. In the process, 45 µL H₂O were added to each well of two compound dilution plates. To minimize potential precipitation, the H₂O was added to each plate only a few minutes before the transfer of the compound solutions into the assay plates. The plates were shaken thoroughly, resulting in compound dilution plates / 10 % DMSO.

[0057] For the assays, 5 ii L solution from each well of the compound dilution plates / 10 % DMSO were transferred into the assay plates. The final volume of the assay was 50 pL. All compounds were tested at 10 final assay concentrations in the range from IE-05 M to 3E-10 M, in duplicate. The final DMSO concentration in the reaction cocktails was 1 % in all cases. The configuration of an IC50 assay plate for 8 test compounds is depicted in the following diagram:Configuration of IC50assay plates,8 compounds / plate

[0058] All protein kinases were expressed in Sf9 insect cells or in E. coli as recombinant GST-fusion proteins or His-tagged proteins, either as full-length or enzymatically active fragments. All kinases were produced from human cDNAs and purified by either glutathione (GSH)-affinity chromatography or immobilized metal affinity chromatography. Affinity tags were removed from several kinases during purification. The purity of the protein kinases was examined by SDS-PAGE / Coomassie staining, the identity was checked by mass spectroscopy. Kinases from external vendors (CAR = Cama Biosciences Inc.; 1NV = Life Technologies (Invitrogen Corporation); MIL = Merck-Millipore (Millipore Corporation), see Table A) were expressed, purified and quality-controlled by virtue of the vendors readings.

[0059] A radiometric protein kinase assay (33PanQinaseTM Activity Assay) was used for measuring the kinase activity of the two protein kinases. All kinase assays were performed inIPTS / 200232236.3 2396-well ScintiPlatesTM from PerkinElmer (Boston, MA, USA) in a 50 pL reaction volume. The reaction cocktail was pipetted in four steps in the following order:

[0060] 25 pL of assay buffer (standard buffer / [g-33P]-ATP)

[0061] 10 pL of ATP solution (in H2O)

[0062] 5 pL of test compound (in 10 % DMSO)

[0063] 10 pL of enzyme / substrate mixture

[0064] The assay for all protein kinases contained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 pM Na-orthovanadate, 1.2 mM DTT, 50 pg / mL PEG20000, ATP (variable concentrations, corresponding to the apparent ATP-Km of the respective kinase), [g-33P]-ATP (approx. 8 x 1005 cpm per well), protein kinase (variable amounts), and substrate (variable amounts). The following amounts of enzyme and substrate were used per well:Table A: Assay parameters for the tested protein kinases.# Kinase Kinase PQ Kinase Kinase ATP Cone. Substrate Substrate Name Lot Cone. Cone. (pM) Name Cone.(ng / 50pl) (nM) * (pg / 50pl) 1 ACVR2A 004,8 60 14.1 3.0 Casein 1.0 2 ACVR2B 002,10 70 22.6 1.0 RB-CTF 1.0

[0065] PanQinase results are shown in Table 2 and Figures 2A-2F. The calculated IC50 for ACTRIIB inhibition was 215 nM - 384 nM for Compound 1, 629 nM - 1.8 pM for Compound 2, and 268 nM - 277 nM for Compound 3.

[0066] The calculated IC50 for ACTRIIA inhibition was 498 nM - 514 nM for Compound 1, 660 nM - 884 nM for Compound 2, and 2.3 pM - 3.1 pM for Compound 3.Table 2: PanQinase activity IC50SACVR2B, 1st ACVR2B, 2nd ACVR2A, 1st ACVR2A, 2nd Compound ID IC50 (M) IC50 (M) IC50 (M) IC50 (M) Compound 1 2.15E-07 3.84E-07 4.98E-07 5.14E-07 Compound 2 6.29E-07 1.80E-06 6.60E-07 8.84E-07 Compound 3 2.77E-07 2.68E-07 2.29E-06 3.05E-06IPTS / 200232236.3 24Cell-based inhibition

[0067] Smad-binding element (SBE) Reporter - HEK293 cells (BPS Bioscience) were cultured in Minimum Essential Medium(MEM) medium with 10% fetal bovine serum (FBS), 1% non-essential amino acid, ImM Na-pyruvate, 1% Pen-strep, and 400 pg / mL Geneticin. Cells were seeded at 35,000 cells per well into white clear-bottom 96-well microplate in 100 pL of growth medium without Geneticin. Cells were incubated at 37°C and 5% CO2 overnight. The next day, media was removed and a serial dilution of compounds was prepared in assay media (growth media + 0.5% FBS without Geneticin). 90 pL of diluted compounds was added to the treatment wells. 90 pL of assay medium without compound was added to the control wells. 100 pL of assay medium was added to cell-free control wells for determining background luminescence. Cells were incubated at 37°C and 5% CO2 for 4-5 hours. 10 pL of diluted Activin A in assay medium was added to wells (final [Activin A] = 50 ng / mL). 10 pL of assay medium was added to unstimulated control wells. Cells were treated overnight. After treatment, cells were lysed and the luciferase assay was performed using ONE-Step luciferase assay system: add 100 pL of One-Step Luciferase reagent per well and rock at room temperature for -15-20 minutes. Luminescence was measured using a luminometer (BioTek SynergyTM 2 microplate reader).

[0068] The assay was performed in triplicate at each concentration. The luminescence intensity data were analyzed using the computer software. Graphpad Prism. In the absence of the compound, the luminescence intensity (Lt) in each data set was defined as 100%. In the absence of cells, the luminescence intensity (Lb) in each data set was defined as 0%. The percent luminescence in the presence of each compound was calculated according to the following equation: % Luminescence = (L-Lb)Z(Lt-Lb), where L= the luminescence intensity in the presence of the compound, Lb= the luminescence intensity in the absence of cells, and Lt = the luminescence intensity in the absence of the compound. The values of percent luminescence versus a series of compound concentrations were then plotted using non-linear regression analysis of Sigmoidal dose-response curve generated with the equation Y=B+(T-B) / l+10((LoglC50-X)xHill Slope), where Y=percent luminescence, B=minimum percent luminescence, T=maximum percent luminescence, X= logarithm of compound and HillIPTS / 200232236.3 25Slope=slope factor or Hill coefficient. The IC50 value was determined by the concentration causing a half-maximal percent activity.

[0069] SBE reporter cell assay results are shown in Table 3 and Figures 3A-3C. The calculated IC50 for inhibition of Activin A-induced gene expression was 290 nM for Compound 1, 2320 nM for Compound 2, and 1010 nM for Compound 3.Table 3: SBE reporter cell activityCompound 1 Luminescence intensity % Luminescence(pM) Repeat 1 Repeat2 Repeat3 Repeat 1 i Repeat2 i Repeat30 65,268 71,477 70,038 95 104 | 102 0.0005 62,246 70,414 76,802 90 102 1110.002 67,614 67.761 73,510 98 | 98 1070.005 69,445 70,403 | 78,243 101 102 1140.01 62,163 67,690 | 67,832 90 98 980.04 73,284 72,541 | 73,509 106 105 1070.12 63.787 61.339 66,373 93 | 89 960.37 26,438 | 28,066 26,488 38 j 41 I 381.11 8,400 8,779 | 8,729 12 | 13 133.33 5,010 4,360 4.296 7 | 6 610 139 129 177 0 0 0no Activin 5,039 4,586 4,886 7 7 7 Background 137 63 95 1 1Compound 2 Luminescence intensity % Luminescence(pM) Repeat 1 1 Repeat 2 | Repeat? Repeat 1 Repeat2 1 Repeat30 53,633 | 49,256 | 49,165 106 97 970.0005 48.163 45,481 | 47,854 95 90 040.002 51.203 46,492 ' 43,182 101 92 850.005 50,585 45,661 49,122 100 1 90 i 970.01 51,836 46.179 38,951 102 1 91 | 770.04 60.886 49.673 50,791 120 | 98 | 1000.12 64,021 j 48,680 55,808 126 96 1 1 100.37 53,613; 52,866 43,963 106 104 871.11 50,414 i 36,776 43,905 99 72! 873.33 15.935 13.763 9,066 31 27! 1810 246 152 | 236 0 | 0 | 0no Activin 3,813 3,585 3.614 7 7 7 Background 178 133 75Compound 3 Luminescence intensity % Luminescence(pM) Repeat 1 i Repeat2 Repeat3 Repeatl | Repeat2 Repeat30 52,555; 48,762 53,229 102 95 103 IPTS / 200232236.3 260.0005 52,383 | 47,687 49,401 102 93 960.002 46,319 | 52,685 47,855 90 102 930.005 52,332 i 50,128 52,624 102 97 1020.01 47,971 i 44,337 48,678 93 86 940.04 50.991!44.002 47,624 99 85 920.12 48.323 40.850 51,169 94 79 990.37 44.081!42.671 43,769 86 83 851.11 30,232 i 27,884 25,047 59 54 483.33 11,803! 11.583 10,052 23 22 1910 12,589 10,500 9,179 24 20 18no Activin 3,689 3,650 3,150 7 7 6Background 178 133 75In vivo inhibition

[0070] Ligand binding to a complex of a Type I receptor and ACTRIIB results in ACTRIIB phosphorylating the Type I receptor, and subsequently the Type I receptor phosphorylating the transcription factors SMAD2 and SMAD3, which enables their translocation to the nucleus and activation of downstream gene transcription. The ability of Compounds 2 and 3 to inhibit ACTRIIB activity in vivo was assessed by examining the extent of SMAD2 and SMAD3 phosphorylation in various mouse tissues following oral dosing of these compounds. As SMAD2 and SMAD3 are difficult to distinguish from each other by antibody binding, they are measured together as a combined SMAD2 / 3 pool.

[0071] Compounds 2 and 3 were evaluated in vivo following oral dosing of diet-induced obese (DIO) mice over three weeks, with various doses and frequencies of dosing. To assess inhibition of ACTRIIB in vivo, various tissues were excised from the DIO mice following sacrifice at the end of the three week period, and Western blots were performed on protein extracts of these tissues to determine the concentration of total SMAD2 / 3 protein and the concentration of phosphorylated SMAD2 / 3 (P-SMAD2 / 3) in each tissue. The ratio of P-SMAD2 / 3 to total SMAD2 / 3 protein indicates the extent of inhibition of Activin signaling in a given tissue. While not a specific measure of ACTRIIB activity alone, this determination indicates the relative potency of signaling pathway blockade in vivo following oral administration of the compounds.

[0072] Male C57BL / 6J-DIO mice were obtained from Gempharmatech (Nanjing, China) at 20 weeks of age, having been maintained on a high fat diet (HFD, 60% kcal% fat). Mice were singly housed per cage and maintained on the HFD, assigned to groups of 8 animals per treatment condition, with an average body weight of 50-52 g at the start of the study in each group. Compounds were prepared as uniform suspensions in the vehicle solution ((3% IPTS / 200232236.3 27DMSO + 2% Solutol + 95% (0.9% Saline)) and stored at 4°C when not in use. Compounds were prepared at a concentration such that the below dose levels were achieved with oral administration of 5 mL / kg per dose. Treatment groups included: Vehicle QD (once per day). Compound 2 at 20 mg / kg QD, Compound 3 at 20 mg / kg QD, and Compound 3 at 10 mg / kg BID (twice per day). Following three weeks of dosing, animals were sacrificed and tissues (including adipose, muscle, and liver) were extracted and snap-frozen. Adipose tissue included both inguinal and peri-renal fat pads, and muscle tissue included pectoralis, gastrocnemius, soleus, tibialis, and quadriceps muscles.

[0073] Tissues were added to RIPA Lysis Buffer (Beyotime) (containing inhibitors) by weight in a ratio of 1: 10 (or 1:4 for adipose tissue). Mechanical homogenization was performed under ice bath conditions, at 60 Hz for 240 s. For adipose tissue, excess fat was removed using a protein defatting column. Protein concentration in the supernatant was measured by BCA assay, and 50 ug of protein was mixed with loading buffer and heated at 95 °C for 10 min. Proteins were loaded per lane, resolved by 4-12% SDS-PAGE and transferred to a nitrocellulose (NC) membrane. Blots were incubated in blocking buffer for 1 h and then incubated with primary antibodies (Anti-SMAD2 / 3 (CST), 1:500, Anti-Phospho-SMAD2 / SMAD3 (CST), 1:500) overnight at 4°C. After incubation with primary antibodies, the blots were washed three times in tris-buffered saline with Tween 20 (TBST) and incubated with goat anti-rabbit secondary antibody (Licor) for 1 h. After incubation with secondary antibodies, the blots were washed three times in TBST. The blots were detected by Odyssey CLx. The signal intensity of the bands was quantified and normalized to the corresponding signal for -Actin.

[0074] Tissue specific Western blot data for Compounds 2 and 3 are shown in Figures 4A-4C and Table 4. Compared to animals treated only with vehicle, the P-SMAD2 / 3 to SMAD2 / 3 ratio change from baseline was reduced by all three compounds in various tissues.Table 4: Mouse tissue Western blot data for P-SMAD2 / 3AdiposetissueGroup Relative Relative density Relative Relative density Relative Density ID density ratio density ratio density ratio ratio*£2 Actin AVE SMA Actin SMAD2 AVE £2 AVESM SMAD2X D2\3 \3 / SMA3 / Actin D2\3 / IPTS / 200232236.3 28AD Actin SMA2X3 D2\3Baseline 35.9 235 0.1528 0.1650 136 2060 0.0660 0.0449 2.3140 4.879 (Day 0 9 sacrifice)33.5 189 0.1772 75.7 3180 0.0238 7.4458Vehicle 74.6 370 0.2016 0.2060 140 2970 0.0471 0.0343 4.2773 7.041774.7 355 0.2104 103 4800 0.0215 9.8061Cmpd 1. 52.1 302 0.1725 0.2202 43.3 3120 0.0139 0.0434 12.430 8.050 0.21 20 8 0 mg / kgQD78.2 292 0.2678 127 1740 0.0730 3.6692Cmpd 2, 72.1 220 0.3277 0.2637 131 1020 0.1284 0.0723 2.5518 7.420 0.08 20 0 mg / kgQD62.9 315 0.1997 33.8 2080 0.0163 12.2882Cmpd 2. 36.2 352 0.1028 0.2692 131 2750 0.0476 0.1268 2.1589 2.136 -1.01 10 9 mg / kgBID67.5 155 0.4355 97.6 474 0.2059 2.1150MuscletissueGroup Relative Relative density Relative Relative density Relative Density ID density ratio density ratio density ratio ratio*Actin AVE SMA Actin SMAD2 AVE Be AVE SM SMAD2\ D2\3 \3 / Actin SMAAD 3 / Actin D2\3 / 2X3 SMAD2\3Baseline 36.4 1160 0.0314 0.0189 57.2 198 0.2889 0.3898 0.1086 0.060 (Day 0 9 sacrifice)4.87 757 0.0064 105.0 214 0.4907 0.0131IPTS / 200232236.3 29Vehicle 117 1350 0.0867 0.0571 81.9 562 0.1457 0.1392 0.5947 0.401452.2 1890 0.0276 84.8 639 0.1327 0.2081Cmpdl, 33 530 0.0623 0.0419 109 112 0.9732 0.9029 0.0640 0.044 -5.86 20 9 mg / kgQD26.9 1250 0.0215 189 227 0.8326 0.0258Cmpd 2, 26.1 1040 0.0251 0.0437 142 188 0.7553 0.5368 0.0332 0.114 -4.71 20 6 mg / kgQD55 882 0.0624 72.9 229 0.3183 0.1959Cmpd 2, 23.7 1120 0.0212 0.0134 69.4 329 0.2109 0.3750 0.1003 0.055 -5.68 10 4 mg / kgBID6.79 1200 0.0057 166 308 0.5390 0.0105LivertissueGroup Relative Relative density Relative Relative density Relative Density ID density ratio density ratio density ratio ratio*Ec Actin Ec AVE SMA Actin SMAD2 AVE Ec AVE SM SMAD2\ D2\3 \3 / Actin SMAAD 3 / Actin D2\3 / 2\3 SMAD2\3Baseline 202 4660 0.0433 0.0353 82.8 3310 0.0250 0.0319 1.7329 1.217 (Day 0 1 sacrifice)92.4 3390 0.0273 103 2650 0.0389 0.7013Vehicle 268 2550 0.1051 0.1089 73.6 2360 0.0312 0.0282 3.3700 3.9178301 2670 0.1127 56.8 2250 0.0252 4.4657Cmpd 1. 256 3130 0.0818 0.0837 84.3 2320 0.0363 0.0395 2.2509 2.127 -1.47 20 4 mg / kgQD260 3040 0.0855 95.6 2240 0.0427 2.0040IPTS / 200232236.3 30Cmpd 2. 189 3620 0.0522 0.0569 69.6 2450 0.0284 0.0262 1.8379 2.199 -1.41 20 5 mg / kgQD223 3620 0.0616 63.5 2640 0.0241 2.5611Cmpd 2. 258 4020 0.0642 0.0556 64.7 2690 0.0241 0.0265 2.6683 2.144 -1.46 10 5 mg / kgBID232 4930 0.0471 90.3 3110 0.0290 1.6207*Density ratio relative to baseline, vehicle adjusted

[0075] This example demonstrates that Compounds 1, 2, and 3 inhibit the kinase activity of ACTRIIB sufficiently to block intracellular signaling by that receptor when used in biochemical and cell-based assays, and in vivo.Example 2: ACTRIIB inhibitors reduce fat mass, liver mass, liver fat, and overall body weight in DIO mice

[0076] This example describes the in vivo phenotypic effects of oral administration of Compounds 2 and 3 in DIO mice at various doses and dose frequencies. Treatment with these Compounds, as compared to treatment with Vehicle alone, over three weeks, leads to reduced fat mass (as measured by NMR), reduced overall body weight, reduced liver mass, and reduced liver fat (as measured by densitometry).

[0077] Compounds 1 and 2 were evaluated in vivo following oral dosing of various doses in diet-induced obese (DIO) mice over two weeks. Male C57BL / 6J-DIO mice were obtained from Gempharmatech (Nanjing, China) at 20 weeks of age, having been maintained on a high fat diet (HFD, 60% kcal% fat). Mice were singly housed per cage and maintained on the HFD, assigned to groups of 8 animals per treatment condition, with an average body weight of 50-52 g at the start of the study in each group. Compounds were prepared as uniform suspensions in the vehicle solution ((3% DMSO + 2% Solutol + 95% (0.9% Saline)) and stored at 4°C when not in use. Compounds were prepared at a concentration such that the below dose levels were achieved with oral administration of 5 mL / kg per dose. Treatment groups included: Vehicle QD (once per day), Compound 1 at 3 mg / kg QD, 10 mg / kg QD, and 30 mg / kg QD, and Compound 2 at 3 mg / kg QD, 10 mg / kg QD, and 30 mg / kg QD. Body weight and food intake were recorded twice a week during the course of the study. BodyIPTS / 200232236.3 31composition (fat mass and lean mass) was measured using a Bruker TD-NMR minispec Whole Body Composition Live Mice Analyzer (LF50) at baseline and weekly for 2 weeks.

[0078] Compounds 2 and 3 were evaluated in vivo following oral dosing of diet-induced obese (DIO) mice over three weeks, with various doses and frequencies of dosing. Male C57BL / 6J-DIO mice were obtained from Gempharmatech (Nanjing, China) at 20 weeks of age, having been maintained on a high fat diet (HFD, 60% kcal% fat). Mice were singly housed per cage and maintained on the HFD, assigned to groups of 8 animals per treatment condition, with an average body weight of 50-52 g at the start of the study in each group. Compounds were prepared as uniform suspensions in the vehicle solution ((3% DMSO + 2% Solutol + 95% (0.9% Saline)) and stored at 4°C when not in use. Compounds were prepared at a concentration such that the below dose levels were achieved with oral administration of 5 mL / kg per dose. Treatment groups included: Vehicle QD, Compound 2 at 20 mg / kg QD, Compound 3 at 20 mg / kg QD, and Compound 3 at 10 mg / kg BID (twice per day). Body weight and food intake were recorded twice a week during the course of the study (day 1, day 4, day 8, day 11, day 15, day 18, day 21). Body composition (fat mass and lean mass) was measured using a Bruker TD-NMR minispec Whole Body Composition Live Mice Analyzer (LF50) at baseline and weekly for 3 weeks. At the end of the study, mice were sacrificed and livers removed and weighed. Liver fat was measured by densitometry of individual organs.

[0079] Compound 3 was evaluated in vivo following oral dosing of diet-induced obese (DIO) mice over 7 days. Male C57BL / 6J-DIO mice were obtained from The Jackson Laboratory (Bar Harbor, ME) at 18 weeks of age, having been maintained on a high fat diet (HFD, 60% kcal% fat). Mice were singly housed in Promethion Core metabolic monitoring cages with beam break activity monitoring (Sable Systems International, Las Vegas, NV) and maintained on the HFD, assigned to groups of 8 animals per treatment condition, with an average body weight of 45-50 g at the start of the study in each group. Compound 3 was prepared as a uniform suspension in the vehicle solution ((3% DMSO + 2% Solutol + 95% (0.9% Saline)) and stored at 4°C when not in use. Treatment groups included: Vehicle QD and Compound 3 at 30 mg / kg QD. Body weight and composition were monitored over 7 days, and at the end of the study mice were sacrificed and individual tissues weighed, including liver, epidydimal white adipose tissue, and gastrocnemius muscle.

[0080] Reductions in fat mass and overall body weight in DIO mice are shown in Figures 5A-5B, Table 5, and Figures 12A-12B. Over two weeks, compared to vehicle, Compound 1 at 3 mg / kg QD reduced body weight by 0.45 g (0.9%) and reduced fat mass by 0.66 g (3.8%); Compound 1 at 10 mg / kg QD reduced body weight by 0.22 g (0.4%) and reduced fat mass by IPTS / 200232236.3 320.88 g (5.0%); Compound 1 at 30 mg / kg QD reduced body weight by 7.0 g (14.2%) and reduced fat mass by 6.1 g (34.6%); Compound 2 at 3 mg / kg QD reduced body weight by 0.19 g (0.4%) and reduced fat mass by 0.52 g (3.0%); Compound 2 at 10 mg / kg QD reduced body weight by 0.69 g (1.4%) and reduced fat mass by 1.11 g (6.4%); and Compound 2 at 30 mg / kg QD reduced body weight by 5.34 g (10.8%) and reduced fat mass by 4.0 g (22.7%). Table 5: Reductions in fat mass and body weight in DIO miceChange from Body BW BW Fat(g) Fat Fat Lean(g) Lean Lean baseline weight vehicle vehicle vehicle vehicle vehicle vehicle over 2 adjusted adjusted adjusted adjusted adjusted adjusted tglweeksVehicle 1.43 1.66 -0.36Cmpd 1 0.99 -0.45 -0.9% 1.00 -0.66 -3.8% -0.45 -0.09 -0.4% 3 mg / kgCmpd 1 1.22 -0.22 -0.4% 0.77 -0.88 -5.0% -0.34 0.01 0.1% 10 mg / kgCmpd 1. -5.60 -7.03 -14.2% -4.42 -6.08 -34.6% -0.67 -0.31 -1.3% 30 mg / kgCmpd 2, 1.25 -0.19 -0.4% 1.13 -0.52 -3.0% -0.50 -0.14 -0.6% 3 mg / kgCmpd 2, 0.74 -0.69 -1.4% 0.55 -1.11 -6.4% -0.57 -0.21 -0.9% 10 mg / kgCmpd 2. -3.91 -5.34 -10.8% -2.34 -4.00 -22.7% -1.27 -0.91 -3.9% 30 mg / kgGrams (g)Change from baseline BW BW change FM FM change LM LM change over 3 weeks vehicle adjusted change vehicle adjusted change vehicle adjusted changeVehicle 2.9 1.93 0.2Compound 2. 1.53 -1.37 1.08 -0.85 0.75 0.5520 mg / kg QDCompound 3. -0.71 -3.61 0.25 -1.68 -0.28 -0.48IPTS / 200232236.3 3320 mg / kg QDCompound 3. -2.89 -5.79 -1.19 -3.12 -0.57 -0.7710 mg / kg BIDBW = body weight; FM = fat mass; LM = lean massPercentageChange from baseline BW BW change FM FM change LM LM change over 3 weeks change vehicle adjusted change vehicle adjusted change vehicle adjustedVehicle 5.6% 10.0% 0.8%Cmpd 2. 3.0% -2.6% 5.6% -4.4% 3.2% 2.3%20 mg / kg QDCmpd 3, -1.4% -7.0% 1.3% -8.7% -1.2% -2.0%20 mg / kg QDCmpd 3. -5.6% -11.2% -6.2% -16.2% -2.4% -3.3%10 mg / kg BIDBW = body weight; FM = fat mass; LM = lean mass

[0081] Over three weeks, compared to vehicle, Compound 2 at 20 mg / kg QD reduced body weight by 1.37 g (2.6%) and reduced fat mass by 0.85 g (4.4%); Compound 3 at 20 mg / kg QD reduced body weight by 3.61 g (7.0%) and fat mass by 1.68 g (8.7%); and Compound 3 at 10 mg / kg BID reduced body weight by 5.79 g (11.2%) and fat mass by 3.12 g (16.2%). Representative images from mice in these treatment groups are shown in Figure 11.

[0082] Over one week, compared to vehicle, Compound 3 at 30 mg / kg QD reduced body weight by 2.8 g (6.5%) and fat mass by 2.6 g (15.5%).

[0083] Reductions in liver mass and liver fat in DIO mice are shown in Figure 6, Table 6, and Figures 13A-13F. Over three weeks, compared to vehicle, Compound 2 at 20 mg / kg QD reduced liver mass by 483 mg (17%) and reduced liver fat by 121 mg (30%); Compound 3 at 20 mg / kg QD reduced liver mass by 1174 mg (42%) and liver fat by 309 mg (75%); and Compound 3 at 10 mg / kg BID reduced liver mass by 1345 mg (48%) and liver fat by 329 mg (80%). Over one week, compared to vehicle, Compound 3 at 30 mg / kg QD reduced liver weight by 480 mg (23.1%).Table 6: Reductions in liver size and liver fat in DIO miceMilligrams (mg)IPTS / 200232236.3 34Change from baseline Liver Liver change vehicle Liver fat Liver fat change vehicle change adjusted change adjusted(Dav 0) to Dav 21Vehicle 239.4 126Compound 2. 20 mg / kg QD -243.8 -483.2 5 -121Compound 3, 20 mg / kg QD -935.0 -1174.4 -183 -309Compound 3. 10 mg / kg BID -1105.4 -1344.7 -203 -329PercentageChange from baseline Liver Liver change vehicle Liver fat Liver fat change vehicle change adjusted change adjusted(Day 0) to Day 21Vehicle 8.5% 30.6%Compound 2. 20 mg / kg QD -8.6% -17.1% 1.2% -29.5%Compound 3, 20 mg / kg QD -33.0% -41.5% -44.6% -75.2%Compound 3. 10 mg / kg BID -39.1% -47.5% -49.6% -80.2%

[0084] This example demonstrates that ACTRIIB inhibitors Compound 2 and Compound 3 reduce fat mass (measured by NMR), overall body weight, liver weight, and liver fat (measured by densitometry) when dosed orally over three weeks in DIO mice. In a range of published experiments by many authors, results in DIO mice have correlated well with results in humans when compounds with varying mechanisms of action have been tested in clinical studies (Muller et al. (2022) NAT. REV. DRUG DISC. 21:201).Example 3: Small molecule ACTRIIB inhibitors reduce fat mass and overall body weight without lean mass hypertrophy in DIO mice

[0085] The monoclonal antibody bimagrumab is an ACTRIIB inhibitor, and has been shown in DIO mice and in humans to reduce fat while increasing lean mass via induction of muscle hypertrophy. Unexpectedly, it was found that oral small molecule ACTRIIB inhibitors selectively reduce fat mass, and do so without increasing lean mass. Without wishing to be bound by theory, this may result from differences in inhibition of ACTRIIB signaling, namely that antibodies provide continuous inhibition throughout the day by virtue of very long half-lives in circulation while small molecules with short half-lives provide inhibition for only a portion of the day. Thus it is possible that whereas fat reduction isIPTS / 200232236.3 35achievable with small molecules, muscle hypertrophy is not. Alternatively, it may be that antibodies achieve greater inhibition specifically in muscle, perhaps as muscle is highly exposed to the vasculature, whereas small molecules that are more lipophilic achieve better exposure in adipose tissue. Nevertheless, this example demonstrates that oral small molecule ACTRIIB inhibitors can reduce fat mass in DIO mice without causing lean mass hypertrophy by comparing such treatment with bimagrumab treatment.

[0086] Male C57BL / 6J-DIO mice were obtained from Gempharmatech (Nanjing, China) at 20 weeks of age, having been maintained on a high fat diet (HFD, 60% kcal% fat). Mice were singly housed per cage and maintained on the HFD, assigned to groups of 8 animals per treatment condition, with an average body weight of 50-52 g at the start of the study in each group. Compounds were prepared as uniform suspensions in the vehicle solution ((3% DMSO + 2% Solutol + 95% (0.9% Saline)) and stored at 4oC when not in use. Compounds were prepared at a concentration such that the below dose levels were achieved with oral administration of 5 mL / kg per dose. Treatment groups included: Vehicle QD, Compound 2 at 20 mg / kg QD, Compound 3 at 20 mg / kg QD, Compound 3 at 10 mg / kg BID, and human bimagrumab (MedChemExpress LLC, Monmouth Junction, NJ) at 20 mg / kg (administered weekly by subcutaneous injection). Body weight and food intake were recorded twice a week during the course of the study (day 1, day 4, day 8, day 11, day 15, day 18, day 21). Body composition (fat mass and lean mass) was measured using a Bruker TD-NMR minispec Whole Body Composition Live Mice Analyzer (LF50) at baseline and weekly for 3 weeks. Four hours following the last dose of compound, mice were sacrificed and tissues (including adipose, muscle, brain, and liver) were extracted and snap-frozen. Homogenate was prepared by homogenizing tissues with 9 volumes (w:v) of PBS: MeOH (V: V=2:1) buffer. Adipose tissue included both inguinal and peri-renal fat pads, and muscle tissue included pectoralis, gastrocnemius, soleus, tibialis, and quadriceps muscles.

[0087] Reductions in fat mass, overall body weight, liver mass, and liver fat in DIO mice are shown in Figure 7 and Table 7. Over three weeks, compared to vehicle, Compound 2 at 20 mg / kg QD reduced body weight by 1.37 g (2.6%), reduced lean mass by 0.55 g (2.3%), reduced liver mass by 483 mg (17%), and reduced liver fat by 121 mg (30%); Compound 3 at 20 mg / kg QD reduced body weight by 3.61 g (7.0%), lean mass by 0.48 g (2.0%), liver mass by 1174 mg (42%) and liver fat by 309 mg (75%); Compound 3 at 10 mg / kg BID reduced body weight by 5.79 g (11.2%), lean mass by 0.77 g (3.3%), liver mass by 1345 mg (48%) and liver fat by 329 mg (80%); and bimagrumab increased body weight by 1.82 g (3.5%), increased lean mass by 3.55 g (15.0%), increased liver mass by 0.19 g (6.8%) and reduced IPTS / 200232236.3 36liver fat by 0.001 g (3.1%). Representative images from mice in these treatment groups are shown in Figure 11.Table 7: Small molecule ACTRIIB inhibitors do not cause lean mass hypertrophy

[0088] The concentrations of Compounds 2 and 3 in their respective treatment groups BW change FM change LM change Liver change Liver fat change vehicle vehicle vehicle vehicle vehicle adjusted adjusted (g) adjusted (g) adjusted (g) adjusted (g)Compound 2. 20 mg / kg QD -1.37 -2.6% -0.85 -4.4% 0.55 2.3% -0.48 -17.1% -0.121 -29.5% Compound 3. 20 mg / kg QD -3.61 -7.0% -1.68 -8.7% -0.48 -2.0% -1.17 -41.5% -0.309 -75.2% Compound 3, 10 mg / kg BID -5.79 -11.2% -3.12 -16.2% -0.77 -3.3% -1.34 -47.5% -0.329 -80.2% Bimagrumab, 20 mg / kg QW 1.82 3.5% -1.21 -6.3% 3.55 15.0% 0.19 6.8% -0.001 -3.1%were determined in individual homogenized mouse tissues following sacrifice at the end of the 3-week study. The measured concentrations of each compound in specific tissues are shown in Table 11.Table 11: Tissue specific ACTRIIB inhibitor concentrationsAdipose Adipose fold Adipose fold Liver Liver fold Liver fold nM above IC50 above IC50 nM above IC50 above IC50 (ACTRIIB) (ACTRIIA) (ACTRIIB) (ACTRIIA) Compound 2. 20 mg / kg QD 16796.7 13.8 21.8 95946.2 79.1 124.3 Compound 3, 20 mg / kg QD 17583.4 64.5 6.6 32829.8 120.4 12.3 Compound 3. 10 mg / kg BID 7038.9 25.8 2.6 13410.1 49.2 5.0Muscle nM Muscle fold Muscle fold Brain nM Brain fold Brain fold above IC50 above IC50 above IC50 above IC50 (ACTRIIB) (ACTRIIA) (ACTRIIB) (ACTRIIA) Compound 2, 20 mg / kg QD 7018.1 5.8 9.1 2871.0 2.4 3.7 Compound 3. 20 mg / kg QD 7651.0 28.1 2.9 6232.1 22.9 2.3 Compound 3, 10 mg / kg BID 2707.1 9.9 1.0 1916.9 7.0 0.7

[0089] The concentrations of Compound 2 and Compound 3, both on an absolute (nM) basis and relative to their IC50s for inhibition of ACTRIIB and ACTRIIA, were substantially higher in adipose and liver tissues as compared to muscle and brain tissues. This may in partIPTS / 200232236.3 37account for these compounds demonstrating a larger reduction in fat mass than bimagrumab, while not demonstrating muscle hypertrophy like bimagrumab. Additionally, or alternatively, muscle hypertrophy may require 24-hour per day inhibition of ACTRIIB and ACTRIIA, which is better achieved with an antibody that has a long circulating half-life, while reduction in fat mass does not require constant pathway inhibition and instead can result from daily treatment with an oral compound, which provides pathway inhibition for a portion of the day given its shorter half-life.

[0090] This example demonstrates that oral small molecule ACTRIIB inhibitors, unlike ACTRIIB inhibiting antibodies, reduce fat mass, overall body weight, liver weight, and liver fat over three weeks in DIO mice without increasing lean mass. I n contrast, ACTRIIB inhibiting antibodies increase lean mass (muscle hypertrophy) along with body fat reduction, but without a reduction in liver mass or (meaningfully) liver fat.Example 4: ACTRIIB inhibitors synergistically reduce fat mass and overall body weight in DIO mice in combination with GLP-1 agonists

[0091] Injectable biologic and oral small molecule agonists of the GLP-1 receptor (referred to as a class as incretins) have been shown in both mouse studies and human clinical trials to reduce food intake and correspondingly reduce fat mass and overall body weight. This example describes the synergistic reduction in fat mass and body weight when an ACTRIIB inhibitor is combined with a GLP-1 agonist in DIO mice.

[0092] Compound 2 was evaluated in vivo following oral dosing of diet-induced obese (DIO) mice over three weeks. Male C57BL / 6J-DIO mice were obtained from Gempharmatech (Nanjing, China) at 20 weeks of age, having been maintained on a high fat diet (HFD, 60% kcal% fat). Mice were singly housed per cage and maintained on the HFD, assigned to groups of 8 animals per treatment condition, with an average body weight of 50-52 g at the start of the study in each group. Compound 2 was prepared as a uniform suspension in the vehicle solution ((3% DMSO + 2% Solutol + 95% (0.9% Saline)) and stored at 4°C when not in use, and was orally administered at 5 mL / kg per dose. Treatment groups included: Vehicle QD, Compound 2 at 20 mg / kg QD, semaglutide (a GLP-1 agonist) at 40 pg / kg QD (injected subcutaneously), and Compound 2 at 20 mg / kg QD (orally) together with semaglutide at 40 pg / kg QD (injected subcutaneously). Body weight and food intake were recorded twice a week during the course of the study (day 1, day 4, day 8, day 11, day 15, day 18, day 21). Body composition (fat mass and lean mass) was measured usingIPTS / 200232236.3 38a Bruker TD-NMR minispec Whole Body Composition Live Mice Analyzer (LF50) at baseline and weekly for 3 weeks.

[0093] Reductions in fat mass and overall body weight in DIO mice are shown in Figure 8 and Table 8. Over three weeks, compared to vehicle, Compound 2 at 20 mg / kg QD reduced body weight by 1.37 g (2.6%) and reduced fat mass by 0.85 g (4.4%); semaglutide at 40 pg / kg QD reduced body weight by 10.9 g (21%) and fat mass by 6.8 g (36%); and the combination of Compound 2 and semaglutide reduced body weight by 19.3 g (37%) and fat mass by 13.6 g (71%). Representative images from mice in these treatment groups are shown in Figure 11.Table 8: Combination of Compound 2 and a GLP-1 agonistGramsBW FM LMChange from FM change LM change changebaseline (Day 0) to BW change vehicle vehicle vehicle change changeDav 7 adjusted adjusted adjustedVehicle 0.58 0.31 0.21 Compound 2,-0.79 -1.37 -0.97 -1.28 0.24 0.03 20 mg / kg QDSemaglutide,-5.69 -6.27 -3.13 -3.44 -1.5 -1.71 40 pg / kg QDCompound 2 plus-8.56 -9.14 -5.53 -5.84 -1.59 -1.8semaglutidePercentBWChange from FM FM change LM LM change changebaseline (Day 0) to BW change change vehicle change vehicle vehicleDav 7 adjusted adjusted adjustedVehicle 1.11 1.75 1.02 Compound 2,-1.53 -2.64 -4.98 -6.73 1.08 0.06 20 mg / kg QDSemaglutide,-10.92 -12.03 -16.2 -17.95 -6.34 -7.36 40 pg / kg QDCompound 2 plus-16.52 -17.63 -29.12 -30.87 -6.67 -7.69semaglutideGramsBWChange from FM FM change LM LM change changebaseline (Dav 0) to BW change vehicle vehicle vehicle change changeDav 14 adjusted adjusted adjustedVehicle 1.65 1.64 0.46 Compound 2,-0.07 -1.72 0.19 -1.45 0.48 0.02 20 mg / kg QDSemaglutide,-7.83 -9.48 -4.99 -6.63 -2.46 -2.9240 pg / kg QDIPTS / 200232236.3 39Compound 2 plus-16.39 -18.04 -11.38 -13.02 -2.77 -3.23 semaglutidePercentBW FM LMChange from FM change LM change changebaseline (Day 0) to BW change vehicle vehicle vehicle change changeDav 14 adjusted adjusted adjustedVehicle 3.15 8.53 2.01 Compound 2,-0.17 -3.32 0.99 -7.54 1.98 -0.03 20 mg / kg QDSemaglutide,-15.03 -18.18 -26 -34.53 -10.36 -12.37 40 pg / kg QDCompound 2 plus-31.59 -34.74 -59.46 -67.99 -11.63 -13.64semaglutideGram (g)Change from BW BW FM FM change LM LM change baseline (Dav 0) to change change change vehicle change vehicle Dav 20 vehicle adjusted adjusted adjustedVehicle 2.9 1.93 0.2Compound 2, 1.53 -1.37 1.08 -0.85 0.75 0.55 20 mg / kg QDSemaglutide, -7.99 -10.89 -4.9 -6.83 -1.4 -1.6 40 pg / kg QDCompound 2 plus -16.37 -19.27 -11.63 -13.56 -1.68 -1.88 semaglutidePercent (%)Change from BW BW FM FM change LM LM change baseline (Day 0) to change change change vehicle change vehicle Dav 20 vehicle adjusted adjusted adjustedVehicle 5.6% 10.0% 0.8%Compound 2, 3.0% -2.6% 5.6% -4.4% 3.2% 2.3% 20 mg / kg QDSemaglutide, -15.4%. -21.0% -25.5% -35.5% -5.9% -6.8% 40 pg / kg QDCompound 2 plus -31.6% -37.2% -60.6% -70.7% -7.1% -7.9% semaglutide

[0094] This example demonstrates that when combined with a GLP-1 agonist, ACTRIIB inhibition reduces fat mass and body weight in DIO mice in a synergistic manner. The total IPTS / 200232236.3 40body weight lost and fat mass lost over 3 weeks with the combination is far greater than would be expected by the addition of the body weight or fat mass lost with Compound 2 as monotherapy and semaglutide as monotherapy. In fact, the combination of Compound 2 and semaglutide lost approximately double the body weight and double the fat mass compared to semaglutide as monotherapy.Example 5: ACTRIIB inhibitors maintain reduced fat mass and overall body weight following discontinuation of GLP-1 agonists

[0095] In animals and in humans treated with agonists of the GLP-1 receptor, discontinuation of that treatment results in rapid regain of both fat and overall body weight that was lost while on therapy. This example describes the maintained fat reduction and body weight reduction with continued ACTRIIB inhibitor treatment when GLP-1 agonist treatment is discontinued in DIO mice.

[0096] Compound 2 was evaluated in vivo following oral dosing of diet-induced obese (DIO) mice over three weeks. Male C57BL / 6J-DIO mice were obtained from Gempharmatech (Nanjing, China) at 20 weeks of age, having been maintained on a high fat diet (HFD, 60% kcal% fat). Mice were singly housed per cage and maintained on the HFD, assigned to groups of 8 animals per treatment condition, with an average body weight of SO-52 g at the start of the study in each group. Compound 2 was prepared as a uniform suspension in the vehicle solution ((3% DMSO + 2% Solutol + 95% (0.9% Saline)) and stored at 4°C when not in use, and was orally administered at 5 mL / kg per dose. Treatment groups included: Vehicle QD, Compound 2 at 20 mg / kg QD, semaglutide (a GLP-1 agonist) at 40 pg / kg QD (injected subcutaneously), and Compound 2 at 20 mg / kg QD (orally) together with semaglutide at 40 pg / kg QD (injected subcutaneously). Body weight and food intake were recorded twice a week during the course of the study (day 1, day 4, day 8, day 11, day 15, day 18, day 21). Body composition (fat mass and lean mass) was measured using a Bruker TD-NMR minispec Whole Body Composition Live Mice Analyzer (LF50) at baseline and weekly for 3 weeks.

[0097] Among the 8 mice in the combination group, semaglutide treatment was discontinued in 2 animals at Day 13. Thus those 2 animals received the combination of Compound 2 plus semaglutide for approximately 2 weeks, and only received Compound 2 forIPTS / 200232236.3 41the remaining week of the study. The other 6 animals in the combination group received the combination for the entire study.

[0098] Reductions in fat mass and overall body weight in DIO mice are shown in Figure 9 and Table 9. Over three weeks, compared to vehicle, Compound 2 at 20 mg / kg QD reduced body weight by 1.37 g (2.6%) and reduced fat mass by 0.85 g (4.4%); semaglutide at 40 pg / kg QD reduced body weight by 10.9 g (21%) and fat mass by 6.8 g (36%); the combination of Compound 2 and semaglutide for a full 3 weeks (in 6 animals) reduced body weight by 18.7 g (36%) and fat mass by 13.1 g (68%); and the combination of Compound 2 and semaglutide for 13 days followed by only Compound 2 treatment for the remainder of the study (in 2 animals) reduced body weight by 20.9 g (41%) and fat mass by 14.9 g (79%). Table 9: Weight and fat loss following discontinuation of a GLP-1 agonistGramsBW change FM change LM change BW FM LMChange from baseline (Dav 0) to Dav 7 vehicle vehicle vehicle change change changeadjusted adjusted adjusted Vehicle 0.58 0.31 0.21 Compound 2, 20 mg / kg QD -0.79 -1.37 -0.97 -1.28 0.24 0.03 Semaglutide, 40 μg / kg QD -5.69 -6.27 -3.13 -3.44 -1.5 -1.71 Compound 2 plus semaglutide (n=6animals on combination for entire 3 -7.85 -8.43 -5.08 -5.39 -1.33 -1.54 weeks)Compound 2 plus semaglutide (n=2animals who discontinued semaglutide -10.69 -11.27 -6.89 -7.2 -2.39 -2.6 on Day 13)PercentBW change FM change LM change, BW FM LMChange from baseline (Day 0) to Day 7 vehicle vehicle vehicle change change changeadjusted adjusted adjusted Vehicle 1.11 1.75 1.02 Compound 2. 20 mg / kg QD -1.53 -2.64 -4.98 -6.73 1.08 0.06 Semaglutide, 40 pg / kg QD -10.92 -12.03 -16.2 -17.95 -6.34 -7.36 Compound 2 plus semaglutide (n=6animals on combination for entire 3 -15.10 -16.21 -26.49 -28.24 -5.62 -6.64 weeks)Compound 2 plus semaglutide (n=2animals who discontinued semaglutide -20.78 -21.89 -36.99 -38.74 -9.85 -10.87 on Day 13)GramsBW change FM change LM change BW FM LMChange from baseline (Dav 0) to Dav 14 vehicle vehicle vehicle change change changeadjusted adjusted adjustedIPTS / 200232236.3 42Vehicle 1.65 1.64 0.46 Compound 2, 20 mg / kg QD -0.07 -1.72 0.19 -1.45 0.48 0.02 Semaglutide, 40 μg / kg QD -7.83 -9.48 -4.99 -6.63 -2.46 -2.92 Compound 2 plus semaglutide (n=6animals on combination for entire 3 -14.3 -15.95 -10.24 -11.88 -2.03 -2.49 weeks)Compound 2 plus seinaglutide (n=2animals who discontinued semaglutide -22.64 -24.29 -14.79 -16.43 -5.01 -5.47 on Day 13)PercentBW change FM change LM change BW FM LMChange from baseline (Day 0) to Day 14 vehicle vehicle vehicle change change adjusted change adjusted adjusted Vehicle 3.15 8.53 2.01 Compound 2. 20 mg / kg QD -0.17 -3.32 0.99 -7.54 1.98 -0.03 Semaglutide, 40 pg / kg QD -15.03 -18.18 -26 -34.53 -10.36 -12.37 Compound 2 plus semaglutide (n=6animals on combination for entire 3 -27.46 -30.61 -53.01 -61.54 -8.52 -10.53 weeks)Compound 2 plus semaglutide (n=2animals who discontinued semaglutide -43.99 -47.14 -78.84 -87.37 -20.94 -22.95 on Day 13)Gram ( g )Change from baseline (Day 0) to Day 20 BW BW change FM FM change LM LM change change vehicle change vehicle change vehicle adjusted adjusted adjusted Vehicle 2.9 1.93 0.2Compound 2, 20 mg / kg QD 1.53 -1.37 1.08 -0.85 0.75 0.55 Semaglutide, 40 pg / kg QD -7.99 -10.89 -4.9 -6.83 -1.4 -1.6 Compound 2 plus semaglutide (n=6 -15.84 -18.74 -11.2 -13.13 -1.62 -1.82 animals on combination for entire 3weeks)Compound 2 plus semaglutide (n=2 -17.95 -20.85 -12.94 -14.87 -1.85 -2.05 animals who discontinued semaglutideon Day 13)Percent (%)Change from baseline (Dav 0) to Dav 20 BW BW change FM FM change LM LM change change vehicle change vehicle change vehicle adjusted adjusted adjusted Vehicle 5.6% 10.0% 0.8% Compound 2. 20 mg / kg QD 3.0% -2.6% 5.6% -4.4% 3.2% 2.3% Semaglutide, 40 pg / kg QD -15.4% -21.0% -25.5% -35.5% -5.9% -6.8%IPTS / 200232236.3 43Compound 2 plus semaglutide (n=6 -30.5% -36.0% -58.1% -68.1% -6.9% -7.7% animals on combination for entire 3weeks)Compound 2 plus semaglutide (n=2 -34.9% -40.5% -68.6% -78.9% -7.7% -8.6% animals who discontinued semaglutideon Day 13)

[0099] These weight loss and fat loss effects occurred despite an immediate and substantial increase in food intake by the 2 animals that discontinued semaglutide treatment. Food intake data are shown in Figures 10A-10B and Table 10. While animals that remained on the combination had a lower food intake than those on semaglutide monotherapy, the animals that discontinued semaglutide had a much higher daily food intake over the last week of the study and on a cumulative basis over 3 weeks consumed roughly the same amount of food as those on semaglutide monotherapy for the entire study.Table 10: Food intake following discontinuation of a GLP-1 agonistDaily average food intake (g)Group Days 1- Days Days 7- Days 11- Days 14- Days 4 T7 11 14 18 18-21 Vehicle2.6 2.82 2.46 2.9 2.71 2.99 Compound 2, 20 mg / kg QD1.88 2.44 2.43 2.73 2.75 3.11 Semaglutide, 40 pg / kg QD0.81 1.56 1.55 2.14 2.16 2.52 Compound 2 plus semaglutide (n=8)0.5 0.67 0.72 1.5 2.17 2.45 Compound 2 plus semaglutide (n=6 animals oncombination for entire 3 weeks) 0.54 0.82 0.91 1.39 1.72 2.20 Compound 2 plus semaglutide (n=2 animals whodiscontinued semaglutide on Day 13) 0.37 0.22 0.15 1.82 3.51 3.21Cumulative food intake (g)Group Days 1- Days Days 1- Days 1- Days 1- Days 1- 4 1-7 11 14 18 21 Vehicle 7.8 16.27 26.12 34.83 45.69 54.66 Compound 2, 20 mg / kg QD 5.63 12.96 22.66 30.84 41.84 51.16 Semaglutide, 40 μg / kg QD 2.43 7.11 13.31 19.74 28.37 35.93IPTS / 200232236.3 44Compound 2 plus seinaglutide (n=8)1.49 3.5 6.37 10.86 19.53 26.88 Compound 2 plus seinaglutide (n=6 animals oncombination for entire 3 weeks) 1.61 4.08 7.71 11.87 18.75 25.34 Compound 2 plus seinaglutide (n=2 animals w’hodiscontinued seinaglutide on Day 13) 1.11 1.77 2.36 7.83 21.88 31.50[000100] This example demonstrates that following discontinuation of a GLP-1 agonist in animals that had been treated with the GLP-1 agonist combined with an ACTRIIB inhibitor, continued treatment with the ACTRIIB inhibitor maintains fat loss and body weight loss despite a substantial increase in consumption of a HFD. In fact, in this study animals that discontinued GLP-1 agonist treatment after two weeks, while increasing their food intake on the HFD and while continuing on ACTRIIB inhibitor treatment, lost more fat and more body weight by the end of the study than animals who remained on the combination for the entire study.Example 6: Small molecule ACTRIIB inhibitors increase oxygen consumption and energy expenditure, and decrease respiratory exchange ratio (RER), in DIO mice [000101] In addition to measuring body weight and fat mass, changes in metabolism can be measured in living animals by monitoring consumption of food, water, and oxygen, and excretion of carbon dioxide as well as activity. This example describes the effects of Compound 3 on these parameters in DIO mice.[000102] Compound 3 was evaluated in vivo following oral dosing of diet-induced obese (DIO) mice over 7 days. Male C57BL / 6J-DIO mice were obtained from The Jackson Laboratory (Bar Harbor, ME) at 18 weeks of age, having been maintained on a high fat diet (HFD, 60% kcal% fat). Mice were singly housed in Promethion Core metabolic monitoring cages with beam break activity monitoring (Sable Systems International, Las Vegas, NV) and maintained on the HFD, assigned to groups of 8 animals per treatment condition, with an average body weight of 45-50 g at the start of the study in each group. Compound 3 was prepared as a uniform suspension in the vehicle solution ((3% DMSO + 2% Solutol + 95% (0.9% Saline)) and stored at 4°C when not in use. Treatment groups included: Vehicle QD and Compound 3 at 30 mg / kg QD. Body weight and composition, food and water consumption, and energy expenditure were measured throughout the study.[000103] Changes in oxygen consumption, energy expenditure, and respiratory exchange ratio (RER) with Compound 3 treatment are shown in Figures 13A-13F. Compared to vehicle, Compound 3 over 7 days reduced average energy balance ((g food * caloric content IPTS / 200232236.3 45of food in kcal / g) - kcal energy expenditure) during dark cycles (12 hour periods per day) by 1.77 kcal / day, reduced average daily oxygen consumption (VO2) by 0.065 ml / min, increased average daily energy expenditure by 0.41 kcal, increased daily energy expenditure normalized (by ANCOVA) for body mass by 1.05 kcal, and reduced average daily RER by 0.019 (representing a change from baseline RER of -0.12 relative to vehicle).[000104] This example demonstrates that ACTRIIB inhibitor treatment increases oxygen consumption and energy expenditure, and reduces RER, in DIO mice. These changes are known to be indicative of a shift in metabolism toward increased fat oxidation versus carbohydrate utilization. These changes in metabolism may underlie the reduction in fat mass, body weight, liver mass, and liver fat associated with ACTRIIB inhibitor treatment. Example 7: ACTRIIB inhibitors reduce fat mass and overall body weight in DIO mice without lean mass hypertrophy.[000105] This example describes the in vivo phenotypic effects of oral administration of Compound 3 in DIO mice at various doses. Treatment with Compound 3, as compared to treatment with Vehicle alone, over three weeks, lead to reduced fat mass (as measured by NMR), reduced overall body weight, and an increase in the ratio of lean body mass to total body weight without lean mass hypertrophy.[000106] Compound 3 was evaluated in vivo following oral dosing of diet-induced obese (DIO) mice over three weeks. Male C57BL / 6J-DIO mice were obtained from Gempharmatech (Nanjing, China) at 20 weeks of age, having been maintained on a high fat diet (HFD, 60% kcal% fat). Mice were singly housed per cage and maintained on the HFD, assigned to groups of 8 animals per treatment condition, with an average body weight of 50-52 g at the start of the study in each group. Compound 3 was prepared as a uniform suspension in the vehicle solution ((3% DMSO + 2% Solutol + 95% (0.9% Saline)) and stored at 4 °C when not in use. Compound 3 was prepared at a concentration such that the below dose levels were achieved with oral administration of 5 mL / kg per dose. Treatment groups included: Vehicle PO QD, Semaglutide (a GLP-1 agonist) at 20 μg / kg / day SQ, Compound 3 at 5 mg / kg PO QD, 15 mg / kg PO QD, or 30 mg / kg PO QD, and Compound 3 at those same doses combined with Semaglutide at the same dose as described above. Body weight and food intake were recorded twice a week during the course of the study (day 1, day 4, day 7, day 11, day 14, day 18, day 21). Body composition (fat mass and lean mass) wasIPTS / 200232236.3 46measured using a Bruker TD-NMR minispec Whole Body Composition Live Mice Analyzer (LF50) at baseline and weekly for 3 weeks.[000107] Reductions in fat mass and overall body weight in DIO mice are shown in Figure 13 and Table 12.[000108] Over three weeks, compared to vehicle, Compound 3 at 5 mg / kg PO QD reduced body weight by 3.2 g (6%), reduced fat mass by 1.8 g (10%), reduced lean mass by 0.7 g (3%), and increased the ratio of lean mass to body weight by 2%; Compound 3 at 15 mg / kg PO QD reduced body weight by 5.5 g (11%), reduced fat mass by 3.6 g (20%), reduced lean mass by 0.6 g (3%), and increased the ratio of lean mass to body weight by 3%; Compound 3 at 30 mg / kg PO QD reduced body weight by 8.7 g (17%), reduced fat mass by 6 g (34%), reduced lean mass by 1.2 g (5%), and increased the ratio of lean mass to body weight by 6%.[000109] Semaglutide monotherapy reduced body weight by 10.3 g (20%), reduced fat mass by 6.8 g (39%), reduced lean mass by 1.9 g (8%), and increased the ratio of lean mass to body weight by 6%.[000110] Compound 3 at 5 mg / kg PO QD plus Semaglutide reduced body weight by 12.6 g (25%), reduced fat mass by 8.5 g (49%), reduced lean mass by 1.8 g (8%), and increased the ratio of lean mass to body weight by 9%; Compound 3 at 15 mg / kg PO QD plus Semaglutide reduced body weight by 15.8 g (31%), reduced fat mass by 11.2 g (63%), reduced lean mass by 2.2 g (9%), and increased the ratio of lean mass to body weight by 14%; and Compound 3 at 30 mg / kg PO QD plus Semaglutide reduced body weight by 18.5 g (36%), reduced fat mass by 13 g (74%), reduced lean mass by 2.2 g (9%), and increased the ratio of lean mass to body weight by 18%.[000111] This example demonstrates that the ACTRIIB inhibitor Compound 3 reduces fat mass (measured by NMR) and overall body weight when dosed orally over three weeks in DIO mice, both as monotherapy and in combination with a GLP-1 agonist, while increasing the ratio of lean mass to body weight but not inducing overall lean mass hypertrophy.Table 12: Reductions in fat mass and body weight in DIO mice with Compound 3 monotherapy and in combination with a GLP-1 agonistBody Body Lean Lean Fat mass Fat mass Lean weight weight mass mass % change % mass % change % change of body Group n from change change from change from weight, baseline from from baseline from baseline end of (g), baseline, baseline, (g), baseline,(g)> study, IPTS / 200232236.3 47vehicle vehicle vehicle vehicle vehicle vehicle vehicle adjusted adjusted adjusted adjusted adjusted adjusted adjusted Semaglutide @ 20 μg / kg / day SQ 8 (10.29) -20% (6.83) -39% (1.87) -8% 6% 5 mg / kg QD PO 8 (3.21) -6% (1.83) -10% (0.65) -3% 2% 15 mg / kg QD PO 8 (5.48) -11% (3.57) -20% (0.64) -3% 3% 30 mg / kg QD PO 8 (8.73) -17% (6.00) -34% (1.20) -5% 6% 5 mg / kg QD PO + Semaglutide 8 (12.59) -25% (8.49) -49% (1.82) -8% 9% 15 mg / kg QD PO + Semaglutide 8 (15.82) -31% (11.16) -63% (2.20) -9% 14% 30 mg / kg QD PO + Semaglutide 8 (18.47) -36% (12.96) -74% (2.24) -9% 18%INCORPORATION BY REFERENCE

[0084] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes. This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference.

[0085] If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.EQUIVALENTS

[0086] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.IPTS / 200232236.3 48

Claims

Claims1. A method of treating or preventing obesity or an obesity-related disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a small molecule inhibitor of an Activin receptor Type II.

2. A method of reducing body weight, fat mass, liver weight, or liver fat in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a small molecule inhibitor of an Activin receptor Type II.

3. A method of increasing oxygen consumption or energy expenditure or decreasing respiratory exchange ratio in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a small molecule inhibitor of an Activin receptor Type II.

4. The method of any one of claims 1-3, wherein the Activin receptor Type II is the Activin receptor Type II A isoform or B isoform.

5. The method of any one of claims 1-4, wherein the small molecule inhibitor is administered to the subject orally.

6. The method of any one of claims 1-5, wherein the method does not substantially increase muscle mass.

7. The method of any one of claims 1-5, wherein the method does not substantially increase lean mass.

8. The method of any one of claims 1-7, wherein the method does not result in lean mass hypertrophy.

9. The method of any one of claims 1-8, wherein the method further comprises administering to the subject a second therapeutic agent.

10. The method of claim 9, wherein the second therapeutic agent is a GLP-1 agonist.

11. The method of claim 10, wherein the GLP-1 agonist is semaglutide.

12. The method of any one of claims 9-11, wherein the method results in synergistic effect with the second therapeutic agent.IPTS / 200232236.3 4913. The method of claim 12, wherein the synergistic effect continues after discontinuing the administration of the second therapeutic agent.

14. The method of claim 11 or 12, wherein the synergistic effect continues after increasing food intake by the subject.

15. The method of any one of claims 1-14, wherein the small molecule inhibitor of an Activin receptor Type II is a compound of Formula (I):(I)wherein:Ar is pyrazole, indole or pyrazole containing substituents; in the pyrazole containing substituents, the substituent is selected from C1-4 hydrocarbon groups, and the number of substituents is 1 or 2; andR is 4-methylpiperazine, piperazine, morpholine,o16. The method of any one of claims 1-14, wherein the small molecule inhibitor of an Activin receptor Type II is a compound of Formula (II):(IDor a pharmaceutically acceptable salt thereof, wherein:Ar is selected from pyrazole, indole, pyrazole containing substituents, isoxazoleIPTS / 200232236.3 50containing substituents, pyrimidine or 3-pyridyl; in the pyrazole containing substituents and the isoxazole containing substituents, the substituent is selected from C1-4 hydrocarbon groups, and the number of substituents is 1 or 2; andR is selected from 4-methylpiperazine, piperazine, morpholine,°,17. The method of any one of claims 1-14, wherein the small molecule inhibitor of an Activin receptor Type IT is a compound of Formula (III):(III)or a pharmaceutically acceptable salt thereof, wherein:Ar is selected from pyrazole, indole, pyrazole containing substituents, isoxazole containing substituents, pyrimidine or 3 -pyridine; in the pyrazole containing substituents and the isoxazole containing substituents, the substituent is selected from C1-4 hydrocarbon groups, and the number of substituents is 1 or 2; andR is selected from 4-methylpiperazine, piperazine,° or18. The method of any one of claims 1-14, wherein the small molecule inhibitor of an Activin receptor Type II is a compound of Formula (IV):IPTS / 200232236.3 51(IV)or a pharmaceutically acceptable salt thereof, wherein:Ar is selected from pyrazole, indole, pyrazole containing substituents, isoxazole containing substituents, pyrimidine or 3-pyridine; in the pyrazole containing substituents and the isoxazole containing substituents, the substituent is selected from C1-4 hydrocarbon groups, and the number of substituents is 1 or 2; andR is selected from 4-methylpiperazine, piperazine, morpholine,°,19. The method of any one of claims 1-14, wherein, the small molecule inhibitor of an Activin receptor Type II is a compound of Formula (V):(V)or a pharmaceutically acceptable salt thereof, wherein:Ar is selected from pyrazole, indole, pyrazole containing substituents, isoxazole containing substituents, pyrimidine, pyridine or phenyl; in the pyrazole containing substituents and the isoxazole containing substituents, the substituent is selected from C1-4 hydrocarbon groups, and the number of substituents is 1 or 2;o'^ L, N. v R is selected from 4-methylpiperazine, piperazine, morpholine,°, and o20. The method of any one of claims 15-19, wherein the Ar is indole.IPTS / 200232236.3 5221. The method of any one of claims 15-20, wherein the indoleis22. The method of any one of claims 15-19, wherein the Ar is pyrazole or pyrazole containing substituents.NH\ A 23. The method of any one of claims 15-19 and 22, wherein the Ar is selected from r24. The method of any one of claims 15-19, 22, and 23, wherein the Ar is A.

25. The method of any one of claims 15-24, wherein the R is 4-methylpiperazine. IT '"26. The method of any one of claims 15-25, wherein the R is A27. The method of any one of claims 1-26, wherein the small molecule inhibitor of an Activin receptor Type II is selected from the group consisting of:IPTS / 200232236.3 53IPTS / 200232236.3 54, or a pharmaceutically acceptable salt thereof.IPTS / 200232236.3 55

Images