Myocardial sarcomere inhibitor

Abstract

Compounds of formula (I): TIFF2024508526000131.tif68165 (in the formula, R 1 , R 2 , R 3 , and R 4 is as defined herein), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. Also provided is a pharma- ceutically acceptable composition comprising a compound of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. Also provided is a method of using a compound of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 156,853, filed March 4, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Provided herein are heterocyclic compounds, pharmaceutical compositions containing such compounds, and methods of using such compounds to treat various cardiac diseases and conditions. [Background technology]

[0003] The present disclosure relates to specific chemical moieties that selectively modulate cardiac sarcomeres, and in particular to specific chemical moieties, pharmaceutical compositions, and methods for treating various cardiac diseases and conditions.

[0004] The cardiac sarcomere is composed of a network of contractile and structural proteins that regulate myocardial function. Components of the cardiac sarcomere present targets for the treatment of various cardiac diseases and conditions, for example, by increasing contractility or by promoting complete relaxation to regulate systolic and diastolic function, respectively. The force and velocity of myocardial contraction are major determinants of organ function and are regulated by the periodic interplay of actin and myosin. Regulation of actin and myosin binding is mediated by a network of myofilament regulatory proteins and intracellular Ca2+. 2+ The troponin complex and tropomyosin are thin filament proteins that govern the availability of actin-binding sites, as well as the intrinsic and regulatory light chains, and myosin-binding protein C regulates the positioning and mechanical properties of myosin.

[0005] Abnormalities in the myocardial sarcomere have been identified as the driving cause of various cardiac diseases and conditions, such as hypertrophic cardiomyopathy (HCM) and heart failure with preserved ejection fraction (HFpEF). Mutations in sarcomere proteins cause disease by rendering the myocardium either "hyper" or "hypo" contractile. Modulators of the myocardial sarcomere can be used to realign contractility and halt or reverse the course of the disease.

[0006] Current drugs that target the myocardial sarcomere, such as inotropes (drugs that enhance the heart's ability to contract), have poor selectivity for cardiac tissue, leading to the recognition of adverse effects that limit their use. These adverse effects include cellular damage caused by increased rates of energy expenditure, exacerbation of relaxation abnormalities, and increased cytosolic Ca in inotropically stimulated myocardium. 2+ and potential arrhythmic side effects that may result from increased cyclic AMP concentrations. Given the limitations of current agents, new approaches are needed to improve cardiac function in HCM and HFpEF.

[0007] There remains a great need for drugs that utilize new mechanisms of action and may provide better outcomes in terms of both short-term and long-term symptom relief, safety, and patient mortality. New drugs with improved therapeutic indexes over current drugs provide a means to achieve these clinical outcomes. Selectivity of drugs directed to myocardial sarcomeres (e.g., by targeting cardiac myosin) has been identified as a key means to achieve this improved therapeutic index. The present disclosure provides such drugs, particularly cardiac sarcomere inhibitors, and methods of their use. These drugs are allosteric inhibitors of cardiac myosin. Advantages of these compounds include a wider therapeutic index, less impact on cardiac relaxation, better pharmacokinetics, and better safety. The drugs of the present disclosure are also useful in that they may allow for once-daily dosing for the methods of use described herein.

[0008] The present disclosure provides chemical entities, pharmaceutical compositions, and methods for the treatment of heart failure, including HCM and HFpEF. The compositions are inhibitors of cardiac sarcomeres, such as inhibitors of cardiac myosin. Summary of the Invention

[0009] In one embodiment, a compound of formula (I): [ka] (In the formula, R 1 is halo or C 1-6 is haloalkyl, R 2 is H, halo, or C 1-6 is alkyl, R 3 teeth, (i) cyclohexyl, optionally with one or more independently selected C 1-6 Alkyl or C 1-6 cyclohexyl as defined above, substituted by a haloalkyl substituent; or (ii) C 1-6 alkyl, R 4 teeth, (i) -C(O)H, (ii) -C(O)NH 2 , [ka] and however, (1)R 3 C 1-6 If it is alkyl, R 4 teeth [ka] and (2)R 3 If is isopropyl, R 1 and R 2 the total number of halo atoms is at least 2; (3)R 3is one or more independently selected C 1-6 When R is cyclohexyl substituted by haloalkyl substituents, 4 is other than -C(O)H), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing is provided.

[0010] In one embodiment, the compound of formula (IA): [ka] (In the formula, R 1 , R 2 , and R 3 is as described herein for compounds of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, are provided herein.

[0011] In one embodiment, a compound of formula (IB): [ka] (In the formula, R 1 , R 2 , and R 3 is as described herein for compounds of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, are provided herein.

[0012] In one embodiment, a compound of formula (IC): [ka] (In the formula, R 1 , R 2 , and R 3 is as described herein for compounds of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, are provided herein.

[0013] In one embodiment, a compound of formula (ID): [ka] (In the formula, R 1 , R 2 , and R 3 is as described herein for compounds of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, are provided herein.

[0014] In some embodiments, a compound selected from the group consisting of the compounds in Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing is provided.

[0015] In some aspects, there is provided a pharmaceutical composition comprising: (i) a compound of formula (I), or a variation or embodiment thereof, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing; and (ii) one or more pharma- ceutically acceptable excipients.

[0016] In some aspects, a method of treating a cardiac disease in a subject in need thereof is provided, comprising administering to the subject a compound of formula (I), or any variation thereof, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, or a pharmaceutical composition containing a compound of formula (I), or any variation thereof, or a stereoisomer or tautomer thereof, or a pharma-ceutically acceptable salt of any of the foregoing. In some embodiments, the cardiac disease is hypertrophic cardiomyopathy (HCM). In some embodiments, the HCM is obstructive or non-obstructive, or is caused by sarcomeric and / or non-sarcomeric mutations. In some embodiments, the cardiac disease is heart failure with preserved ejection fraction (HFpEF). In some embodiments, the cardiac disease is selected from the group consisting of diastolic dysfunction, primary or secondary restrictive cardiomyopathy, myocardial infarction, angina pectoris, and left ventricular outflow tract obstruction. In some embodiments, the cardiac disease is hypertensive heart disease, congenital heart disease, cardiac ischemia, coronary heart disease, diabetic heart disease, congestive heart failure, right heart failure, cardiorenal syndrome, or infiltrative cardiomyopathy. In some embodiments, the cardiac disease is cardiac aging and / or age-related diastolic dysfunction or a condition associated therewith. In some embodiments, the cardiac disease is left ventricular hypertrophy and / or concentric left ventricular remodeling or a condition associated therewith.

[0017] In another aspect, there is provided a method of treating a disease or condition associated with HCM in a subject in need thereof, the method comprising administering to the subject a compound of formula (I), or any variation thereof, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, or a pharmaceutical composition containing a compound of formula (I), or any variation thereof, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. In some embodiments, the disease or condition is selected from the group consisting of Fabry disease, Danon disease, mitochondrial cardiomyopathy, and Noonan syndrome.

[0018] In some aspects, methods are provided for treating a disease or condition associated with secondary left ventricular wall thickening in a subject in need thereof, the methods comprising administering to the subject a compound of formula (I), or any variation thereof, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, or a pharmaceutical composition containing a compound of formula (I), or any variation thereof, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. In some embodiments, the disease or condition is selected from the group consisting of hypertension, valvular heart disease (such as aortic stenosis and mitral regurgitation), metabolic syndrome (such as diabetes and obesity), end-stage renal disease, scleroderma, sleep apnea, amyloidosis, Fabry disease, Friedreich's ataxia, Danon disease, Noonan syndrome, and Pompe disease.

[0019] In other aspects, methods are provided for treating diseases or conditions associated with small left ventricular lumen and lumen obstruction, high-output left ventricular contraction, myocardial ischemia, or cardiac fibrosis. Methods are also provided for treating muscular dystrophy (e.g., Duchenne muscular dystrophy) or glycogen storage disease.

[0020] Also provided is a method of inhibiting cardiac sarcomere, the method involving contacting cardiac sarcomere with a compound of formula (I), or any variation thereof, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, or a pharmaceutical composition containing a compound of formula (I), or any variation thereof, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. [Brief description of the drawings]

[0021] [Figure 1] Figure 2 shows the experimental X-ray powder diffraction (XRPD) pattern of the amorphous form of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] definition As used in this document, the following words and phrases are generally intended to have the meanings set forth below, unless the context in which they are used indicates otherwise.

[0023] Throughout this application, unless the context indicates otherwise, references to compounds of formula (I) include all subgroups of formula (I) defined herein, such as formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID) or (I-D1), and include all substructures, subgenera, preferences, embodiments, examples and specific compounds defined and / or described herein. References to compounds of formula (I) and subgroups thereof, such as formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), include ionic forms, polymorphs, pseudopolymorphs, amorphous forms, solvates, cocrystals, chelates, isomers, tautomers, oxides (e.g., N-oxides, S-oxides), esters, prodrugs, isotopes, and / or protected forms thereof. In some embodiments, references to compounds of formula (I) and subgroups thereof, such as formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), include polymorphs, solvates, cocrystals, isomers, tautomers, and / or oxides thereof. In some embodiments, references to compounds of formula (I) and subgroups thereof, such as formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), include polymorphs, solvates, and / or co-crystals thereof. In some embodiments, references to compounds of formula (I) and subgroups thereof, such as formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), include isomers, tautomers, and / or oxides thereof. In some embodiments, references to compounds of formula (I) and subgroups thereof, such as formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), include solvates thereof.

[0024] "Alkyl" includes straight and branched carbon chains having the indicated number of carbon atoms, e.g., 1 to 20 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms. For example, C 1-6 Alkyl includes both straight and branched chain alkyls of 1 to 6 carbon atoms. When an alkyl residue having a specific number of carbons is named, it is intended that all branched and straight chain versions having that number of carbons are included. Thus, for example, "propyl" includes n-propyl and isopropyl, and "butyl" includes n-butyl, sec-butyl, isobutyl, and t-butyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl.

[0025] If a range of values ​​is specified (for example, C 1-6 alkyl), each value within that range, and all ranges therebetween. For example, "C 1-6 "Alkyl" includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 2-6 , C 3-6 , C 4-6 , C 5-6 , C 1-5 , C 2-5 , C 3-5 , C 4-5 , C 1-4 , C 2-4 , C 3-4 , C 1-3 , C 2-3 , and C 1-2 Contains alkyl.

[0026] "Halogen" or "halo" refers to fluoro, chloro, bromo, or iodo.

[0027] "Haloalkyl" refers to an alkyl moiety, as defined herein, in which one or more of the hydrogen atoms in the alkyl moiety are replaced by one or more independently selected halo moieties. Examples of haloalkyl moieties include, but are not limited to, -CH 2 F, - CHF 2 , -CF 3 , -CH 2 -CH 2 Cl, -CH 2 -CHCl 2 , -CH 2 -CCl 3 , and -CHF-CH 2 Examples include Cl.

[0028] "Cyclohexyl" is [ka] Refers to a part.

[0029] Unless otherwise specified, the compounds disclosed and / or described herein include all possible enantiomers, diastereomers, mesoisomers and other stereoisomeric forms, optionally including racemic mixtures, optically pure forms, and intermediate mixtures thereof. Enantiomers, diastereomers, mesoisomers, and other stereoisomeric forms may be prepared using chiral synthons or chiral reagents or resolved using conventional techniques. Unless otherwise specified, when the compounds disclosed and / or described herein contain olefinic double bonds or other centers of geometric asymmetry, the compounds are intended to include both E and Z isomers. When the compounds described herein contain moieties capable of tautomerization, the compounds are intended to include all possible tautomers, unless otherwise specified.

[0030] "Protecting group" has the meaning customarily associated with it in organic synthesis, i.e., a group that selectively blocks one or more reactive sites in a polyfunctional compound, allowing a chemical reaction to be selectively performed at an otherwise unprotected reactive site, and that can be easily removed after the selective reaction is complete. Various protecting groups are disclosed, for example, in TH Greene and PG M Huts, Protective Groups in Organic Synthesis, Third Edition, John Wiley & Sons, New York (1999). For example, a "hydroxy-protected form" contains at least one hydroxyl group protected with a hydroxyl-protecting group. Similarly, amines and other reactive groups can be similarly protected.

[0031] The term "pharmaceutically acceptable salt" refers to any salt of the compounds herein that is known to be non-toxic and commonly used in the pharmaceutical literature. In some embodiments, a pharmaceutically acceptable salt of a compound retains the biological effectiveness of the compounds described herein and is not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts can be found in Berge et al., Pharmaceutical Salts, J. Pharmaceutical Sciences, January 1977, 66(1), 1-19. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethylsulfonic acid, p-toluenesulfonic acid, stearic acid, and salicylic acid. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines such as naturally occurring substituted amines, cyclic amines, and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharma- ceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts.

[0032] When the compound described herein is obtained as an acid addition salt, the solution of the acid salt can be basified to obtain the free base.On the other hand, when the compound is a free base, the addition salt, particularly the pharma-ceutically acceptable addition salt, can be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid according to the conventional procedure for preparing an acid addition salt from a basic compound (see, for example, Berge et al., Pharmaceutical Salts, J. Pharmaceutical Sciences, January 1977, 66(1), 1-19).Those skilled in the art will recognize various synthetic methods that can be used to prepare pharma-ceutically acceptable addition salts.

[0033] A "solvate" is formed by the interaction of a solvent and a compound. Suitable solvents include, for example, water and alcohols (e.g., ethanol). Solvates include hydrates having any ratio of compound to water, such as monohydrates, dihydrates, and hemihydrates.

[0034] The term "substituted" means that the specified group or moiety has one or more substituents, for example, alkyl or haloalkyl substituents. The term "unsubstituted" means that the specified group has no substituents. When the term "substituted" is used to describe a structural system, it is intended that the substitution occur at any position in that system where valence allows. When a group or moiety has multiple substituents, it is understood that the substituents can be the same or different from one another. In some embodiments, a substituted group or moiety has 1-5 substituents. In some embodiments, a substituted group or moiety has 1 substituent. In some embodiments, a substituted group or moiety has 2 substituents. In some embodiments, a substituted group or moiety has 3 substituents. In some embodiments, a substituted group or moiety has 4 substituents. In some embodiments, a substituted group or moiety has 5 substituents.

[0035] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description is meant to include instances where the event or circumstance occurs and instances where it does not occur. For example, "optionally substituted cyclohexyl" encompasses both unsubstituted and substituted cyclohexyl, as defined herein. As will be understood by those of skill in the art, with respect to any group that contains one or more substituents, it is not intended that such group introduce any substitution or substitution pattern that is sterically impractical, synthetically impractical, and / or inherently unstable. It will also be understood that when a group or moiety is optionally substituted, the present disclosure includes both embodiments in which the group or moiety is substituted and embodiments in which the group or moiety is not substituted.

[0036] The compounds disclosed and / or described herein may be in isotopically enriched form, e.g. 2 H, 3 H, 11 C. 13 C and / or 14The compound may be enriched in C content. In one embodiment, the compound contains at least one deuterium atom. Such deuterated forms can be made, for example, by the means described in U.S. Patent Nos. 5,846,514 and 6,334,997. Such deuterated compounds can improve the efficacy and increase the duration of action of the compounds disclosed and / or described herein. Deuterium substituted compounds can be synthesized using a variety of methods, such as those described in Dean, D., Recent Advances in the Synthesis and Applications of Radiolabeled Compounds for Drug Discovery and Development, Curr. Pharm. Des., 2000;6(10); Kabalka, G. et al., The Synthesis of Radiolabeled Compounds via Organometallic Intermediates, Tetrahedron, 1989,45(21),6601-21; and Evans, E., Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981,64(1-2),9-32.

[0037] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions.

[0038] The terms "patient," "individual," and "subject" refer to an animal, such as a mammal, bird, or fish. In some embodiments, the patient or subject is a mammal. Mammals include, for example, mice, rats, dogs, cats, pigs, sheep, horses, cows, and humans. In some embodiments, the patient or subject is a human, e.g., a human who has been or will be the object of treatment, observation, or experiment. The compounds, compositions, and methods described herein can be useful in both human therapy and veterinary applications.

[0039] As used herein, the term "treatment" refers to the ability to modulate cardiac sarcomeres. As used herein, "modulation" refers to a change in activity relative to the activity in the absence of the chemical moiety, as a direct or indirect response to the presence of a chemical moiety described herein. The change may be an increase or decrease in activity, and may result from a direct interaction of the chemical moiety with the target, or from the interaction of the chemical moiety with one or more other factors that in turn affect the activity of the target. For example, the presence of the chemical moiety may increase or decrease the target activity, for example, by directly binding to the target, by causing another factor to increase or decrease the target activity (directly or indirectly), or by increasing or decreasing the amount of the target present in the cell or organism (directly or indirectly).

[0040] The term "therapeutically effective amount" or "effective amount" refers to an amount of a compound disclosed and / or described herein that is sufficient to affect a treatment as defined herein when administered to a patient in need of such treatment. A therapeutically effective amount of a compound may be an amount sufficient to treat a disease responsive to modulation of cardiac sarcomeres. A therapeutically effective amount varies depending, for example, on the subject and condition being treated, the subject's weight and age, the severity of the condition, the particular compound, the dosing regimen to be followed, the timing of administration, and the mode of administration, all of which can be readily determined by one skilled in the art. A therapeutically effective amount can be ascertained experimentally, for example, by assessing the blood concentration of the chemical moiety, or theoretically, by calculating the bioavailability.

[0041] "Treatment" (and related terms such as "treat," "treated," "treating") includes one or more of inhibiting a disease or disorder, delaying or preventing the onset of clinical symptoms of a disease or disorder, and / or alleviating a disease or disorder (i.e., causing a reduction or regression of clinical symptoms). The term encompasses both complete and partial reduction of a condition or disease, and complete or partial reduction of clinical symptoms of a disease or disorder. Thus, the compounds described and / or disclosed herein may prevent the worsening of an existing disease or disorder, assist in the management of a disease or disorder, or reduce or eliminate a disease or disorder.

[0042] "ATPase" refers to an enzyme that hydrolyzes ATP. ATPases include proteins that include molecular motors such as myosin.

[0043] As used herein, "selective binding" or "selectively binds" refers to preferential binding to a target protein in one type of muscle or muscle fiber as opposed to another type. For example, a compound selectively binds to fast skeletal troponin C if it preferentially binds to troponin C in fast skeletal muscle fibers or sarcomere troponin complexes compared to troponin C in slow muscle fibers or sarcomere troponin complexes, or troponin C in cardiac sarcomere troponin complexes.

[0044] It will be understood that embodiments described herein as "comprising" include "consisting of" and "consisting essentially of" embodiments.

[0045] compound Compounds and their salts (such as pharma- ceutically acceptable salts) are described in detail herein, including the Summary and the accompanying claims. Also provided are all uses of the compounds described herein, including any and all stereoisomers, including geometric isomers (cis / trans), E / Z isomers, enantiomers, diastereomers, and mixtures thereof in any ratio, including racemic mixtures, salts, and solvates, of the compounds described herein, as well as methods of making such compounds. Any compound described herein may be referred to as a drug.

[0046] In one embodiment, a compound of formula (I): [ka] (In the formula, R 1 is halo or C 1-6 is haloalkyl, R 2 is H, halo, or C 1-6 is alkyl, R 3 teeth, (i) cyclohexyl, optionally with one or more independently selected C 1-6 Alkyl or C 1-6 cyclohexyl as defined above, substituted by a haloalkyl substituent; or (ii) C 1-6 alkyl, R 4 teeth, (i) -C(O)H, (ii) -C(O)NH 2 , [ka] and however, (1)R 3 But, C 1-6 If it is alkyl, R 4 teeth [ka] and (2)R 3 is isopropyl, R 1 and R 2 the total number of halo atoms is at least 2; (3)R 3 is one or more independently selected C 1-6 When R is cyclohexyl substituted by haloalkyl substituents, 4 is other than -C(O)H), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing is provided.

[0047] In some embodiments, provided herein is a compound of Formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R 4 is -C(O)H. In some embodiments, provided herein is a compound of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein the compound is of formula (IA): [ka] or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0048] In some embodiments, provided herein is a compound of Formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R 4 is -C(O)NH 2 In some embodiments, provided herein is a compound of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein the compound is of formula (IB): [ka] or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0049] In some embodiments, provided herein is a compound of Formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R 4 teeth, [ka] In some embodiments, provided herein is a compound of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein the compound is of formula (IC): [ka] or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0050] In some embodiments, provided herein is a compound of Formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R 4 teeth, [ka] In some embodiments, provided herein is a compound of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein the compound is of formula (ID): [ka] or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0051] In some embodiments, provided herein is a compound of formula (I), e.g., a compound of (IA), (IB), (IC), or (ID), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R 3 is cyclohexyl, and cyclohexyl is optionally selected from one or more independently selected C 1-6Alkyl or C 1-6 In some embodiments, R 3 is unsubstituted cyclohexyl. In some embodiments, R 3 is cyclohexyl, and cyclohexyl is one or more independently selected C 1-6 Alkyl or C 1-6 Substituted by haloalkyl substituents.

[0052] In some embodiments, R 3 is cyclohexyl, and cyclohexyl is optionally selected from one or more independently selected C 1-6 In some embodiments, R 3 is cyclohexyl, and cyclohexyl is optionally selected from one or more independently selected C 1-3 In some embodiments, R 3 is cyclohexyl, which is optionally substituted by one or more independently selected methyl or ethyl substituents. In some embodiments, R 3 is cyclohexyl, which is optionally substituted with one or more methyl substituents. In some embodiments, R 3 is cyclohexyl, which is optionally substituted with one or more ethyl substituents. In some embodiments, R 3 is cyclohexyl, which is optionally substituted with one methyl substituent. In some embodiments, R 3 is cyclohexyl, which is optionally substituted with one ethyl substituent. In some embodiments, R 3 teeth, [ka] In some embodiments, R 3 teeth, [ka] It is.

[0053] In some embodiments, R 3 is cyclohexyl, and cyclohexyl is one or more independently selected C 1-6 In some embodiments, R 3 is cyclohexyl, and cyclohexyl is one or more independently selected C 1-3 In some embodiments, R 3 teeth, [ka] It is.

[0054] In some embodiments, R 3 teeth, [ka] It is.

[0055] In some embodiments, provided herein is a compound of Formula (I) or Formula (IA), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R 3 teeth, [ka] and R x is C 1-6 alkyl, and the compound is of formula (I-A1): [ka] or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. x is methyl.

[0056] In some embodiments, provided herein is a compound of Formula (I) or Formula (IB), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R 3 teeth, [ka] and R x is C 1-6 Alkyl or C 1-6 haloalkyl, and the compound is of formula (I-B1): [ka] or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. In some embodiments, provided herein is a compound of formula (I-B1), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R x is C 1-6 In some embodiments, R x is methyl.

[0057] In some embodiments, provided herein is a compound of Formula (I) or Formula (IC), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R 3 teeth, [ka] and R x is C 1-6 Alkyl or C 1-6 haloalkyl, and the compound is of formula (I-C1): [ka] or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. In some embodiments, provided herein is a compound of formula (I-C1), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein Rx is C 1-6 In some embodiments, R x is methyl.

[0058] In some embodiments, provided herein is a compound of Formula (I) or Formula (ID), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R 3 teeth, [ka] and R x is C 1-6 Alkyl or C 1-6 haloalkyl, and the compound is of formula (I-D1): [ka] or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. In some embodiments, provided herein is a compound of formula (I-D1), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R x is C 1-6 In some embodiments, R x is methyl.

[0059] In some of the foregoing embodiments, R 3 teeth, [ka] and [ka] The moiety is attached to the remainder of the molecule in a (1r,4r) stereochemical configuration. 3 teeth, [ka] In some embodiments, R 3 teeth, [ka] It is.

[0060] In some embodiments, provided herein is a compound of formula (I), e.g., a compound of formula (IC), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R 3 is C 1-6 In some embodiments, R 3 is C 1-5 In some embodiments, R 3 is C 1-4 In some embodiments, R 3 is C 1-3 In some embodiments, R 3 is C 3-6 In some embodiments, R 3 is C 3-5 In some embodiments, R 3 teeth, [ka] In some embodiments, R 3 teeth, [ka] It is.

[0061] In some of the foregoing embodiments, R 3 is C 1-6 is alkyl, R 3 C 1-6 The alkyl moiety is attached to the remainder of the molecule in the S stereochemical configuration. 3 teeth, [ka] It is.

[0062] In some embodiments, provided herein is a compound of Formula (I) or Formula (IC), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R 3 is isopropyl, and the compound is of formula (I-C2): [ka] or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. In some embodiments, provided herein is a compound of formula (I-C2), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein R 1 and R 2 The total number of halo atoms in R is at least 2. 1 and R 2 The total number of halo atoms in R is 2. 1 and R 2 The total number of halo atoms in R is at least 3. 1 and R 2 The total number of halo atoms in R is 3. 1 and R 2 The total number of halo atoms in R is at least 4. 1 and R 2 The total number of halo atoms in is four.

[0063] In some embodiments, provided herein is a compound of Formula (I), e.g., a compound of Formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing; 1 is halo. In some embodiments, R 1 is fluoro or chloro. In some embodiments, R 1 is fluoro. In other embodiments, R 1 is chloro.

[0064] In some embodiments, provided herein is a compound of Formula (I), e.g., a compound of Formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing; 1 is C 1-6 In some embodiments, R 1 is C 1-6 In some embodiments, R 1 is C 1-2 In some embodiments, R 1 -CF 3 It is.

[0065] In some embodiments, provided herein is a compound of Formula (I), e.g., a compound of Formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing; 2 is H.

[0066] In some embodiments, provided herein is a compound of Formula (I), e.g., a compound of Formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing; 2 is halo. In some embodiments, R 2 is fluoro or chloro. In some embodiments, R 2 is fluoro. In other embodiments, R 2 is chloro.

[0067] In some embodiments, provided herein is a compound of Formula (I), e.g., a compound of Formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing; 2 is C 1-6 In some embodiments, R 2 is C 1-3 In some embodiments, R 2 is C 1-3 In some embodiments, R 2 is C 1-2 In some embodiments, R 2 is methyl.

[0068] In some embodiments, provided herein is a compound of Formula (I), e.g., a compound of Formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing; 1 is halo or C 1-6 haloalkyl, R 2 is H. In some embodiments, R 1 is halo or C 1-6 haloalkyl, R 2 is halo. In some embodiments, R 1 is halo or C 1-6 haloalkyl, R 2 is chloro or fluoro. In some embodiments, R 1 is halo or C 1-6 haloalkyl, R 2 is C 1-6 In some embodiments, R 1 is halo or C 1-6 haloalkyl, R 2 is methyl.

[0069] In some embodiments, provided herein is a compound of Formula (I), e.g., a compound of Formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing; 1 is a halo, R 2 is H. In some embodiments, R 1 is fluoro or chloro, R 2 is H. In some embodiments, R 1 is fluoro and R 2 is H. In some embodiments, R 1 is chloro and R 2 is H. In some embodiments, R 1 is C 1-6 haloalkyl, R 2 is H. In some embodiments, R 1 -CF 3 and R 2 is H.

[0070] In some embodiments, R 1 is a halo, R 2 is halo. In some embodiments, R 1 is fluoro or chloro, R 2 is halo. In some embodiments, R 1 is fluoro or chloro, R 2 is fluoro or chloro. In some embodiments, R 1 is fluoro and R 2 is fluoro. In some embodiments, R 1 is chloro and R 2 is chloro. In some embodiments, R 1 is fluoro and R 2 is chloro. In some embodiments, R 1 is chloro and R 2 is fluoro. In some embodiments, R 1is C 1-6 haloalkyl, R 2 is halo. In some embodiments, R 1 -CF 3 and R 2 is a halo.

[0071] In some embodiments, R 1 is a halo, R 2 is C 1-6 In some embodiments, R 1 is fluoro or chloro, R 2 is C 1-6 In some embodiments, R 1 is fluoro or chloro, R 2 is methyl. In some embodiments, R 1 is fluoro and R 2 is methyl. In some embodiments, R 1 is chloro and R 2 is methyl. In some embodiments, R 1 is C 1-6 haloalkyl, R 2 is C 1-6 In some embodiments, R 1 -CF 3 and R 2 is C 1-6 It is an alkyl.

[0072] In some embodiments, provided herein are compounds set forth in Table 1 and salts thereof. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0073] In some embodiments, provided herein is a compound of formula (I), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, wherein the compound is 5-(4-chlorobenzyl)-8-(4-ethylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide, 8-(4-ethylcyclohexyl)-5-(4-fluorobenzyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide, 5-(4-chloro-3-fluorobenzyl)-8-(4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide, 5-(3,4-difluorobenzyl)-8-(4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde, 5-(3,4-difluorobenzyl)-8-(4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide, 5-(4-fluoro-3-methylbenzyl)-8-(4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide, 5-(3-chloro-4-fluorobenzyl)-8-(4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide, 5-(4-chloro-3-fluorobenzyl)-8-isopropyl-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione, 5-(3-chloro-4-fluorobenzyl)-8-isopropyl-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione, 5-(4-chlorobenzyl)-8-(4-methylcyclohexyl)-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione, 5-(3,4-difluorobenzyl)-8-(4-ethylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide, 5-(3,4-difluorobenzyl)-8-(4-methylcyclohexyl)-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione, 8-isopropyl-2-(pyridazin-3-yl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione, 8-(sec-butyl)-5-(3,4-difluorobenzyl)-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione, 5-(3,4-difluorobenzyl)-8-(pentan-3-yl)-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione, 8-(sec-butyl)-5-(4-fluoro-3-methylbenzyl)-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione, 5-(4-fluorobenzyl)-8-(4-methylcyclohexyl)-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione, 5-(4-chlorobenzyl)-8-(4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide, 5-(4-fluorobenzyl)-8-(4-methylcyclohexyl)-2-(pyridazin-4-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione, and 8-(4-(difluoromethyl)cyclohexyl)-5-(4-fluorobenzyl)-2-(pyridazin-4-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0074] In some variations, any of the compounds of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or any variation thereof, or any of the compounds described herein, such as the compounds in Table 1, may be deuterated (i.e., one or more hydrogen atoms are replaced by one or more deuterium atoms). In some of these variations, the compound is deuterated at a single site. In other variations, the compound is deuterated at multiple sites. Deuterated compounds can be prepared from deuterated starting materials in a manner similar to the preparation of the corresponding non-deuterated compounds. Other methods known in the art may be used to replace hydrogen atoms with deuterium atoms.

[0075] Any formula shown herein, for example, formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), is intended to represent a compound having the structure shown by the structural formula, as well as certain variations or forms. In particular, compounds of any formula shown herein may have asymmetric centers and therefore may exist in different enantiomeric or diastereomeric forms. All optical isomers and stereoisomers of a compound of the general formula, as well as mixtures thereof in any ratio, are considered to be within the scope of the formula. Thus, any formula shown herein is intended to represent a racemate, one or more enantiomeric forms, one or more diastereomeric forms, one or more atropisomeric forms, and mixtures thereof in any ratio. Where a compound in Table 1 is shown in a particular stereochemical configuration, any alternative stereochemical configuration of the compound, as well as mixtures of stereoisomers of the compound in any ratio, are also provided herein. For example, if a compound of Table 1 has a stereocenter in the "S" stereochemical configuration, then enantiomers of that compound in the "R" stereochemical configuration are also provided herein. Similarly, if a compound of Table 1 has a stereocenter in the "R" configuration, then enantiomers of that compound in the "S" stereochemical configuration are also provided herein. Also provided are mixtures of compounds having both "S" and "R" stereochemical configurations. Additionally, if a compound of Table 1 has more than one stereocenter, then any enantiomers or diastereomers of the compound are also provided. For example, if a compound of Table 1 contains a first stereocenter and a second stereocenter having "R" and "R" stereochemical configurations, respectively, then stereoisomers of the compound having the first and second stereocenters having "S" and "S" stereochemical configurations, respectively, and "S" and "R" stereochemical configurations, respectively, are also provided.When a compound of Table 1 contains a first stereocenter and a second stereocenter having the stereochemical configurations of "S" and "S", respectively, stereoisomers of the compound having the first and second stereocenters having the stereochemical configurations of "R" and "R", respectively, "S" and "R", respectively, and "R" and "S", respectively, are also provided. When a compound of Table 1 contains a first stereocenter and a second stereocenter having the stereochemical configurations of "S" and "R", respectively, stereoisomers of the compound having the first and second stereocenters having the stereochemical configurations of "R" and "S", respectively, "R" and "R", respectively, and "S" and "S", respectively, are also provided. Similarly, where a compound of Table 1 contains a first stereocenter and a second stereocenter having the stereochemical configurations of "R" and "S", respectively, stereoisomers of the compound having the first and second stereocenters having the stereochemical configurations of "S" and "R", respectively, "R" and "R", respectively, and "S" and "S", respectively, are also provided. Furthermore, certain structures may exist as geometric isomers (i.e., cis and trans isomers), as tautomers, or as atropisomers. Furthermore, any formula shown herein is intended to refer to any one of the hydrates, solvates, and amorphous and polycrystalline forms of such compounds, and mixtures thereof, even if such forms are not explicitly recited. In some embodiments, the solvent is water and the solvate is a hydrate.

[0076] Representative examples of compounds detailed herein, including intermediates and final compounds, are set forth in the Tables and elsewhere herein. In one aspect, it is understood that any compound may be used in the methods detailed herein, including intermediate compounds, which, where applicable, may be isolated and administered to an individual or subject.

[0077] The compounds provided herein may exist as salts even if the salt is not provided, and as will be appreciated by one of skill in the art, the compositions and methods provided herein are understood to encompass all salts and solvates of the compounds provided herein, as well as non-salt and non-solvated forms of the compounds. In some embodiments, the salts of the compounds provided herein are pharma- ceutically acceptable salts.

[0078] In one variation, the compounds herein are synthetic compounds prepared for administration to an individual or subject. In another variation, compositions are provided that include the compounds in substantially pure form. In another variation, pharmaceutical compositions are provided that include the compounds detailed herein and a pharma- ceutically acceptable carrier. In another variation, methods of administering the compounds are provided. The purified forms, pharmaceutical compositions, and methods of administering the compounds are suitable for any of the compounds or forms thereof detailed herein.

[0079] The R provided in this document 1 , R 2 , R 3 , and R 4 Any variation or embodiment of R is expressly incorporated herein by reference as if each combination were individually and specifically set forth in the same manner. 1 , R 2 , R 3 , and R 4 can be combined with any other variations or embodiments of the present invention.

[0080] As used herein, when any variable occurs more than one time in a chemical formula, its definition at each occurrence is independent of its definition at every other occurrence.

[0081] Formula (I) includes all subformulas thereof, for example, formula (I) includes compounds of formula (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1).

[0082] Certain compound names provided herein, including Table 1, are provided by ChemBioDraw Professional 15.0.0.106. Those skilled in the art will appreciate that compounds may be named or identified using a variety of commonly recognized naming systems and symbols. By way of example, compounds may be named or identified by common, systematic, or non-systematic names. Naming systems and symbols commonly recognized in the chemical arts include, for example, Chemical Abstract Service (CAS), ChemBioDraw Ultra, and International Union of Pure and Applied Chemistry (IUPAC).

[0083] In some embodiments, the disclosed compounds or pharma- ceutically acceptable salts thereof may have advantages related to one or more of hERG profile, toxicity profile, safety window, selectivity, off-target profile, favorable drug / drug interaction profile, bioavailability, PK parameters including clearance and half-life, mechanism of action, CYP inhibition and time-dependent inhibition profile, permeability and / or efflux, solubility, metabolism, unbound fraction, suitable human dose, and ease of large-scale synthesis. composition

[0084] Compositions are also provided, such as pharmaceutical compositions, that include the compounds disclosed and / or described herein, and one or more additional agents, pharmaceuticals, adjuvants, carriers, excipients, and the like. Suitable agents and pharmaceuticals include those described herein. In some embodiments, the pharmaceutical composition includes a pharma- ceutically acceptable excipient or adjuvant and at least one chemical component described herein. Examples of pharma- ceutically acceptable excipients include, but are not limited to, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, croscarmellose sodium, glucose, gelatin, sucrose, and magnesium carbonate. In some embodiments, compositions are provided, such as pharmaceutical compositions, that include one or more compounds described herein, or stereoisomers or tautomers thereof, or pharma- ceutically acceptable salts of any of the foregoing.

[0085] In some embodiments, a pharma- ceutically acceptable composition is provided that includes a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. In some aspects, the composition may contain a synthetic intermediate that may be used in the preparation of the compounds described herein. The compositions described herein may contain any other suitable active or inactive agents.

[0086] Any of the compositions described herein may be sterile or contain components that are sterile. Sterilization can be achieved by methods known in the art. Any of the compositions described herein may contain one or more compounds or conjugates that are substantially pure.

[0087] Also provided is a packaged pharmaceutical composition comprising a pharmaceutical composition described herein and instructions for using the composition to treat a patient suffering from a disease or condition described herein.

[0088] How to use The compounds and pharmaceutical compositions herein can be used to treat or prevent a disease or condition in an individual or subject.

[0089] When used in a prophylactic manner, the compounds disclosed and / or described herein may prevent a disease or disorder from occurring or reduce the extent of a disease or disorder that may occur in an individual or subject at risk of developing the disease or disorder.

[0090] Without being bound by theory, it is believed that the compounds and pharmaceutical compositions disclosed herein act by inhibiting myosin. This inhibition potentially reduces the number of independent myosin heads that interact with actin filaments, reducing the amount of contraction. Reducing cardiac muscle contraction may be important for the treatment of cardiac diseases where excessive contraction is a problem. In some embodiments, a method of treating or preventing cardiac disease in an individual or subject is provided, comprising administering to an individual or subject in need thereof a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. In some embodiments, a method of treating or preventing cardiac disease in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of at least one chemical moiety described herein. In some embodiments, there are provided methods of treating cardiac disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one chemical moiety described herein. In some embodiments, there are provided methods of treating established or diagnosed cardiac disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one chemical moiety described herein. In some embodiments, there are provided methods of preventing cardiac disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one chemical moiety described herein.

[0091] Also provided herein is the use of a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, in the manufacture of a medicament for the treatment of a cardiac disease in a subject. In some aspects, a compound or composition described herein is provided for use in a method of treating the human or animal body by therapy. In some embodiments, provided herein is a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in a method of treating the human or animal body by therapy. In some embodiments, this document provides a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in treating or preventing cardiac disease. In some embodiments, this document provides a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in treating cardiac disease. In some embodiments, provided herein is a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in treating established or diagnosed cardiac disease.In some embodiments, this document provides a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in preventing heart disease. In some embodiments, this document provides a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in treating a disease or condition associated with HCM. In some embodiments, provided herein is a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in treating a disease or condition associated with secondary left ventricular wall thickening. In some embodiments, provided herein is a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in ameliorating a symptom associated with cardiac disease. In other embodiments, provided herein is a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in reducing the risk of conditions associated with cardiovascular disease.In other embodiments, provided herein is a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in treating a disease or condition associated with a small left ventricular lumen, lumen obstruction, high-output left ventricular contraction, obstruction of blood flow from the left ventricle, cardiac hypertrophy, low cardiac stroke ejection, impaired left ventricular relaxation, high left ventricular filling pressures, myocardial ischemia, or cardiac fibrosis. In certain embodiments, provided herein is a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in treating a disease or condition associated with small left ventricular cavity and cavity obstruction, high-output left ventricular contraction, myocardial ischemia, or cardiac fibrosis. In some embodiments, provided herein is a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in treating a muscular dystrophy. In some embodiments, this document provides a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in treating a glycogen storage disease. In other embodiments, this document provides a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in modulating cardiac sarcomere, such as inhibiting cardiac sarcomere.In yet other embodiments, provided herein is a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, for use in enhancing cardiac myosin.

[0092] In some embodiments, the subject is a mammal. In some embodiments, the subject is a mouse, rat, dog, cat, pig, sheep, horse, cow, or human. In some embodiments, the subject is a human. In some embodiments, the subject has established or diagnosed cardiac disease. In some embodiments, the subject has established or diagnosed hypertrophic cardiomyopathy (HCM). In some embodiments, the subject is at risk of developing cardiac disease. In some embodiments, the subject has a mutation that increases the risk of cardiac disease. In some embodiments, the subject has a mutation that increases the risk of hypertrophic cardiomyopathy (HCM). In some embodiments, the mutation is a sarcomere mutation. In some embodiments, the mutation is in myosin heavy chain beta (MHC-β), cardiac troponin T (cTnT), tropomyosin alpha-1 chain (TPM1), myosin-binding protein C cardiac type (MYBPC3), cardiac troponin I (cTnI), myosin essential light chain (ELC), titin (TTN), myosin regulatory light chain 2 ventricular / cardiac isoform (MLC-2), cardiac alpha actin, muscle LIM protein (MLP), or protein kinase AMP activating non-catalytic subunit gamma 2 (PRKAG2). In some embodiments, the mutation is in MHC-β. In some embodiments, the subject has established or diagnosed hypertrophic cardiomyopathy without a confirmed genetic etiology.

[0093] In some embodiments, the subject is at high risk for a progressive condition. In some embodiments, the subject is at high risk for atrial fibrillation, ventricular tachyarrhythmia, stroke, and / or sudden death. In some embodiments, the subject has reduced exercise capacity. In some embodiments, the reduced exercise capacity is reduced compared to an age-matched control population. In some embodiments, the subject is eligible for surgical intervention or percutaneous ablation therapy to treat cardiac disease.

[0094] In some embodiments, the cardiac disease is hypertrophic cardiomyopathy (HCM). In some embodiments, the cardiac disease is obstructive HCM. In some embodiments, the cardiac disease is non-obstructive HCM. In some embodiments, the HCM is associated with a sarcomere mutation. In some embodiments, the HCM is associated with a non-sarcomere mutation. In some embodiments, the cardiac disease is obstructive or non-obstructive HCM caused by a sarcomere mutation and / or a non-sarcomere mutation. In some embodiments, the sarcomere mutation is a mutation in myosin heavy chain beta (MHC-β), cardiac troponin T (cTnT), tropomyosin alpha-1 chain (TPM1), myosin binding protein C cardiac type (MYBPC3), cardiac troponin I (cTnI), myosin essential light chain (ELC), titin (TTN), myosin regulatory light chain 2 ventricular / cardiac isoform (MLC-2), cardiac alpha actin, or muscle LIM protein (MLP). In some embodiments, the sarcomeric mutation is a mutation in MHC-β. In some embodiments, the non-sarcomeric mutation is a mutation in protein kinase AMP-activated non-catalytic subunit gamma 2 (PRKAG2).

[0095] In some embodiments, provided herein is a method of treating a disease or condition associated with HCM, comprising administering to an individual or subject in need thereof a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound in Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. In some embodiments, the disease or condition is Fabry disease, Danon disease, mitochondrial cardiomyopathy, or Noonan syndrome.

[0096] Also provided herein is the use of a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, in the manufacture of a medicament for the treatment of a disease or condition associated with HCM.

[0097] In some embodiments, the cardiac disease is heart failure with preserved ejection fraction (HFpEF). In some embodiments, the cardiac disease is diastolic dysfunction. In some embodiments, the cardiac disease is a cardiomyopathy. In some embodiments, the cardiac disease is primary or secondary restrictive cardiomyopathy. In some embodiments, the cardiac disease is a condition or symptom caused by coronary artery disease. In some embodiments, the cardiac disease is myocardial infarction or angina pectoris. In some embodiments, the cardiac disease is left ventricular outflow tract obstruction. In some embodiments, the cardiac disease is hypertensive heart disease. In some embodiments, the cardiac disease is congenital heart disease. In some embodiments, the cardiac disease is cardiac ischemia and / or coronary heart disease. In some embodiments, the cardiac disease is diabetic heart disease. In other embodiments, the cardiac disease is congestive heart failure. In some embodiments, the cardiac disease is right heart failure. In other embodiments, the cardiac disease is cardiorenal syndrome. In some embodiments, the cardiac disease is infiltrative cardiomyopathy. In some embodiments, the cardiac disease is, or is associated with, cardiac aging or age-related diastolic dysfunction. In some embodiments, the cardiac disease is, or is associated with, left ventricular hypertrophy and / or concentric left ventricular remodeling.

[0098] In some embodiments, there is provided a method of treating a disease or condition associated with secondary left ventricular wall thickening in an individual or subject, comprising administering to an individual or subject in need thereof a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. In some embodiments, the disease is hypertension, valvular heart disease (aortic stenosis, mitral regurgitation), metabolic syndrome (diabetes, obesity), end stage renal disease, scleroderma, sleep apnea, amyloidosis, Fabry's disease, Friedreich's ataxia, Danon's disease, Noonan's syndrome, or Pompe's disease.

[0099] Also provided herein is the use of a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, in the manufacture of a medicament for the treatment of a disease or condition associated with secondary left ventricular wall thickening.

[0100] In some embodiments, there is provided a method of alleviating a symptom associated with cardiac disease in a subject, comprising administering to an individual or subject in need thereof a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing; wherein the symptom is reduced cardiac elasticity. or reduced diastolic left ventricular relaxation, abnormal left atrial pressure (e.g., abnormally high left atrial pressure), paroxysmal or permanent atrial fibrillation, increased left atrial and pulmonary capillary wedge pressure, increased left ventricular diastolic pressure, collapsing, relaxation of the ventricle during diastole, ventricular fibrosis, left ventricular hypertrophy, left ventricular mass, increased left ventricular wall thickness, left ventricular midchamber obstruction, increased mitral valve systolic anterior motion, left ventricular outflow tract obstruction, chest pain, exertional dyspnea, pre-collapse, abnormal exercise capacity, and fatigue.

[0101] In some embodiments, there is provided a method of reducing the risk of a condition associated with cardiac disease in a subject, comprising administering to an individual or subject in need thereof a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharmacologic acceptable salt of any of the foregoing; The symptoms are selected from one or more of: impaired or reduced elasticity, poor or reduced relaxation of the left ventricular during diastole, abnormal left atrial pressure (e.g., abnormally high left atrial pressure), paroxysmal or permanent atrial fibrillation, increased left atrial and pulmonary capillary wedge pressure, increased left ventricular diastolic pressure, collapse, ventricular relaxation during diastole, ventricular fibrosis, left ventricular hypertrophy, left ventricular mass, increased left ventricular wall thickness, left ventricular midchamber obstruction, increased mitral valve systolic anterior motion, left ventricular outflow tract obstruction, chest pain, exertional dyspnea, pre-collapse, abnormal exercise capacity, and fatigue.

[0102] In some embodiments, there is provided a method of treating a disease or condition associated with a small left ventricular cavity, lumen obstruction, high output left ventricular contraction, obstruction of blood outflow from the left ventricle, cardiac hypertrophy, low cardiac stroke ejection, impaired left ventricular relaxation, high left ventricular filling pressure, myocardial ischemia, or cardiac fibrosis in an individual or subject, comprising administering to an individual or subject in need thereof a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0103] In some embodiments, there is provided a method of treating a disease or condition associated with small left ventricular cavity and lumen obstruction, high output left ventricular contraction, myocardial ischemia, or cardiac fibrosis in an individual or subject, comprising administering to an individual or subject in need thereof a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0104] Also provided herein is the use of a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, in the manufacture of a medicament for the treatment of a disease or condition associated with small left ventricular lumen and lumen obstruction, high-output left ventricular contraction, myocardial ischemia, or cardiac fibrosis.

[0105] In some embodiments, there is provided a method of treating muscular dystrophy (e.g., Duchenne muscular dystrophy) in an individual or subject, comprising administering to the individual or subject in need thereof a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. Also provided herein is the use of a compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, in the manufacture of a medicament for the treatment of muscular dystrophy (e.g., Duchenne muscular dystrophy).

[0106] In some embodiments, there is provided a method of treating glycogen storage disease in an individual or subject, comprising administering to an individual or subject in need thereof a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. Also provided herein is the use of a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, in the manufacture of a medicament for the treatment of glycogen storage disease.

[0107] Also provided are methods for modulating cardiac sarcomeres in an individual or subject, comprising administering to an individual or subject in need thereof a therapeutically effective amount of at least one chemical moiety described herein. In some embodiments, provided are methods for inhibiting cardiac sarcomeres, comprising contacting the cardiac sarcomere with at least one chemical moiety described herein, such as a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. Additionally provided herein is the use of at least one chemical moiety described herein, such as a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, in the manufacture of a medicament for inhibiting cardiac sarcomere in an individual or subject.

[0108] Also provided is a method for enhancing cardiac myosin in an individual or subject, comprising administering to the individual or subject in need thereof a therapeutically effective amount of at least one chemical moiety described herein, e.g., a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound in Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing. Additionally provided herein is the use of at least one chemical moiety described herein, e.g., a compound of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, in the manufacture of a medicament for enhancing cardiac myosin in an individual or subject.

[0109] In some embodiments, the method provided herein further comprises monitoring the effectiveness of the treatment. Examples of indicators include, but are not limited to, improvements in one or more of the following: New York Heart Association (NYHA) functional class, exercise capacity, cardiac elasticity, diastolic relaxation of the left ventricle, left atrial pressure, paroxysmal or permanent atrial fibrillation, left atrial and pulmonary capillary wedge pressure, left ventricular diastolic pressure, fainting, ventricular relaxation during diastole, ventricular fibrosis, left ventricular hypertrophy, left ventricular mass, left ventricular wall thickness, left ventricular mid-cavity obstruction, mitral valve systolic anterior motion, left ventricular outflow tract obstruction, chest pain, exertional dyspnea, pre-fainting, exercise capacity abnormalities, and fatigue. These indicators can be monitored by techniques known in the art, including self-report, ECG including Holter ECG, echocardiography, cardiac MRI, CT, biopsy, cardiopulmonary exercise testing (CPET), and actigraphy.

[0110] In some embodiments, the compounds reduce cardiomyocyte contractility. In some embodiments, the compounds reduce cardiomyocyte contractility by more than 40%, e.g., 45%, 50%, 60%, 70%, 80%, or more than 90%. In some embodiments, the compounds reduce cardiomyocyte contractility by 40%-90%, e.g., 40%-80%, 40%-70%, 50%-90%, 50%-80%, or 50%-70%. In some embodiments, the compounds do not significantly alter calcium translocation in cardiomyocytes. In some embodiments, the compounds reduce ATPase activity in cardiomyocytes. Methods for measuring contractility, ATPase activity, and calcium translocation are known in the art, e.g., by calcium labeling, electrophysiological recording, and microscopic imaging. In some embodiments, the compounds do not significantly inhibit or induce cytochrome P450 (CYP) proteins.

[0111] In some embodiments, the elimination half-life (t 1 / 2 , ln(2) / k, where the elimination rate constant k is calculated as the absolute value of the slope of the linear regression of the log of concentration versus time for the last three data points in the concentration-time profile, is less than or equal to 30 hours in humans. In some embodiments, the compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, is provided. In some embodiments, the compound of Formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or a compound of Table 1, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, is less than or equal to 10 hours in humans. 1 / 2 In some embodiments, t 1 / 2 In some embodiments, t is about 10 hours to about 30 hours, about 10 hours to about 25 hours, about 15 hours to about 30 hours, or about 15 hours to about 25 hours. 1 / 2 is about 12, 15, 18, 21, 24, 27, or 30 hours. In some embodiments, the elimination half-life of the compounds provided herein is such that the compounds are suitable for once daily administration.

[0112] In some embodiments, the subject has a left ventricular wall thicker than normal before treatment. In some embodiments, the subject has a left ventricular wall thickness greater than 15 mm, such as greater than 18 mm, 20 mm, 22 mm, 25 mm, or 30 mm before treatment. In some embodiments, the left ventricular wall thickness is reduced by more than 5% after treatment, for example, by more than 8%, 10%, 12%, 15%, 20%, or 30%. The left ventricular wall thickness can be measured by methods known in the art, such as echocardiography, CT scan, or cardiac MRI.

[0113] In some embodiments, the subject has abnormal cardiac fibrosis before treatment. In some embodiments, the abnormal cardiac fibrosis is reduced by more than 5% after treatment, for example, by more than 8%, 10%, 12%, 15%, 20%, or 30%. Cardiac fibrosis can be measured by methods known in the art, such as biopsy or cardiac MRI.

[0114] In some embodiments, the subject has reduced exercise capacity prior to treatment. In some embodiments, the subject's exercise capacity increases by more than 5%, e.g., more than 8%, 10%, 12%, 15%, 20% or 30%, after treatment. In some embodiments, exercise capacity is measured by cardiopulmonary exercise testing (CPET). CPET measures changes in oxygen consumption (VO 2 max) is measured. CPET and VO 2 Methods for measuring VO max are well known in the art (Malhotra et al., JACC: Heart Failure, 2016, 4(8):607-616; Guazzi et al., J Amer College Cardiol, 2017, 70 (13):1618-1636; Rowin et al., JACC: Cariovasc Imaging, 2017, 10(11):1374-1386). In some embodiments, VO 2 Max is 1.2mL / kg / m after treatment. 2 , 1.4 mL / kg / m 2 , 1.5 mL / kg / m 2 , 1.7 mL / kg / m 2 , 2 mL / kg / m2 , 2.2 mL / kg / m 2 , 2.5 mL / kg / m 2 , 3 mL / kg / m 2 , 3.2 mL / kg / m 2 , or 3.5 mL / kg / m 2 More than 1 mL / kg / m 2 will be improved beyond that.

[0115] In some embodiments, the subject has a New York Heart Association (NYHA) functional class of II, III or IV before treatment. In some embodiments, the subject has a New York Heart Association (NYHA) functional class of III or IV before treatment. In some embodiments, the subject has a New York Heart Association (NYHA) functional class of IV before treatment. In some embodiments, the subject remains in the same NYHA functional class or has a decreased NYHA functional class after treatment.

[0116] In some embodiments, the VO 2 Maximum is 1.2 mL / kg / m 2 , 1.4 mL / kg / m 2 , 1.5 mL / kg / m 2 , 1.7 mL / kg / m 2、 or 2 mL / kg / m 2 More than 1 mL / kg / m 2 In some embodiments, the subject has an improvement in NYHA functional class beyond VO 2 Maximum is 2.5 mL / kg / m 2 , 3 mL / kg / m 2 , 3.2 mL / kg / m 2 , or 3.5 mL / kg / m 2 and the subject remains in the same NYHA functional class after treatment or has a decline in NYHA functional class.

[0117] In some embodiments, the subject's daily functioning and / or activity level is improved following treatment. Improved daily functioning and / or activity levels can be measured, for example, by journaling or actigraphy, such as a FitBit or a FitBit-like monitor.

[0118] In some embodiments, the subject has one or more of reduced shortness of breath, reduced chest pain, reduced arrhythmia burden such as atrial fibrillation and ventricular arrhythmias, reduced incidence of heart failure, and reduced ventricular outflow obstruction after treatment.

[0119] Dosage The compounds and compositions disclosed and / or described herein are administered in therapeutically effective dosages, e.g., dosages sufficient to effect treatment of a medical condition. Human dosage levels of the chemical moieties described herein have not yet been optimized, but generally range from about 0.01 to 100 mg / kg body weight daily, in some embodiments from about 0.05 to 10.0 mg / kg body weight, and in some embodiments from about 0.10 to 1.4 mg / kg body weight. Thus, when administered to a 70 kg human, the dosage range is, in some embodiments, from about 0.7 to 7000 mg per day, in some embodiments, from about 3.5 to 700.0 mg per day, and in some embodiments, from about 7 to 100.0 mg per day. The amount of chemical moiety administered depends, for example, on the subject and medical condition being treated, the severity of the disease, the mode and schedule of administration, and the judgment of the prescribing physician. For example, exemplary dosage ranges for oral administration are from about 5 mg to about 500 mg per day, and exemplary dosages for intravenous administration are from about 5 mg to about 500 mg per day, each depending on the pharmacokinetics of the compound.

[0120] A daily dose is the total amount administered in one day. A daily dose can be administered daily, every other day, weekly, every two weeks, monthly, or at various intervals, but is not limited to these. In some embodiments, a daily dose is administered for a period ranging from one day to the subject's lifetime. In some embodiments, a daily dose is administered once a day. In some embodiments, a daily dose is administered in multiple divided doses, such as two, three, or four divided doses. In some embodiments, a daily dose is administered in two divided doses.

[0121] Administration of the compounds and compositions disclosed and / or described herein can be via any accepted mode of administration for therapeutic agents, including, but not limited to, oral, sublingual, subcutaneous, parenteral, intravenous, intranasal, topical, transdermal, intraperitoneal, intramuscular, pulmonary, intravaginal, rectal, or ocular administration. In some embodiments, the compounds or compositions are administered orally or intravenously. In some embodiments, the compounds or compositions disclosed and / or described herein are administered orally.

[0122] Pharmaceutically acceptable compositions include solid, semi-solid, liquid and aerosol dosage forms, such as tablets, capsules, powders, solutions, suspensions, suppositories, and aerosol forms. The compounds disclosed and / or described herein can also be administered in sustained or controlled release dosage forms (e.g., controlled / sustained release pills, depot injections, osmotic pumps, or transdermal (including electrotransport) patch forms) for extended administration and / or pulse administration at a predetermined rate. In some embodiments, the compositions are provided in unit dosage forms suitable for single administration of precise doses.

[0123] The compounds disclosed and / or described herein may be administered alone or in combination with one or more conventional pharmaceutical carriers or excipients (e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, croscarmellose sodium, glucose, gelatin, sucrose, magnesium carbonate). If desired, the pharmaceutical compositions may contain small amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents, and the like (e.g., sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate). In general, pharmaceutical compositions contain about 0.005% to 95% by weight, or about 0.5% to 50% by weight, of the compounds disclosed and / or described herein, depending on the intended mode of administration. Actual methods for preparing such dosage forms will be known or apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0124] In some embodiments, the composition takes the form of a pill or tablet, and thus the composition may contain one or more of a diluent (e.g., lactose, sucrose, dicalcium phosphate), a lubricant (e.g., magnesium stearate), and / or a binder (e.g., starch, gum arabic, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives) along with the compound disclosed and / or described herein. Other solid dosage forms include powders, marumes, solutions or suspensions (e.g., in propylene carbonate, vegetable oils, or triglycerides) enclosed in a gelatin capsule.

[0125] Liquid pharma- ceutically administrable compositions can be prepared, for example, by dissolving, dispersing, or suspending the compounds disclosed and / or described herein and optional pharmaceutical excipients in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycol, ethanol, etc.) to form a solution or suspension. Injectables can be prepared in conventional forms, as liquid solutions or suspensions, as emulsions, or in solid forms suitable for dissolving or suspending in liquid prior to injection. The percentage of compound in such parenteral compositions depends, for example, on the physical properties of the compound, the activity of the compound, and the needs of the subject. However, percentages of active ingredient between 0.01% and 10% in solution can be used, and may be higher if the composition is solid and will be subsequently diluted to another concentration. In some embodiments, the composition contains about 0.2-2% of the compounds disclosed and / or described herein in solution.

[0126] Pharmaceutical compositions of the compounds disclosed and / or described herein may be administered to the respiratory tract as an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, either alone or in combination with an inert carrier such as lactose. In such cases, the particles of the pharmaceutical composition may have diameters of less than 50 microns, or in some embodiments, less than 10 microns.

[0127] Additionally, pharmaceutical compositions may include a compound disclosed and / or described herein, as well as one or more additional drugs, pharmaceutical agents, adjuvants, etc. Suitable drugs and pharmaceutical agents include those described herein.

[0128] kit Also provided are articles of manufacture and kits containing any of the compounds or pharmaceutical compositions provided herein. The articles of manufacture may include a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic. The containers may hold the pharmaceutical compositions provided herein. The label on the container may indicate that the pharmaceutical composition is used to prevent, treat, or inhibit a condition described herein, and may indicate instructions for either in vivo or in vitro use. In one aspect, this document provides a kit that contains the compound or composition described herein and instructions for use.The kit can include instructions for use in an individual or subject who needs to treat heart disease.The kit can further include any material or device that can be used to administer the compound or composition, such as a vial, a syringe, or an IV bag.The kit can include a sterile package.

[0129] combination The compounds and compositions described and / or disclosed herein may be administered alone or in combination with other therapies and / or therapeutic agents useful in the treatment of the aforementioned disorders, diseases, or conditions.

[0130] The compounds and compositions described and / or disclosed herein may be combined with one or more other therapeutic agents for treating cardiac diseases such as HCM or HFpEF. In some embodiments, the one or more therapeutic agents include therapeutic agents that slow the progression of heart failure and prevent cardiac remodeling by downregulating cardiac neurohormonal stimulation (e.g., ACE inhibitors, angiotensin receptor blockers (ARBs), beta-blockers, aldosterone receptor antagonists, or neuroendopeptidase inhibitors). In some embodiments, the one or more therapeutic agents include therapeutic agents that improve cardiac function by stimulating cardiac contractility (e.g., positive inotropic agents such as the beta-adrenergic agonist dobutamine or the phosphodiesterase inhibitor milrinone). In other embodiments, the one or more therapeutic agents include therapeutic agents that reduce cardiac preload (e.g., diuretics such as furosemide) or therapeutic agents that reduce afterload (vasodilators of any class, including but not limited to calcium channel blockers, phosphodiesterase inhibitors, endothelin receptor antagonists, renin inhibitors, or smooth muscle myosin modulators).

[0131] The compounds and compositions described and / or disclosed herein may be combined with one or more other therapeutic agents for treating HCM or HFpEF, in some embodiments, the compounds and / or compositions may be combined with a beta-blocker, verapamil, and / or disopyramide.

[0132] General synthesis method Compounds of formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1) are described below with reference to exemplary synthetic schemes for their general preparation and specific examples that follow. Those skilled in the art will recognize that to obtain various compounds herein, starting materials can be suitably selected such that the ultimately desired substituents are retained throughout the reaction scheme, with or without protection as necessary, to yield the desired products. Alternatively, it may be necessary or desirable to employ, in place of the ultimately desired substituent, a suitable group that is retained throughout the reaction scheme and can be replaced with the desired substituent as appropriate. Furthermore, those skilled in the art will recognize that certain functional groups (amino, carboxy, or side chain groups) can be protected from reaction conditions using protecting groups, and that such groups are removed under standard conditions when appropriate. Unless otherwise specified, the variables are as defined above for formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1).

[0133] If it is desired to obtain a particular enantiomer of a compound, this can be achieved from the mixture of corresponding enantiomers by using any suitable conventional procedure for separating or resolving enantiomers.Thus, for example, diastereomeric derivatives can be produced by reacting a mixture of enantiomers, for example a racemate, with a suitable chiral compound.The diastereomers can then be separated by any convenient means, for example by crystallization, and the desired enantiomer can be recovered.In another resolution process, chiral high performance liquid chromatography can be used to separate the racemate.Alternatively, if desired, a particular enantiomer can be obtained by using a suitable chiral intermediate in one of the steps described.

[0134] Chromatography, recrystallization, and other conventional separation procedures may also be used to obtain a specific isomer of a compound, an intermediate, or a final product where it is desired to otherwise purify the product of a reaction.

[0135] General methods for preparing the compounds described herein are shown in the following exemplary methods. The variables in the schemes provided herein are defined as in formula (I), (IA), (I-A1), (IB), (I-B1), (IC), (I-C1), (I-C2), (ID), or (I-D1), or any variation thereof. Other compounds described herein can be prepared by similar methods.

[0136] In some embodiments, compounds provided herein can be synthesized according to Scheme 1. [ka]

[0137] In Scheme 1, R 1 and R 2 is as defined elsewhere herein for a compound of formula (I), or a variation or embodiment thereof, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing; R x is C 1-6 Alkyl or C 1-6 haloalkyl, R y is H or -NH 2 It is.

[0138] In some embodiments, compounds provided herein can be synthesized according to Scheme 2. [ka]

[0139] In Scheme 2, R 1 and R 2is as defined elsewhere herein for a compound of formula (I), or a variation or embodiment thereof, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing; R y is H or -NH 2 It is.

[0140] In some embodiments, compounds provided herein can be synthesized according to Scheme 3. [ka]

[0141] In Scheme 3, R 1 and R 2 is as defined elsewhere herein for a compound of formula (I), or a variation or embodiment thereof, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

[0142] Specific non-limiting examples are provided in the Examples section below. EXAMPLES

[0143] The following examples are provided to illustrate, but not limit, the compositions, uses, and methods provided herein. Compounds are prepared using the general methods described above.

[0144] The following abbreviations may be used throughout the examples: TEA (trimethylamine), DCM (dichloromethane), (Boc). 2O (di-tert-butyl decarbonate), EA (ethyl acetate), PE (petroleum ether), DMF (N,N-dimethylformamide), DIEA (N-ethyl-N-isopropylpropan-2-amine), HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), HOAt (1-hydroxy-7-azabenzotriazole), HOBt (hydroxybenzotriazole), EDCI (1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide), MeOH (methanol), EtOH (ethanol), IPA (iPrOH, propan-2-ol), NMP (1-methylpyrrolidin-2-one), STAB (sodium triacetoxyborohydride), ACN (acetonitrile), TFA (trifluoroacetic acid), DPPA (diphenylphosphoryl azide), DBU (1,8-diazabicyclo(5.4.0)undec-7-ene), THF (tetrahydrofuran), PPh 3 (triphenylphosphane), SM (starting material), Hex (hexane), NCS (N-chlorosuccinimide), rt (room temperature), DCE (dichloroethane), FA (formic acid), CHCl 3 (chloroform), BnBr (benzyl bromide), HCl (hydrochloric acid), equiv (equivalent), RT (retention time), SFC (supercritical fluid chromatography), and DSC (bis(2,5-dioxopyrrolidin-1-yl)carbonate).

[0145] The XRPD diffractograms were collected using the following parameters:

[0146] XRPD equipment: Rigaku MiniFlex600 6G benchtop X-ray diffraction system, X-ray generator: supplying 600W of power (40kV / 15mA), sealed X-ray tube: Toshiba A-21-Cu tube with normal focus, X-ray radiation: CuKα, software: Smartlab Studio II x64 ver.4.5.352.0 (data collection and analysis), entrance Soller slit: 2.5°, divergence slit: 1.25°, length limiting slit: 10, sample stage: ASC-8PM_MF, filter: Cu_beta_X1.5, receiving Soller slit: 2.5°, scattering slit: 8.0mm, receiving slit: 0.3mm, detector: high speed D / tex Utra2 MF RAC, power: 40 kV / 15 mA, scan speed: 2°θ / min, step: 0.01°θ, scan range: 3°~30°θ or 3°~40°θ, specimen holder: zero-background Si specimen holder with 0.2 mm indentation.

[0147] Example 1 Synthesis of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Compound 4) Step 1: Synthesis of 1-(tert-butyl) 3-ethyl 3-((3,4-difluorobenzyl)amino)azetidine-1,3-dicarboxylate: [ka] To a solution of 1-tert-butyl 3-ethyl 3-aminoazetidine-1,3-dicarboxylate (4.0 g, 16.4 mmol, 1.0 equiv) and 3,4-difluorobenzaldehyde (2.4 g, 19.6 mmol, 1.2 equiv) in DCE (40.0 mL) at 0° C., STAB (7.0 g, 32.8 mmol, 2.0 equiv) and AcOH (2.0 g, 32.8 mmol, 2.0 equiv) were added. The resulting mixture was stirred at room temperature overnight, the pH was adjusted to 8 with ammonium hydroxide, water (50.0 mL) was added, and extracted twice with DCM (50.0 mL). The combined organic layers were washed twice with brine (50 mL) and anhydrous NaCl was added.2 SO 4 Drying over rt and concentration under reduced pressure gave 6.0 g of 1-tert-butyl 3-ethyl 3-(3,4-difluorobenzyl)amino)azetidine-1,3-dicarboxylate as a yellow oil. LRMS (ES) m / z 315 (M+H-56).

[0148] Step 2: Synthesis of 1-(tert-butyl) 3-ethyl 3-(2-bromo-N-(3,4-difluorobenzyl)acetamido)azetidine-1,3-dicarboxylate: [ka] To a solution of 1-tert-butyl 3-ethyl 3-[[(3,4-difluorophenyl)methyl]amino]azetidine-1,3-dicarboxylate (6.0 g, 16.2 mmol, 1.0 equiv) in DCM (60.0 mL) at 0 °C was added a solution of K 2 CO 3 A solution of (3.4 g, 24.3 mmol, 1.50 equiv) was added followed by dropwise addition of bromoacetyl bromide (3.9 g, 19.4 mmol, 1.2 equiv) over 10 min. The resulting mixture was stirred at room temperature overnight and extracted twice with DCM (50.0 mL). The combined organic layers were washed twice with brine (100 mL) and diluted with anhydrous Na 2 SO 4 Drying over rt and concentration under reduced pressure gave 8.0 g of 1-(tert-butyl) 3-ethyl 3-(2-bromo-N-(3,4-difluorobenzyl)acetamido)azetidine-1,3-dicarboxylate as a yellow oil. LRMS (ES) m / z 435 (M+H-56).

[0149] Step 3: Synthesis of tert-butyl 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate: [ka] To a solution of 1-(tert-butyl) 3-ethyl 3-(2-bromo-N-(3,4-difluorobenzyl)acetamido)azetidine-1,3-dicarboxylate (8.0 g, 16.3 mmol, 1.0 equiv.) in ACN (80 mL) was added TEA (4.9 g, 48.4 mmol, 3.0 equiv.) and trans-(1r,4r)-4-methylcyclohexan-1-amine (2.8 g, 24.7 mmol, 1.5 equiv.). The resulting mixture was stirred at room temperature for 1 h, gradually warmed to 80° C., and stirred at 80° C. overnight. The mixture was cooled to room temperature, concentrated under reduced pressure and triturated with a mixture of PE and EA (7 / 1, 80 mL) to give 7 g (approximately 80% purity) of tert-butyl 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate as an off-white solid. LRMS (ES) m / z 422 (M+H-56).

[0150] Step 4: Synthesis of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione: [ka] To a stirred solution of tert-butyl 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (7.0 g, 14.7 mmol, 1.0 equiv) in DCM (70.0 mL) was added TFA (18.0 mL). The resulting mixture was stirred at room temperature for 3 h, diluted with water (100.0 mL), adjusted to pH 13-14 with aqueous NaOH (2 N), and extracted twice with DCM (100 mL). The combined organic layers were washed twice with brine (100.0 mL) and diluted with anhydrous Na 2 SO 4Drying over rt and concentration under reduced pressure gave 4.5 g (approximately 80% purity) of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione as a yellow semi-solid. LRMS (ES) m / z 378 (M+H).

[0151] Step 5: Synthesis of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (compound 4): [ka] A solution of tert-butyl 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (1.5 g, 4.0 mmol, 1.0 equiv) in ethyl formate (15.0 mL) was stirred at 80° C. overnight. The mixture was cooled to room temperature, concentrated under reduced pressure, and purified by C18 column chromatography in water (0.05% NH 4 HCO 3 ) / CH 3 Elution with a mixture of CN (3:2) afforded 1.3 g (81%) of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde as an amorphous white solid. The experimental X-ray powder diffraction (XRPD) pattern of this amorphous white solid is shown in Figure 1. LRMS (ES) m / z 406 (M+H); 1H NMR (300 MHz, DMSO-d6) δ 7.96 (s, 1H), 7.47 - 7.29 (m,2H), 7.10 (ddd, J = 9.4, 4.4, 2.0 Hz, 1H), 4.82 (s, 2H), 4.50 (d, J = 9.6 Hz, 1H),4.15-4.28 (m, J = 3H), 4.01 (s, 2H), 3.96 (d, J = 10.8 Hz, 1H), 1.80 - 1.69 (m,2H), 1.65 - 1.48 (m, 4H), 1.35 (d, J = 10.9 Hz, 1H), 1.13 - 0.93 (m, 2H), 0.88 (d,J = 6.5 Hz, 3H).

[0152] Example 2 Synthesis of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide (Compound 5) Step 1: Synthesis of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide (compound 5): [ka] To a stirred solution of tert-butyl 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (1.5 g, 4.0 mmol, 1.0 equiv) in THF (15.0 mL) at 0° C. was added TEA (1.2 g, 11.9 mmol, 3.0 equiv) and isocyanatotrimethylsilane (685 mg, 6.0 mmol, 1.5 equiv) dropwise over 5 min. The resulting mixture was stirred at room temperature overnight, concentrated under reduced pressure and triturated first with a mixture of PE and EA (5 / 1; 20 mL) and then with hexanes (20 mL) to give 1.4 g (84%) of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide as an off-white solid. LRMS (ES) m / z 421 (M+H); 1 H NMR (400 MHz, DMSO-d6) δ 7.55 - 7.30 (m, 2H), 7.09 (dd,J = 8.0, 4.3 Hz, 1H), 6.00 (s, 2H), 4.82 (s, 2H), 4.20 (t, J = 9.1 Hz, 3H), 3.99(s, 2H), 3.84 (d, J = 9.3 Hz, 2H), 1.74 (d, J = 12.9 Hz, 2H), 1.58 (dtt, J = 20.6,12.1, 6.1 Hz, 4H), 1.42 - 1.29 (m, 1H), 1.04 (qd, J = 12.2, 4.5 Hz, 2H), 0.88 (d,J = 6.5 Hz, 3H).

[0153] The compounds in the following table were prepared in a similar manner to compound 5. [Table 2-1] [Table 2-2]

[0154] Example 3 Synthesis of 5-(4-chloro-3-fluorobenzyl)-8-isopropyl-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione (Compound 8) Step 1: Synthesis of 1-(tert-butyl) 3-ethyl 3-(4-chloro-3-fluorobenzyl)amino)azetidine-1,3-dicarboxylate: [ka] To a solution of 1-tert-butyl 3-ethyl 3-aminoazetidine-1,3-dicarboxylate (2.0 g, 8.2 mmol, 1.0 equiv) and 4-chloro-3-fluorobenzaldehyde (2.0 g, 12.3 mmol, 1.5 equiv) in DCE (20.0 mL) at 0° C., ACOH (984 mg, 16.4 mmol, 2.0 equiv) and STAB (3.5 g, 16.5 mmol, 2.0 equiv) were added portionwise. The resulting mixture was stirred at room temperature overnight, the pH was adjusted to 8 with ammonium hydroxide, water (30.0 mL) was added, and extracted twice with DCM (30.0 mL). The combined organic layers were washed twice with brine (30.0 mL) and diluted with anhydrous NaCl. 2 SO 4 Drying over rt and concentration under reduced pressure gave 3.0 g of 1-tert-butyl 3-ethyl 3-(4-chloro-3-fluorobenzyl)amino)azetidine-1,3-dicarboxylate as a yellow oil. LRMS (ES) m / z 331 (M+H-56).

[0155] Step 2: Synthesis of 1-(tert-butyl) 3-ethyl 3-(2-bromo-N-(4-chloro-3-fluorobenzyl)acetamido)azetidine-1,3-dicarboxylate: [ka] To a stirred solution of 1-tert-butyl 3-ethyl 3-(4-chloro-3-fluorobenzyl)amino)azetidine-1,3-dicarboxylate (3.0 g, 7.8 mmol, 1.0 equiv) in DCM (30.0 mL) at 0 °C was added a solution of K 2 CO3 A solution of (1.7 g, 12.3 mmol, 1.50 equiv) was added followed by the dropwise addition of bromoacetyl bromide (1.9 g, 9.4 mmol, 1.2 equiv) over 5 min. The resulting mixture was stirred at room temperature overnight and extracted twice with DCM (40.0 mL). The combined organic layers were washed twice with brine (40 mL) and diluted with anhydrous Na 2 SO 4 Drying over rt, concentrating under reduced pressure and purifying by C18 column chromatography eluting with water (0.5% ammonium carbonate) and ACN (1 / 4) gave 3.9 g (89%) of 1-(tert-butyl) 3-ethyl 3-(2-bromo-N-(4-chloro-3-fluorobenzyl)acetamido)azetidine-1,3-dicarboxylate as a yellow oil. LRMS (ES) m / z 451 (M+H-56).

[0156] Step 3: Synthesis of tert-butyl 5-(4-chloro-3-fluorobenzyl)-8-isopropyl-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate: [ka] To a solution of 1-(tert-butyl) 3-ethyl 3-(2-bromo-N-(4-chloro-3-fluorobenzyl)acetamido)azetidine-1,3-dicarboxylate (1.5 g, 3.0 mmol, 1.0 equiv.) in ACN (20 mL) was added TEA (898 mg, 8.9 mmol, 3.0 equiv.) and isopropylamine (262 mg, 4.4 mmol, 1.5 equiv.). The resulting mixture was stirred at room temperature for 1 h, gradually warmed to 80° C., and stirred at 80° C. overnight. The mixture was cooled to room temperature and water was added with stirring. The precipitated solid was collected by filtration and washed with water (50.0 mL) to give 1.1 g (85%) of tert-butyl 5-(4-chloro-3-fluorobenzyl)-8-isopropyl-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate as an off-white solid. LRMS (ES) m / z 384 (M+H-56).

[0157] Step 4: Synthesis of 5-(4-chloro-3-fluorobenzyl)-8-isopropyl-2,5,8-triazaspiro[3.5]nonane-6,9-dione: [ka] To a stirred solution of tert-butyl 5-(4-chloro-3-fluorobenzyl)-8-isopropyl-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (1.1 g, 2.4 mmol, 1.0 equiv.) in DCM (12.0 mL) was added TFA (3.0 mL). The resulting mixture was stirred at room temperature for 3 h, diluted with water (20.0 mL), adjusted to pH 13-14 with aqueous NaOH (2N), and extracted twice with DCM (20 mL). The combined organic layers were washed twice with brine (20.0 mL) and diluted with anhydrous Na 2 SO 4 Drying over rt and concentration under reduced pressure gave 950 mg of 5-(4-chloro-3-fluorobenzyl)-8-isopropyl-2,5,8-triazaspiro[3.5]nonane-6,9-dione as a yellow solid. LRMS (ES) m / z 340 (M+H).

[0158] Step 5: Synthesis of 5-(4-chloro-3-fluorobenzyl)-8-isopropyl-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione (compound 8): [ka] To a stirred solution of 5-(4-chloro-3-fluorobenzyl)-8-isopropyl-2,5,8-triazaspiro[3.5]nonane-6,9-dione (950 mg, 2.8 mmol, 1.0 equiv.) and 3-bromopyridazine (662.2 mg, 4.2 mmol, 1.5 equiv.) in dioxane (10 mL) was added Cs 2 CO 3(1.8 g, 5.5 mmol, 2.0 equiv.) and Pd-PEPPSI-IPentCl2-methylpyridine (o-picoline) (117.6 mg, 0.14 mmol, 0.05 equiv.) were added. The resulting mixture was stirred overnight at 90° C. under nitrogen atmosphere. The mixture was cooled to room temperature, filtered to remove solids, and purified by C18 column chromatography eluting with water (0.05% ammonium carbonate) / ACN (2:1) to give 695 mg (59%) of 5-(4-chloro-3-fluorobenzyl)-8-isopropyl-2,5,8-triazaspiro[3.5]nonane-6,9-dione as a yellow solid. LRMS (ES) m / z 418 (M+H); 1 H NMR (400 MHz, DMSO-d6) δ 8.59 (dd, J = 4.6, 1.3 Hz,1H), 7.53 (t, J = 8.0 Hz, 1H), 7.43 - 7.34 (m, 2H), 7.14 (dd, J = 8.3, 2.0 Hz, 1H),6.84 (dd, J = 9.0, 1.4 Hz, 1H), 4.95 (s, 2H), 4.60 (h, J = 6.8 Hz, 1H), 4.47 (d,J = 9.5 Hz, 2H), 4.18 (d, J = 9.5 Hz, 2H), 4.03 (s, 2H), 1.15 (d, J = 6.8 Hz, 6H).

[0159] The compounds in the following table were prepared in a similar manner to compound 8. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0160] Example 4 Synthesis of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione (Compound 12) Step 1: Synthesis of tert-butyl 3-cyano-3-((3,4-difluorobenzyl)amino)azetidine-1-carboxylate: [ka] To a solution of tert-butyl 3-oxoazetidine-1-carboxylate (3.1 g, 18.2 mmol, 1.3 equiv) in THF (12.0 mL) was added acetic acid (1.0 g, 16.8 mmol, 1.2 equiv) and (3,4-difluorophenyl)methanamine (2.0 g, 14.0 mmol, 1.0 equiv) in water (6.0 mL). After stirring at room temperature for 5 min, the mixture was added with a solution of sodium cyanide (5.7 g, 116.9 mmol, 1.0 equiv) in water (2.8 mL). The mixture was heated at 60° C. in an oil bath for 15 h, cooled to room temperature, neutralized by adding a saturated aqueous solution of sodium bicarbonate, and extracted with ethyl acetate (30.0 mL×2). The combined organic layers were washed with brine, dried over magnesium sulfate, and concentrated under reduced pressure. To the resulting yellow solid was added diethyl ether (30.0 mL), sonicated for 1 min, cooled to 0° C., and filtered. The resulting white precipitate was washed with ice-cold diethyl ether (15.0 mL) and dried overnight to give 3.4 g (76%) of tert-butyl 3-cyano-3-((3,4-difluorobenzyl)amino)azetidine-1-carboxylate. LRMS (ES) m / z 297.1 (M+H-27).

[0161] Step 2: Synthesis of tert-butyl 3-(2-chloro-N-(3,4-difluorobenzyl)acetamido)-3-cyanoazetidine-1-carboxylate: [ka] To a solution of tert-butyl 3-cyano-3-((3,4-difluorobenzyl)amino)azetidine-1-carboxylate (3.1 g, 9.7 mmol, 1.0 equiv) and N,N-diisopropylethylamine (5.0 mL, 29.0 mmol, 3.0 equiv) in DCM (125.0 mL) cooled to 0° C. was added chloroacetyl chloride (1.9 mL, 24.2 mmol, 2.5 equiv) dropwise over 20 min. The mixture was stirred at 0° C. for 15 min, warmed to room temperature, stirred for 2 h, diluted with saturated sodium bicarbonate, and extracted with DCM. The combined organic layers were dried over sodium sulfate, concentrated, and purified by silica gel column chromatography (0% to 40% EtOAc / hexanes gradient) to give 3.3 g (86%) of tert-butyl 3-(2-chloro-N-(3,4-difluorobenzyl)acetamido)-3-cyanoazetidine-1-carboxylate. LRMS (ES) m / z 400.1 (M+H).

[0162] Step 3: Synthesis of tert-butyl 3-cyano-3-(N-(3,4-difluorobenzyl)-2-(((1r,4r)-4-methylcyclohexyl)amino)acetamido)azetidine-1-carboxylate: [ka] To a solution of tert-butyl 3-(2-chloro-N-(3,4-difluorobenzyl)acetamido)-3-cyanoazetidine-1-carboxylate (1.5 g, 3.8 mmol, 1.0 equiv.) in acetonitrile (15.0 mL) was added (1r,4r)-4-methylcyclohexan-1-amine (641.6 mg, 5.7 mmol, 1.5 equiv.) and DIPEA (2.0 mL, 11.3 mmol, 3.0 equiv.). The solution was heated at 75 °C for 2 h, concentrated, and purified by silica gel chromatography using a gradient of 30-100% EtOAc in hexanes as eluent to give 1.3 g (70%) of tert-butyl 3-cyano-3-(N-(3,4-difluorobenzyl)-2-((1r,4r)-4-methylcyclohexyl)amino)acetamido)azetidine-1-carboxylate. LRMS(ES)m / z 477.3(M+H). Step 4: Synthesis of tert-butyl 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate: [ka]

[0163] To a solution of tert-butyl 3-cyano-3-(N-(3,4-difluorobenzyl)-2-(((1r,4r)-4-methylcyclohexyl)amino)acetamido)azetidine-1-carboxylate (1.3 g, 2.6 mmol, 1.0 equiv.) in ethanol (15.0 mL) was added acetic acid (2.3 mL, 40.0 mmol, 15.0 equiv.). The reaction was heated at 75° C. for 15 h, then at 90° C. for 3 h, cooled to room temperature, and sonicated for 10 min. The precipitate was collected by filtration, washed with ice-cold ethanol, and dried to give 953.9 mg (76%) of tert-butyl 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate. LRMS(ES) m / z 422.2(M+H-56). Step 5: Synthesis of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate: [ka]

[0164] To a solution of tert-butyl 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (953.9 mg, 2.0 mmol, 1.0 equiv) in DCM (2.0 mL) was added TFA (2.0 mL) at room temperature. The mixture was stirred for 30 min, concentrated, and dried under high vacuum to give 981.0 mg (99%) of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate. LRMS (ES) m / z 378.20 (M+H). Step 6: Synthesis of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione (compound 12): [ka]

[0165] To a solution of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate (302.8 mg, 0.62 mmol, 1.0 equiv) in IPA (4.0 mL) was added potassium carbonate (171.5 mg, 1.2 mmol, 2.0 equiv) and methyl 6-chloropyridazine-3-carboxylate (159.5 mg, 0.92 mmol, 1.5 equiv). The reaction vial was capped and heated at 120° C. for 30 min and cooled to room temperature. To the mixture was added LiOH solution (1 M, 1.8 mL, 1.8 mmol, 3.0 equiv) and the mixture was stirred for 10 min, diluted with water, acidified to pH 3 with HCl (1.0 M), and extracted with DCM. The combined organic layers were dried over sodium sulfate and concentrated. Diphenyl ether (4.0 mL) was added to the resulting solid and the mixture was heated at 180 °C for 5 min, cooled to room temperature and purified by silica gel column chromatography using a gradient of 0-10% MeOH in DCM to give 75.1 mg (27%) of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-2-(pyridazin-3-yl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione. LRMS (ES) m / z 456.2 (M+H). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.58 (dd, J = 4.5,1.3 Hz, 1H), 7.41 - 7.32 (m, 3H), 7.12 - 7.06 (m, 1H), 6.83 (dd, J = 9.0, 1.4 Hz,1H), 4.92 (s, 2H), 4.45 (d, J = 9.5 Hz, 2H), 4.20 - 7.11 (m, 1H), 4.18 (d, J = 9.5Hz, 2H), 4.04 (s, 2H), 1.73 (d, J = 13.1 Hz, 2H), 1.64 - 1.49 (m, 4H), 1.40 - 1.28(m, 1H), 1.01 (qd, J = 12.1, 4.6 Hz, 2H), 0.87 (d, J = 6.5 Hz, 3H).

[0166] The compounds in the following table were prepared in a similar manner to compound 12. [Table 4]

[0167] Example 5 Synthesis of 5-(4-chlorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide (Compound 18) Step 1: Synthesis of tert-butyl 3-((4-chlorobenzyl)amino)-3-cyanoazetidine-1-carboxylate: [ka] To a solution of tert-butyl 3-oxoazetidine-1-carboxylate (20.0 g, 116.9 mmol, 1.0 equiv) in THF (60.0 mL) was added acetic acid (8.0 mL, 140.2 mmol, 1.2 equiv) and (4-chlorophenyl)methanamine (17.1 mL, 140.2 mmol, 1.2 equiv) in water (45.0 mL). After stirring at room temperature for 5 minutes, the mixture was added with a solution of sodium cyanide (5.7 g, 116.9 mmol, 1.0 equiv) in water (10.0 mL). The mixture was heated at 65° C. in an oil bath for 15 hours, cooled to room temperature, neutralized by adding a saturated aqueous solution of sodium bicarbonate, and extracted with ethyl acetate (150.0 mL×3). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. To the resulting yellow solid was added diethyl ether / hexanes (140.0 mL, 2:1) and the solution was sonicated for 5 min, cooled to 0° C., and filtered. The resulting white precipitate was washed with ice-cold diethyl ether (50.0 mL) and dried overnight to give 29.7 g (92%) of tert-butyl 3-((4-chlorobenzyl)amino)-3-cyanoazetidine-1-carboxylate. LRMS (ES) m / z 295.2 (M+H-27).

[0168] Step 2: Synthesis of tert-butyl 3-(2-chloro-N-(4-chlorobenzyl)acetamido)-3-cyanoazetidine-1-carboxylate: [ka] To a solution of tert-butyl 3-((4-chlorobenzyl)amino)-3-cyanoazetidine-1-carboxylate (14.0 g, 43.5 mmol, 1.0 equiv) and N,N-diisopropylethylamine (22.8 mL, 130.1 mmol, 3.0 equiv) in DCM (150.0 mL) cooled to 0° C. was added 2-chloroacetyl chloride (8.7 mL, 108.8 mmol, 2.5 equiv) in DCM (50.0 mL) dropwise over 25 min. The mixture was stirred at 0° C. for 15 min, warmed to room temperature, and stirred for 20 h. The reaction mixture was diluted with saturated sodium bicarbonate and extracted with DCM. The combined organic layers were dried over sodium sulfate, concentrated, and purified by silica gel column chromatography (0% to 40% EtOAc / hexanes gradient) to give 14.1 g (81%) of tert-butyl 3-(2-chloro-N-(4-chlorobenzyl)acetamido)-3-cyanoazetidine-1-carboxylate. LRMS (ES) m / z 398.1 (M+H).

[0169] Step 3: Synthesis of tert-butyl 3-(N-(4-chlorobenzyl)-2-(((1r,4r)-4-methylcyclohexyl)amino)acetamido)-3-cyanoazetidine-1-carboxylate: [ka] To a solution of tert-butyl tert-butyl 3-(2-chloro-N-(4-chlorobenzyl)acetamido)-3-cyanoazetidine-1-carboxylate (11.0 g, 27.6 mmol, 1 equiv.) in acetonitrile (90.0 mL) was added (1r,4r)-4-methylcyclohexane-1-amine (3.6 g, 30.4 mmol, 1.0 equiv.) and DIPEA (9.6 mL, 69.1 mmol, 2.5 equiv.). The solution was heated at reflux for 3.5 h and additional (1r,4r)-4-methylcyclohexane-1-amine (362.1 mg, 3.0 mmol, 0.1 equiv.) was added. The mixture was stirred and heated for 1 h, cooled, diluted with water, and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated to give 12.6 g (96%) of tert-butyl 3-(N-(4-chlorobenzyl)-2-(((1r,4r)-4-methylcyclohexyl)amino)acetamido)-3-cyanoazetidine-1-carboxylate. LRMS (ES) m / z 475.2 (M+H).

[0170] Step 4: Synthesis of tert-butyl 5-(4-chlorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate: [ka] To a solution of tert-butyl 3-(N-(4-chlorobenzyl)-2-(((1r,4r)-4-methylcyclohexyl)amino)acetamido)-3-cyanoazetidine-1-carboxylate (12.6 g, 26.4 mmol, 1.0 equiv.) in ethanol (88.0 mL) was added acetic acid at 0 h (15.1 mL, 264.7 mmol, 10.0 equiv.), 14.5 h (7.6 mL, 132.4 mmol, 5.0 equiv.), and 20.5 h (7.6 mL, 132.4 mmol, 5.0 equiv.). The reaction was stirred at reflux for a total of 21 h, cooled to room temperature, and sonicated for 10 min. The solid was filtered, washed with ice-cold ethanol, and dried. The filtrate was concentrated, dissolved in ethanol, and the sonication / filtration repeated three more times (four times total) to give 9.8 g (78%) of tert-butyl 5-(4-chlorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate; LRMS (ES) m / z 420.1 (M+H-56).

[0171] Step 5: Synthesis of 5-(4-chlorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate: [ka] To a solution of tert-butyl 5-(4-chlorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (15.1 g, 31.8 mmol, 1.0 equiv) in DCM (55.0 mL) was added TFA (55.0 mL) at room temperature. The mixture was stirred for 30 min, concentrated, and dried under high vacuum to give 15.6 g (99%) of 5-(4-chlorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate. LRMS (ES) m / z 376.2 (M+H).

[0172] Step 6: Synthesis of 5-(4-chlorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide (compound 18): [ka] To a solution of 5-(4-chlorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate (15.6 g, 31.8 mmol, 1.0 equiv.) in acetonitrile (102.0 mL) was added acetic acid (175.0 μL, 3.1 mmol, 0.1 equiv.) and sodium cyanate (4.0 g, 61.2 mmol, 1.9 equiv.). The mixture was heated at 55° C. for 80 min, cooled to room temperature, diluted with water and saturated sodium bicarbonate, and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated to give a precipitate that was sonicated in ether (100.0 mL). The resulting precipitate was collected by filtration, washed with ice-cold ether, and dried to give a white solid, which was sonicated again in ethanol (100.0 mL). The precipitate was again collected by filtration, washed with ice-cold ether, and dried to give 8.7 g (65%) of 5-(4-chlorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide as a white solid. LRMS (ES) m / z 419.2 (M+H). 1 H NMR (400 MHz, DMSO-d 6) δ 7.39 (d, J = 8.4Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 6.01 (s, 2H), 4.82 (s, 2H), 4.22 - 4.12 (m, 1H),4.19 (d, J = 9.3 Hz, 2H), 3.99 (s, 2H), 3.83 (d, J = 9.3 Hz, 2H), 1.77 - 1.69 (m,2H), 1.63 - 1.48 (m, 4H), 1.41 - 1.27 (m, 1H), 1.03 (qd, J = 12.3, 4.1 Hz, 2H),0.87 (d, J = 6.4 Hz, 3H).

[0173] The compounds in the following table were prepared in a similar manner to compound 18. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0174] Example 6 Synthesis of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Comparison A) [ka] Step 1: Synthesis of 1-(tert-butyl) 3-ethyl 3-((4-chlorobenzyl)amino)azetidine-1,3-dicarboxylate [ka] To a solution of 1-tert-butyl 3-ethyl 3-aminoazetidine-1,3-dicarboxylate (10.6 g, 43.3 mmol, 1.0 equiv) and 4-chlorobenzaldehyde (6.1 g, 43.3 mmol, 1.0 equiv) in DCE (120.0 mL) at 0° C. was added AcOH (5.0 mL). After warming to room temperature and stirring at room temperature, the mixture was added with additional AcOH (5.0 mL) twice at 4 h, 8.5 h, and 9 h (total 20.0 mL). To this mixture was added STAB (11.0 g, 52.0 mmol, 1.2 equiv). The mixture was stirred at room temperature overnight, diluted with aqueous sodium bicarbonate, and extracted three times with DCM. The combined organic layers were dried over sodium sulfate and concentrated. The resulting oil was dissolved in warm hexanes and EA (dropwise added until the mixture became a homogeneous solution) with stirring at 60° C. The mixture was then cooled to 0° C. and the precipitate was collected by filtration and washed with cold hexane. The filtrate was concentrated and the process was repeated again. The combined solids were dried under vacuum to give 13.8 g (86%) of 1-(tert-butyl) 3-ethyl 3-((4-chlorobenzyl)amino)azetidine-1,3-dicarboxylate as a white solid. LRMS (ES) m / z 313.1 (M+H-56).

[0175] Step 2: Synthesis of tert-butyl 3-((4-chlorobenzyl)amino)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate [ka] N 2To a stirred solution of 1-(tert-butyl) 3-ethyl 3-((4-chlorobenzyl)amino)azetidine-1,3-dicarboxylate (10.0 g, 27.1 mmol, 1.0 equiv) and 2,4-difluoroaniline (3.0 mL, 29.8 mmol, 1.1 equiv) in THF (200.0 mL) at 0° C. was added LHMDS solution (54.2 mL, 1 M in THF, 54.2 mmol, 2.0 equiv) dropwise over 20 min. The mixture was stirred at 0° C. for 20 min, quenched with water (50 mL), acidified to pH 5 using aqueous HCl (0.5 N) and extracted twice with EA (100 mL). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated to give 11.3 g of tert-butyl 3-((4-chlorobenzyl)amino)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate, which was used in the next step without purification. LRMS (ES) m / z 396.1 (M+H-56).

[0176] Step 3: Synthesis of tert-butyl 3-(2-chloro-N-(4-chlorobenzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate [ka] To a solution of tert-butyl 3-((4-chlorobenzyl)amino)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate (11.3 g, 23.2 mmol, 1.0 equiv.) in THF (20 mL) cooled to 0° C. was added TEA (4.8 mL, 34.7 mmol, 1.5 equiv.) and 2-chloroacetyl chloride (1.5 mL, 27.8 mmol, 1.2 equiv.). Upon stirring at 0° C. for 30 min, additional TEA (4.8 mL, 34.7 mmol, 1.5 equiv.) and 2-chloroacetyl chloride (1.8 mL, 23.2 mmol, 1.0 equiv.) were added to the mixture and the mixture was stirred at 0° C. for 30 min. To this mixture was added additional 2-chloroacetyl chloride (0.6 mL, 6.9 mmol, 0.3 equiv.). The mixture was gradually warmed to room temperature, stirred for 45 min, cooled to 0° C. and quenched with aqueous sodium bicarbonate. The mixture was extracted three times with EA. The combined organic layers were washed with Bring, dried over sodium sulfate and concentrated to give 13.2 g of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate, which was used in the next step without further purification. LRMS (ES) m / z 528.1 (M+H).

[0177] Step 4: Synthesis of tert-butyl 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate [ka] N 2 To a stirred solution of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate (13.2 g, 20.1 mmol, 1.0 equiv.) in DMF (40.0 mL) was added K 2 CO 3(4.2 g, 30.1 mmol, 1.5 equiv) was added. The resulting mixture was stirred at room temperature for 3.5 h, diluted with water and extracted twice with EA. The combined organic layers were washed twice with water and once with brine, dried over sodium sulfate and concentrated to give 11.7 g of tert-butyl 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate. LRMS (ES) m / z 436.1 (M+H-56).

[0178] Step 5: Synthesis of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate [ka] To a stirred solution of tert-butyl 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (14.7 g, 29.8 mmol, 1.0 equiv) in DCM (50.0 mL) was added TFA (25.0 mL). The resulting mixture was stirred at room temperature for 3 h and concentrated to dryness to give 15.1 g of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate, which was used in the next step without further purification. LRMS (ES) m / z 392.1 (M+H).

[0179] Step 6: Synthesis of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde [ka] To a solution of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate (15.1 g, 29.9 mmol, 1 equiv.) and N,N-diisopropylethylamine (15.7 mL, 89.7 mmol, 3 equiv.) in ACN (50 mL) was added 2,2,2-trifluoroethyl formate (5.8 mL, 59.8 mmol, 2 equiv.). The reaction was stirred at room temperature for 1 h, diluted with water, and extracted with DCM. The combined organic layers were dried over sodium sulfate, concentrated, and purified by silica gel chromatography using a 0-100% EtOAc gradient followed by a 0-10% MeOH, DCM gradient. The solid was suspended in EtOH / MTBE 1:1, heated to 80° C., cooled on ice, filtered, washed with MTBE, and dried to give 8.3 g (66%) of 5-(4-chlorobenzyl)-8-(2,4-difluorophenyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde; LRMS (ES) m / z 420.1 (M+H). 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.98 (s, 1H), 7.65(td, J = 8.8, 6.0 Hz, 1H), 7.48 - 7.41 (m, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.34 (d,J = 8.4 Hz, 2H), 7.22 (td, J = 8.4, 2.4 Hz, 1H), 4.91 (s, 2H), 4.55 (d, J = 9.7Hz, 1H), 4.43 (d, J = 1.6 Hz, 2H), 4.37 (d, J = 9.8 Hz, 1H), 4.28 (d, J = 10.8 Hz,1H), 4.08 (d, J = 10.8 Hz, 1H).

[0180] Example 7 Synthesis of 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Comparison B) [ka] Synthesis of 1-(tert-butyl) 3-ethyl 3-((4-(trifluoromethyl)benzyl)amino)azetidine-1,3-dicarboxylate [ka] To a solution of 1-tert-butyl 3-ethyl 3-aminoazetidine-1,3-dicarboxylate (15.0 g, 61.4 mmol, 1.0 equiv) and 4-(trifluoromethyl)benzaldehyde (11.8 g, 67.5 mmol, 1.1 equiv) in DCE (60.0 mL) at 0° C., AcOH (7.4 g, 122.8 mmol, 2.0 equiv) and STAB (19.5 g, 92.1 mmol, 1.5 equiv) were added portionwise. The resulting mixture was stirred at room temperature overnight, the pH was adjusted to 8 with ammonium hydroxide, water (100.0 mL) was added, and extracted twice with DCM (300.0 mL). The combined organic layers were washed twice with brine and diluted with anhydrous Na 2 SO 4 Drying over and concentration under reduced pressure gave 29.6 g of 1-(tert-butyl) 3-ethyl 3-((4-(trifluoromethyl)benzyl)amino)azetidine-1,3-dicarboxylate, which was used for the next step without purification. LRMS (ES) m / z 347.1 (M+H-56).

[0181] Synthesis of tert-butyl 3-((2,4-difluorophenyl)carbamoyl)-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate [ka] N 2To a stirred solution of 1-(tert-butyl) 3-ethyl 3-((4-(trifluoromethyl)benzyl)amino)azetidine-1,3-dicarboxylate (29.6 g, 44.1 mmol, 1.0 equiv) and 2,4-difluoroaniline (4.9 mL, 48.5 mmol, 1.1 equiv) in THF (200.0 mL) at 0° C. was added a solution of LHMDS (88.3 mL, 1 M in THF, 88.3 mmol, 2.0 equiv) dropwise over 20 min. The mixture was stirred at 0° C. for 20 min, quenched with water (50 mL), acidified to pH 5 using aqueous HCl (3 N) and extracted twice with EA (100 mL). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated to give 36.2 g of tert-butyl 3-((2,4-difluorophenyl)carbamoyl)-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate, which was used in the next step without purification. LRMS (ES) m / z 430.1 (M+H-56).

[0182] Synthesis of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate [ka] To a solution of tert-butyl 3-((2,4-difluorophenyl)carbamoyl)-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate (36.2 g, 40.6 mmol, 1.0 equiv.) in THF (100 mL) cooled to 0° C. was added TEA (8.5 mL, 60.9 mmol, 1.5 equiv.) and 2-chloroacetyl chloride (3.9 mL, 48.7 mmol, 1.2 equiv.). Upon stirring at 0° C. for 30 min, additional TEA (8.5 mL, 60.9 mmol, 1.5 equiv.) and 2-chloroacetyl chloride (3.3 mL, 40.6 mmol, 1.0 equiv.) were added to the mixture and the mixture was stirred at 0° C. for 30 min. To this mixture was added additional 2-chloroacetyl chloride (1.0 mL, 12.2 mmol, 0.3 equiv.). The mixture was gradually warmed to room temperature, stirred for 45 min, cooled to 0° C. and quenched with aqueous sodium bicarbonate. The mixture was extracted three times with EA. The combined organic layers were washed with Bring, dried over sodium sulfate and concentrated to give 42.0 g of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate, which was used in the next step without further purification. LRMS (ES) m / z 562.1 (M+H).

[0183] Synthesis of tert-butyl 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carboxylate [ka] N 2 To a stirred solution of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-carboxylate (42.0 g, 33.4 mmol, 1.0 equiv.) in DMF (80.0 mL) was added K 2 CO 3(7.0 g, 50.1 mmol, 1.5 equiv) was added. The resulting mixture was stirred at room temperature for 3.5 h, diluted with water and extracted twice with EA. The combined organic layers were washed twice with water and once with brine, dried over sodium sulfate, concentrated and purified by silica gel using a gradient of 0-60% EA in hexanes as eluent to give 12.0 g (37% for 4 steps) of tert-butyl 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carboxylate. LRMS (ES) m / z 469.7 (M+H-56).

[0184] Synthesis of 8-(2,4-difluorophenyl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate [ka] To a stirred solution of tert-butyl 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (12.0 g, 22.8 mmol, 1.0 equiv) in DCM (12.0 mL) was added TFA (40.0 mL). The resulting mixture was stirred at room temperature for 3 h and concentrated to dryness to give 12.3 g of 8-(2,4-difluorophenyl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate, which was used in the next step without further purification. LRMS (ES) m / z 426.1 (M+H).

[0185] Synthesis of 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde [ka] To a stirred solution of 8-(2,4-difluorophenyl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacete (12.3 g, 22.8 mmol, 1.0 equiv.) in THF (50.0 mL) was added sodium cyanate (4.4 g, 68.4 mmol, 3.0 equiv.) and a few drops of acetic acid. The mixture was stirred at room temperature for 30 min, concentrated, and purified by silica column chromatography using a gradient of 0-10% MeOH in DCM as eluent to give 8.8 g (73% for two steps) of 8-(2,4-difluorophenyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde. LRMS(ES) m / z 469.1(M+H); 1 H NMR (400 MHz, methanol-d4) δ 7.70 (d, J = 8.1 Hz, 2H),7.61 - 7.53 (m, 3H), 7.20 (ddd, J = 10.4, 8.8, 2.8 Hz, 1H), 7.12 (dddd, J = 9.1,8.0, 2.8, 1.4 Hz, 1H), 5.15 (s, 2H), 4.53 - 4.49 (m, 4H), 4.20 (d, J = 9.5 Hz, 2H).

[0186] Example 8 Synthesis of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Comparative item E) Step 1: Synthesis of tert-butyl 3-cyano-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate: [ka] To a solution of tert-butyl 3-oxoazetidine-1-carboxylate (25 g, 146.0 mmol, 1.0 equiv.) in THF (90 mL) was added acetic acid (10.5 g, 175.2 mmol, 1.2 equiv.) and (4-(trifluoromethyl)phenyl)methanamine (31.7 g, 181.1 mmol, 1.2 equiv.) in water (40.0 mL). After stirring at room temperature for 5 min, the mixture was added with a solution of sodium cyanide (7.2 g, 146.0 mmol, 1.0 equiv.) in water (10 mL). The mixture was heated in an oil bath at 60° C. for 18 h, cooled to room temperature, neutralized by adding a saturated aqueous solution of sodium bicarbonate, and extracted with ethyl acetate (150 mL×2). The combined organic layers were washed with brine, dried over magnesium sulfate, and concentrated under reduced pressure. To the resulting yellow solid was added diethyl ether / hexane (200 mL, 1:2) and the solution was sonicated for 1 min, cooled to 0° C. and filtered. The resulting white precipitate was washed with ice-cold diethyl ether (50 mL) and dried overnight to give tert-butyl 3-cyano-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate (43.9 g, 85% yield). LRMS (ES) m / z 329.2 (M+H-27). 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.71 (d, J =8.0 Hz, 2H), 7.60 (d, J = 8.0 Hz, 2H), 4.16 (d, J = 8.8 Hz, 2H), 3.92 (t, J = 7.2Hz, 1H), 3.84 (d, J = 9.2 Hz, 2H), 3.81 (dd, J = 7.3 Hz, 2H), 1.39 (s, 9H).

[0187] Step 2: Synthesis of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-cyanoazetidine-1-carboxylate: [ka] To a solution of tert-butyl 3-cyano-3-((4-(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate (3.0 g, 8.4 mmol, 1.0 equiv) and triethylamine (1.3 g, 12.7 mmol, 1.5 equiv) in DCM (0.2 M) cooled to 0° C. was added chloroacetyl chloride (0.95 g, 8.4 mmol, 1.0 equiv). The mixture was stirred at 0° C. for 15 min, warmed to room temperature, and stirred for 2 h. To the mixture was added additional 2-chloroacetyl chloride (0.95 g, 8.4 mmol, 1.0 equiv) and triethylamine (1.3 g, 12.7 mmol, 1.5 equiv). The reaction was stirred for 2 h, quenched with a saturated aqueous solution of ammonium chloride, and the layers were separated. The aqueous layer was extracted once with DCM. The combined organic layers were dried over magnesium sulfate, concentrated, and purified by silica gel column chromatography (5% to 70% EtOAc / Hexane, R f = 0.24 (20% EtOAc / Hexanes)) to give 3.4 g (92%) of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-cyanoazetidine-1-carboxylate. LRMS (ES) m / z 432.1 (M+H). 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.77 (d, J = 8.1Hz, 2H), 7.60 (d, J = 8.0 Hz, 2H), 4.95 (s, 2H), 4.52 (s, 2H), 4.15 (s, 4H), 1.35(s, 9H).

[0188] Step 3: Synthesis of tert-butyl 3-cyano-3-(2-((((1r,4r)-4-(difluoromethyl)cyclohexyl)amino)-N-(4-(trifluoromethyl)benzyl)acetamido)azetidine-1-carboxylate: [ka] To a solution of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-cyanoazetidine-1-carboxylate (0.8 g, 1.9 mmol, 1 equiv.) in acetonitrile (15 mL) was added (1r,4r)-4-(difluoromethyl)cyclohexane-1-amine hydrochloride (0.51 g, 2.8 mmol, 1.5 equiv.) and DIPEA (1.2 g, 9.3 mmol, 5 equiv.). The solution was heated at 65° C. for 4 h, at which point LCMS indicated the reaction was complete. The reaction was diluted with ethyl acetate and water (1:1, 80 mL) and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate, concentrated and purified by silica gel chromatography using a gradient of 25% to 100% ethyl acetate in hexanes as eluent to give 0.65 g (64%) of tert-butyl 3-cyano-3-(2-((1r,4r)-4-(difluoromethyl)cyclohexyl)amino)-N-(4-(trifluoromethyl)benzyl)acetamido)azetidine-1-carboxylate as a pale yellow oil. f =0.55 (100% ethyl acetate, silica). LRMS (ES) m / z 545.0 (M+H).

[0189] Step 4: Synthesis of tert-butyl 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carboxylate: [ka] To a solution of tert-butyl 3-cyano-3-(2-(((1r,4r)-4-(difluoromethyl)cyclohexyl)amino)-N-(4-(trifluoromethyl)benzyl)acetamido)azetidine-1-carboxylate (0.27 g, 0.50 mmol, 1.0 equiv) in ethanol (2 mL) was added acetic acid (0.18 g, 3.0 mmol, 6.0 equiv). The reaction was heated at 70° C. for 15 h, cooled to room temperature, and diluted with hexanes (1.0 mL). The precipitate was collected by filtration, washed with ethanol-hexane (1:2, 2 mL) and dried to give 186 mg (69%) of tert-butyl 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carboxylate as a pale yellow solid. LRMS (ES) m / z 490.2 (M+H-56). 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.71 (d, J =8.1 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 5.89 (td, J = 56.7, 3.8 Hz, 1H), 4.93 (s,2H), 4.29 - 4.15 (m, 3H), 4.02 (s, 2H), 3.93 (d, J = 9.5 Hz, 2H), 1.91 - 1.55 (m,7H), 1.35 (s, 9H), 1.38 - 1.20 (m, 2H).

[0190] Step 5: Synthesis of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate: [ka] To a solution of tert-butyl 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (200 mg, 0.37 mmol, 1.0 equiv) in DCM (1.5 mL) was added TFA (1.5 mL) at room temperature. The mixture was stirred at room temperature for 1 h, concentrated under reduced pressure, and dried under high vacuum to give 190 mg (94%) of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate, which was used without further purification. LRMS (ES) m / z 446.2 (M+H).

[0191] Step 6: Synthesis of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde (Comparative E): [ka] To a solution of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate (54.0 mg, 0.10 mmol) in acetonitrile (0.6 mL) was added DIPEA (37.0 mg, 0.29 mmol, 3.0 equiv.) and 2,2,2-trifluoroethyl formate (124.0 mg, 0.97 mmol, 10.0 equiv.). The mixture was heated in a microwave reactor at 110 °C for 20 min, concentrated, and purified by HPLC using a gradient of 10% to 100% ACN in water (both with 0.1% HCOOH) as eluent to give 21.0 mg (46%) of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde as a foam. LRMS (ES) m / z 473.9 (M+H). 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.96 (s, 1H), 7.71(d, J = 8.0 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 5.89 (td, J = 56.4, 4.5 Hz, 1H),4.94 (s, 2H), 4.51 (d, J = 9.6 Hz, 1H), 4.30 - 4.15 (m, 3H), 4.04 (s, 2H), 3.96(d, J = 10.7 Hz, 1H), 1.95 - 1.49 (m, 7H), 1.41 - 1.06 (m, 2H).

[0192] Biological Example B-1: Myofibril Assay To evaluate the effect of compounds on the ATPase activity of full-length cardiac myosin in the context of native sarcomeres, a skinned myofibril assay was performed. Bovine cardiac myofibrils were obtained by homogenizing bovine cardiac left ventricular tissue in the presence of a detergent such as TritonX-100. Such treatment removes most of the membrane and soluble cytoplasmic proteins but leaves the cardiac sarcomeric actomyosin apparatus intact. Myofibril preparations were enriched in Ca 2+ The ability to hydrolyze ATP in a regulated manner is maintained by the Ca2+ complex that activates the ATPase activity of such myofibril preparations in the presence and absence of a compound to a defined percentage of the maximum percentage (i.e., 25%, 75%). 2+ Small molecule drugs were assayed at concentrations ranging from 0.1 to 1.0 mM. Small molecule drugs were assessed for their ability to inhibit the steady-state ATPase activity of bovine cardiac myofibrils using a pyruvate kinase and lactate dehydrogenase (PK / LDH) coupled enzyme system. This assay regenerates myosin-generated ADP to ATP by oxidation of NADH, which results in a change in absorbance at 340 nm. Prior to testing small molecule drugs, calcium responsiveness of bovine cardiac myofibrils was assessed and calcium levels were assessed to inhibit 50% (pCa 50 ) or 75%(pCa 75 ) were selected as the final conditions for evaluating the inhibitory activity of small molecule drugs. All enzyme activities were measured in a buffer containing 12 mM PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), 2 mM magnesium chloride at pH 6.8 (PM12 buffer). The final assay conditions were 1 mg / mL bovine cardiac myofibrils, 4 U / mL pyruvate kinase, 6 U / mL lactate dehydrogenase, 50 μM ATP, 0.1 mg / mL BSA (bovine serum albumin), 10 ppm antifoam, 1 mM DTT, 0.5 mM NADH, 1.5 mM PEP, 0.6 mM EGTA, and sufficient CaCl to achieve 50% or 75% activation of myofibrillar ATPase activity. 2 The results for the compounds tested are shown in Table A. The compounds tested were prepared according to the synthetic procedures described herein. [Table 6]

[0193] Comparators C and D have the following structures: [Table 7]

[0194] The preparation of Comparator C and Comparator D is described in WO2020 / 047447A1.

[0195] Biological Example B-2: Pharmacokinetic Single Dose Study Mouse single dose study Male C57BL / 6 mice (18-25 g, 6-8 weeks old) were obtained from Zhejiang Vital River Laboratory Animal Technology Co.,Ltd. All animals for IV dosing had free access to food and water. IV dosing was performed through the tail vein. IV dosing solutions for test samples were prepared in 10%DMA / 20%PG / 70%HPβCD solution (40% w / v HPβCD aqueous solution) at a concentration of 0.1 mg / mL. Oral dosing suspensions were prepared by suspending test samples in 0.5%HPMC / 0.1%Tween®80 in water at a concentration of 0.2 mg / mL. Concentrations of IV and PO doses were measured at the end of the study. Pharmacokinetic (PK) parameters were calculated using the nominal dose values ​​when measurements were within 20% of the nominal values. Groups of 15 mice received IV doses with a volume of 5 mL / kg. Another group of 15 mice received oral gavage of the test article at 1 mg / kg. The oral dose volume was 5 mL / kg. Sparse blood samples were collected from groups of 3 mice via retroorbital bleeding and K 2Plasma was obtained by placing the blood in EDTA microtainer tubes and keeping on ice until centrifugation. Each designated group of mice was bled at two time points. The time points were pre-dose (PO only), 5 (IV only), 15, 30 min, 1, 2, 4, 6, 8, and 24 h post-dose. Blood samples were centrifuged and the collected plasma was stored at -80°C until analysis. Plasma samples were analyzed for test sample concentrations using an LC / MS / MS method. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing an internal standard (IS). The mixture was vortexed and centrifuged. 10 μL of the resulting solution was injected onto a reversed-phase C18 column and the resulting peaks were detected on an LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantitation (BLQ) were treated as zero for PK calculations. Composite PK parameters were estimated from up to two sampling points per mouse and up to three mice per sampling point, and the sparse data option in WinNonlin was used for noncompartmental analysis of concentration-time data (Phoenix WinNonLin software, version 64; Pharsight, Mountain View, CA). The elimination rate constant (k) was calculated as the absolute value of the slope of the linear regression of the logarithm of concentration versus time for the last three data points of the concentration-time profile. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. Area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC t Values ​​were calculated from the time of administration to the last measurable concentration. AUC ∞ value, the corresponding AUC t Plasma clearance (CL) was calculated as the sum of the ratios of dose / AUC ∞ The mean residence time (MRT) was estimated by moment analysis. The steady state (V ss ) The volume of distribution is MRT ∞ × CL. As observed, the maximum concentration (C max ) and C max Time to reach (t max) was recorded. Bioavailability was measured using the dAUC ∞、po / dAUC ∞、iv ×100% was calculated and dAUC was the dose-normalized AUC value. Data for the tested compounds are provided in Table B. The tested compounds were prepared according to the synthetic procedures described herein. [Table 8]

[0196] Single dose study in rats Male Sprague Dawley rats were obtained from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. The animals in the IV group had free access to water and food. The animals in the PO group were fasted overnight before dosing and given food 2 hours after dosing. The IV dose solution was prepared in 10% DMA / 50% PG / 40% HPβCD solution (40% w / v HPβCD aqueous solution) at a concentration of 1 mg / mL. The oral dose suspension was prepared by suspending the test sample in 0.5% HPMC / 0.1% Tween® 80 in water at a concentration of 0.2 mg / mL. The concentrations of the IV and PO doses were measured at the end of the study. When the measured values ​​were within 20% of the nominal values, the pharmacokinetic parameters were calculated using the nominal dose values. Three rats were administered IV via bolus injection via the tail vein. Three rats per dose group were administered by oral gavage. Blood samples were collected from the jugular vein cannula pre-dose, 5 (IV only), 15, 30 min, and 1, 2, 4, 6, and 24 h post-dose. Blood volumes were replaced with an equal volume of sterile 0.9% saline. Blood samples were centrifuged and collected plasma was stored at -80°C for subsequent analysis. Plasma samples were analyzed for test sample concentrations using an LC / MS / MS method. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing an internal standard. The mixture was vortexed and centrifuged. 10 μL of the resulting solution was injected onto a reversed-phase C18 column and the resulting peaks were detected on an LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantification (BLQ) were treated as zero for pharmacokinetic calculations. Pharmacokinetic parameters were estimated from individual animals using noncompartmental analysis of concentration-time data (Phoenix WinNonLin software, version 64; Pharsight, Mountain View, CA). The elimination rate constant (k) was calculated as the absolute value of the slope of the linear regression of the logarithm (log) of concentration versus time for the last three data points of the concentration-time profile. The apparent elimination half-life (t 1 / 2) values ​​were calculated as ln(2) / k. Area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC t Values ​​were calculated from the time of administration to the last measurable concentration. AUC ∞ value, the corresponding AUC t and the ratio of the final detectable concentration divided by k (AUC t - ∞ Plasma clearance (CL) was calculated as the sum of dose / AUC ∞ The mean residence time (MRT) was estimated by moment analysis. The steady state (V ss ) The volume of distribution is MRT ∞ × CL. As observed, the maximum concentration (C max ) and C max Time to reach (t max ) was recorded. The bioavailability was 、 Dose-normalized AUC for individual rats ∞,po / average dAUC ∞、iv ×100% ratio, and dAUC was the dose-normalized AUC value. Data for the tested compounds are provided in Table C. The tested compounds were prepared according to the synthetic procedures described herein. [Table 9]

[0197] Dog single dose study Non-naive male beagle dogs (8 months to 3 years old, weighing 8 to 13 kg) were used in this study. All animals for IV dosing had free access to food and water, and all animals for PO were fasted overnight before dosing and fed approximately 6 hours after dosing. For animals in the PO group, pentagastrin (6.0 μg / kg, im) was administered 20 minutes before administering the PO formulation and 1.5 hours after the first pentagastrin administration. The dose was 0.024 mL / kg, with a concentration of 250 μg / mL in DMSO / 1N NaOH / PBS. 10 mL of 0.001 N HCl was used to flush the gavage catheter of each animal. The IV dosing solution was prepared in 10% DMA / 50% PG / 40% HPβCD solution (40% w / v HPβCD in water) at a concentration of 1.0 mg / mL. Oral dose suspensions were prepared by suspending compounds in 0.5% HPMC / 0.1% Tween® 80 in distilled water at a concentration of 0.2 mg / mL. IV and PO dose concentrations were measured at the end of the study. If measurements were within 20% of the nominal values, PK parameters were calculated using the nominal dose values. Blood samples were collected by venipuncture of peripheral veins, excluding the dosing vein, pre-dose, 5, 15, 30 minutes, 1, 2, 4, 6, 8, 24, and 48 hours post-dose. Blood samples were centrifuged and the resulting plasma was frozen for bioanalysis. Plasma samples were stored at -80°C prior to analysis. Plasma samples were analyzed for compound concentration using an LC / MS / MS method. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing an internal standard. The mixture was vortexed and centrifuged. Ten μL of the resulting solution was injected onto a reversed-phase C18 column and the resulting peaks were detected on an LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantification (BLQ) were treated as zero for PK calculations. PK parameters were estimated from individual animals using noncompartmental analysis of concentration-time data (Phoenix WinNonLin software, version 64; Pharsight, Mountain View, CA).The elimination rate constant (k) was calculated as the absolute value of the slope of the linear regression of the logarithm (log) of concentration versus time for the last three data points of the concentration-time profile. 1 / 2 ) values ​​were calculated as ln(2) / k. Area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC t Values ​​were calculated from the time of administration to the last measurable concentration. AUC ∞ value, the corresponding AUC t Plasma clearance (CL) was calculated as the sum of the ratios of dose / AUC ∞ The mean residence time (MRT) was calculated from the ∞ ) was estimated by moment analysis. V ss MRT ∞ × CL. As observed, the maximum concentration (C max ) and C max Time to reach (t max ) were recorded. Because this was a crossover study, bioavailability was measured using dAUC ∞、po / dAUC ∞、iv × 100% was calculated, where dAUC was the dose-normalized AUC value from the same animal given IV and PO doses. Data for the tested compounds are provided in Table D. The tested compounds were prepared according to the synthetic procedures described herein. [Table 10]

[0198] Monkey single dose study Non-naive male cynomolgus monkeys (2-5 years old, weighing 2-5 kg) used in this study were obtained from Topgene Biotechnology. All animals for IV dosing had free access to food and water, and all animals for PO were fasted overnight prior to dosing and fed approximately 6 hours after dosing. IV dose solutions were prepared in 10% DMA / 50% PG / 40% HPβCD solution (40% w / v HPβCD aqueous solution) at a concentration of 1.0 mg / mL. Oral dose suspensions were prepared by suspending compounds in 0.5% HPMC / 0.1% Tween® 80 in distilled water at a concentration of 0.2 mg / mL. Concentrations of IV and PO doses were measured at the end of the study. PK parameters were calculated using the nominal dose values ​​if the measured values ​​were within 20% of the nominal values. Blood samples were collected by venipuncture of peripheral veins, excluding the dosing vein, pre-dose, 5, 15, 30 min, 1, 2, 4, 6, 8, 24, and 48 h post-dose. Blood samples were centrifuged and the resulting plasma was frozen for bioanalysis. Plasma samples were stored at -80°C prior to analysis. Plasma samples were analyzed for compound concentration using an LC / MS / MS method. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing an internal standard. The mixture was vortexed and centrifuged. 10 μL of the resulting solution was injected onto a reversed-phase C18 column and the resulting peaks were detected on an LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantitation (BLQ) were treated as zero for PK calculations. PK parameters were estimated from individual animals using noncompartmental analysis of concentration-time data (Phoenix WinNonLin software, version 64; Pharsight, Mountain View, CA). The elimination rate constant (k) was calculated as the absolute value of the slope of the linear regression of the logarithm (log) of concentration versus time for the last three data points of the concentration-time profile. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. Area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC t Values ​​were calculated from the time of administration to the last measurable concentration. AUC ∞value, the corresponding AUC t Plasma clearance (CL) was calculated as the sum of the ratios of dose / AUC ∞ The mean residence time (MRT) was calculated from the ∞ ) was estimated by moment analysis. V ss MRT ∞ × CL. As observed, the maximum concentration (C max ) and C max Time to reach (t max ) were recorded. Because this was a crossover study, bioavailability was measured using dAUC ∞、po / dAUC ∞、iv × 100% was calculated, where dAUC was the dose-normalized AUC value from the same animal given IV and PO doses. Data for the tested compounds are provided in Table E. The tested compounds were prepared according to the synthetic procedures described herein. [Table 11]

[0199] Single-dose CL and V in humans ss Value prediction Allometric scaling for prediction of human clearance and volume of distribution was based on simple interspecies allometric scaling of intravenous pharmacokinetic parameters for mouse, rat, dog, and cynomolgus monkey (Boxenbaum, J Pharmacokinet Biopharm 10:201-27, 1982). Prediction of human CL was made by extrapolating plasma intravenous clearance of preclinical species. The "rule of exponents" (Mahmood & Balian, Life Sci 59:579-85, 1996) was considered in this prediction, proposing that if the simple allometric exponent is between 0.71 and 0.99, a correction factor based on the maximum lifetime (MLP) of this species can be applied, and if the simple allometric exponent is greater than 1.0, a correction factor based on brain weight (BrW) can be applied, or a protein binding correction can be applied, if available. In a similar manner, simple allometric scaling was used to predict human volume of distribution. This method has been used successfully for a variety of drugs (Ward & Smith, Drug Metab Dispos 32:612-19, 2004; McGinnity et al., Curr Drug Metab 8:463-79, 2007). Prediction data for selected compounds is provided in Table F. [Table 12]

[0200] Key to Table F: SA = simple allometric growth, ROE = exponential rule, fucorrect = correction for functional groups not bound to serum, MLPcorrect = maximum lifetime correction, BrWcorrect = brain weight correction.

[0201] Dosing the Cassette for Dogs In this study, non-naïve male beagle dogs (8 months to 3 years old, weighing 8 to 14 kg) procured from Jiangsu Johnsen Bioresource CO. and / or Beijing Rixinkeji CO.,LTD and / or Beijing Marshall Biotechnology CO.,LTD were used. All animals for IV dosing had free access to food and water. IV dose solutions were prepared in 10%DMA / 50%PG / 40%HPβCD solution (40% w / v HPβCD aqueous solution) at a concentration of 0.2 mg / mL. The concentration of the IV dose was measured at the end of the study. If the measured values ​​were within 20% of the nominal values, the PK parameters were calculated using the nominal dose values. Blood samples were collected by venipuncture of peripheral veins, excluding the dosed vein, at pre-dose, 5, 15, 30 min, 1, 2, 4, 6, 8, and 24 h post-dose. Blood samples were centrifuged, and the resulting plasma was frozen for bioanalysis. Plasma samples were stored at -80°C prior to analysis. Plasma samples were analyzed for compound concentrations using an LC / MS / MS method. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing an internal standard. The mixture was vortexed and centrifuged. 10 μL of the resulting solution was injected onto a reversed-phase C18 column and the resulting peaks were detected on an LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantification (BLQ) were treated as zero for PK calculations. PK parameters were estimated from individual animals using noncompartmental analysis of concentration-time data (Phoenix WinNonLin software, version 64; Pharsight, Mountain View, CA). The elimination rate constant (k) was calculated as the absolute value of the slope of the linear regression of the logarithm (log) of concentration versus time for the last three data points of the concentration-time profile. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. Area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC t Values ​​were calculated from the time of administration to the last measurable concentration. AUC ∞ value, the corresponding AUC tPlasma clearance (CL) was calculated as the sum of the ratios of dose / AUC ∞ The mean residence time (MRT) was calculated from the ∞ ) was estimated by moment analysis. V ss MRT ∞ × CL. As observed, the maximum concentration (C max ) and C max Time to reach (t max ) were recorded. Because this was a crossover study, bioavailability was measured using dAUC ∞、po / dAUC ∞、iv × 100% was calculated, where dAUC was the dose-normalized AUC value from the same animal given IV and PO doses. Data for the tested compounds are provided in Table G. The tested compounds were prepared according to the synthetic procedures described herein. [Table 13]

[0202] Sarcasset administration Non-naive male beagle dogs (2-5 years old, weighing 2-5 kg) procured from Topgene Biotechnology were used in this study. All animals for IV dosing had free access to food and water. IV dose solutions were prepared in 10% DMA / 50% PG / 40% HPβCD solution (40% w / v HPβCD aqueous solution) at a concentration of 0.2 mg / mL. The concentration of the IV dose was measured at the end of the study. If the measured values ​​were within 20% of the nominal values, PK parameters were calculated using the nominal dose values. Blood samples were collected by venipuncture of peripheral veins, excluding the dosed vein, pre-dose, 5, 15, 30 min, 1, 2, 4, 6, 8, and 24 h post-dose. Blood samples were centrifuged and the resulting plasma was frozen for bioanalysis. Plasma samples were stored at -80 °C prior to analysis. Plasma samples were analyzed for compound concentrations using an LC / MS / MS method. Briefly, a 50 μL aliquot of each plasma sample was mixed with 100 μL of acetonitrile containing an internal standard. The mixture was vortexed and centrifuged. 10 μL of the resulting solution was injected onto a reversed-phase C18 column and the resulting peaks were detected on an LC / MS / MS equipped with a turbo ion spray ionization source. Sample concentrations below the limit of quantification (BLQ) were treated as zero for PK calculations. PK parameters were estimated from individual animals using noncompartmental analysis of concentration-time data (Phoenix WinNonLin software, version 64; Pharsight, Mountain View, CA). The elimination rate constant (k) was calculated as the absolute value of the slope of the linear regression of the logarithm (log) of concentration versus time for the last three data points of the concentration-time profile. The apparent elimination half-life (t 1 / 2 ) values ​​were calculated as ln(2) / k. Area under the concentration-time curve (AUC) values ​​were estimated using the linear trapezoidal method. AUC t Values ​​were calculated from the time of administration to the last measurable concentration. AUC ∞ value, the corresponding AUC t Plasma clearance (CL) was calculated as the sum of the ratios of dose / AUC ∞ The mean residence time (MRT) was calculated from the ∞) was estimated by moment analysis. V ss MRT ∞ × CL. As observed, the maximum concentration (C max ) and C max Time to reach (t max ) were recorded. Because this was a crossover study, bioavailability was measured using dAUC ∞、po / dAUC ∞、iv × 100%, where dAUC was the dose-normalized AUC value from the same animal given the IV and PO doses. Data for the tested compounds are provided in Table H. The tested compounds were prepared according to the synthetic procedures described herein. [Table 14]

[0203] Cassette-administered CL and V in humans ss Value prediction For PK data derived from cassette IV administration, prediction of human clearance and volume of distribution was performed using single-species allometry, where values ​​were predicted from plasma intravenous clearance of dog and monkey PK by applying protein binding corrections (Tang, Drug Metab Dispos 33:1294-96, 2005; Patel, Journal of Pharmaceutical Research International, 22(3): 1-7, 2018). Prediction data for selected compounds are provided in Tables J and K. [Table 15] [Table 16]

[0204] Biological Example B-3: Echocardiographic assessment of acute pharmacodynamic effects on cardiac contractility in rats In vivo cardiac function assessment by echocardiography was performed in male Sprague Dawley rats under isoflurane (1-3%) anesthesia. 2-DM mode images of the left ventricle were acquired in a parasternal long axis view before, during, and after compound administration. In vivo shortening fraction was determined by M-mode image analysis with the following calculation: ((end diastolic diameter-end systolic diameter) / end diastolic diameter x 100). Three pre-dose baseline M-mode images were taken at 1-min intervals before compound administration. Compounds are formulated in 0.5% hydroxypropyl methylcellulose 2910 (HPMC2910):0.1% Tween® 80 suspension and delivered as a single dose (5 mL / kg) by oral gavage. Rats were lightly anesthetized for M-mode echocardiographic measurements at 1 and 4 hours post-dose. Concurrent with the echocardiographic measurements, blood samples were taken to determine the corresponding compound plasma concentrations. The resulting plasma concentrations were used to calculate the IC 50 and IC 10 The IC value was estimated. 50 and IC 10 Values ​​are the concentrations at which the fractional shortening was 50% and 10% of the pre-dose baseline contractility, respectively. [Table 17]

[0205] Biological Example B-4: Determination of time-dependent inhibition of CYP450 enzymes in vitro Evaluation of the time-dependent inhibitory potential of test compounds against major human cytochrome P450 isoenzymes using human liver microsomes was also performed using standard methods (Grimm et al, Drug Metab. Dispos., Jul;37(7):1355-70. doi:10.1124 / dmd.109.026716,2009). Pooled human microsomes and selective CYP probe substrates were used for in vitro evaluation of test compounds at 25 and 50 μM as time-dependent inhibitors of seven human hepatic cytochrome P450 isoenzymes (CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4). LC-MS / MS was used to quantify metabolite formation. Inhibition of each P450 enzyme in human liver microsomes was measured as the percentage of decrease in activity of marker metabolite formation as measured by LC-MS / MS compared to uninhibited controls (=100% activity) at time 0 and after 30 min incubation. The occurrence of any time-dependent inhibition was then expressed as the fold change in enzyme activity at time 0 compared to the activity after 30 min incubation. [Table 18-1] [Table 18-2] [Table 18-3] [Table 18-4]

[0206] NA: Not available, CYP3A4-T: CYP3A4 activity measured by testosterone probe substrate, CYP3A4-M: CYP3A4 activity measured by midazolam probe substrate

[0207] For 3A4, in the % activity measurements using both midarzolam and testosterone as probes, compounds 13, 8, 7, and 4 show no signs of time-dependent inhibition, as the enzyme activity did not change by more than 1.2-fold. However, comparator C showed a fold activity change of more than 1.5-fold for both probes at 25 μM and 50 μM concentrations. Compounds 18, 5, and 6 also showed some change in fold activity of more than 1.2-fold at one of the concentrations tested or at one of the probe substrate tests, suggesting that there may be some change in 3A4 activity when tested in a time-dependent format for these compounds. When tested in this format, all compounds tested showed no change in the activity of 1A2 and 2B6. For 2C9, compound 7 showed only a 1.3-fold change in activity at 25 μM concentration and a 1.3-fold change in activity at 50 μM concentration. For 2C19, compound 5 showed a 1.3-fold change in activity at a concentration of 50 μM, and compound 4 showed a 1.4-fold change in activity at a concentration of 25 μM, but no change in activity of compound 4 was observed at a concentration of 50 μM. For 2D6, compound 13 showed a 1.4-fold change in activity at a concentration of 50 μM, and compounds 6 and 4 showed a 1.3-fold change in activity at a concentration of 25 μM, but no change in activity of these compounds was observed at a concentration of 50 μM. For 2C8, compound 18 showed a 1.3-fold change in activity at a concentration of 50 μM, and compound 5 showed a 2.0-fold change in activity at a concentration of 50 μM.

[0208] The foregoing written description of the compounds, uses, and methods described herein will enable one of ordinary skill in the art to make and use the compounds, uses, and methods described herein, but one of ordinary skill in the art will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiments, methods, and examples herein. Thus, the compounds, uses, and methods provided herein should not be limited by the above embodiments, methods, or examples, but encompass all embodiments and methods within the scope and spirit of the compounds, uses, and methods provided herein.

[0209] All references disclosed herein are incorporated by reference in their entirety.

Claims

1. Compounds of formula (I): 【Chemistry 84】 (In the formula, R １ is halo or C １－６ is haloalkyl, R ２ is H, halo, or C １－６ is alkyl, R ３ teeth, (i) cyclohexyl, optionally including one or more independently selected C １－６ Alkyl or C １－６ said cyclohexyl substituted by a haloalkyl substituent; or (ii) C １－６ alkyl, R ４ teeth, (i)-C(O)H, (ii)-C(O)NH ２ 、 【Chemistry 85】 and however, (1) R ３ But, C １－６ When R is alkyl, ４ teeth 【Chemistry 86】 and (2) R ３ When is isopropyl, R １ and R ２ the total number of halo atoms is at least 2; (3) R ３ is one or more independently selected C １－６ When R is cyclohexyl substituted by a haloalkyl substituent, ４ is other than -C(O)H), or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

2. R ４ is -C(O)H or -C(O)NH2, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

3. R ４ but, 【Chemistry 87】 2. The compound of claim 1, wherein:

4. R ３ is cyclohexyl, said cyclohexyl optionally being selected from one or more independently selected C １－６ Alkyl or C １－６ substituted by haloalkyl substituents, or R 3 is 【Chemistry 89】 or R 3 is 【Chemistry 90】 2. The compound of claim 1, wherein:

5. R ３ But, C １－６ alkyl, or R 3 is 【Chemistry 91】 or R 3 is 【Chemistry 92】 2. The compound of claim 1, wherein:

6. R １ 2. The compound of claim 1, wherein R 1 is halo, or R 1 is chloro or fluoro, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

7. R １ But, C １－６ 2. The compound of claim 1, wherein R 1 is haloalkyl or R 1 is -CF 3 , or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

8. R ２ is H, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

9. R ２ 2. The compound of claim 1, wherein R is halo, or R 2 is chloro or fluoro, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

10. R ２ But, C １－６ 2. The compound of claim 1, wherein R2 is alkyl or R2 is methyl, or a stereoisomer or tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

11. 【Chemical 101】 【Chemistry 102】 or a tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

12. The compound is Compound 4: 【Chemistry 93】 2. The compound of claim 1, wherein:

13. The compound is Compound 13: 【Chemistry 94】 2. The compound of claim 1, wherein:

14. The compound is Compound 18: 【Chemistry 95】 2. The compound of claim 1, wherein:

15. 15. A pharmaceutical composition comprising: (i) a compound according to any one of claims 1 to 14, or a tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing; and (ii) one or more pharma- ceutically acceptable excipients.

16. 15. A pharmaceutical composition for use in treating a cardiac disease in a subject in need thereof, comprising a compound according to any one of claims 1 to 14, or a tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

17. The pharmaceutical composition of claim 16, wherein the cardiac disease is hypertrophic cardiomyopathy.

18. 18. The pharmaceutical composition of claim 17, wherein the hypertrophic cardiomyopathy is obstructive or non-obstructive or caused by (i) sarcomere mutations, (ii) non-sarcomere mutations, or (iii) both sarcomere and non-sarcomere mutations.

19. 17. The pharmaceutical composition of claim 16, wherein the cardiac disease is heart failure with preserved ejection fraction.

20. 17. The pharmaceutical composition of claim 16, wherein the cardiac disease is selected from the group consisting of diastolic dysfunction, primary or secondary restrictive cardiomyopathy, myocardial infarction and angina pectoris, left ventricular outflow tract obstruction, hypertensive heart disease, congenital heart disease, cardiac ischemia, coronary heart disease, diabetic heart disease, congestive heart failure, right heart failure, cardiorenal syndrome, and infiltrative cardiomyopathy.

21. 17. The pharmaceutical composition of claim 16, wherein the cardiac disease is or is associated with one or more conditions selected from the group consisting of cardiac aging, age-related diastolic dysfunction, left ventricular hypertrophy, and concentric left ventricular remodeling.

22. 15. A pharmaceutical composition for use in treating a disease or condition associated with hypertrophic cardiomyopathy in a subject in need thereof, comprising a compound according to any one of claims 1 to 14, or a tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing.

23. 23. The pharmaceutical composition of claim 22, wherein the disease or condition is selected from the group consisting of Fabry disease, Danon disease, mitochondrial cardiomyopathy, and Noonan syndrome.

24. 15. A pharmaceutical composition for use in treating a disease or condition associated with secondary left ventricular wall thickening in a subject in need of such treatment, comprising a compound according to any one of claims 1 to 14, or a tautomer thereof, or a pharma- ceutical acceptable salt of any of the foregoing.

25. 25. The pharmaceutical composition of claim 24, wherein the disease or condition is selected from the group consisting of hypertension, valvular heart disease, aortic stenosis, mitral regurgitation, metabolic syndrome, diabetes, obesity, end stage renal disease, scleroderma, sleep apnea, amyloidosis, Fabry disease, Friedreich's ataxia, Danon disease, Noonan syndrome, and Pompe disease.

26. 15. A pharmaceutical composition for use in the treatment of a disease or condition associated with small left ventricular lumen and lumen obstruction, high output left ventricular contraction, myocardial ischemia, or cardiac fibrosis in a subject in need of such treatment, comprising a compound according to any one of claims 1 to 14, or a tautomer thereof, or a pharma- ceutical acceptable salt of any of the foregoing.

27. 15. A pharmaceutical composition for use in the treatment of a disease or condition selected from muscular dystrophy and glycogen storage disease in a subject in need of such treatment, comprising a compound according to any one of claims 1 to 14, or a tautomer thereof, or a pharma- ceutical acceptable salt of any of the foregoing.

28. A pharmaceutical composition for use in a method of inhibiting cardiac sarcomere, the pharmaceutical composition comprising a compound according to any one of claims 1 to 14, or a tautomer thereof, or a pharma- ceutically acceptable salt of any of the foregoing, the method comprising contacting the cardiac sarcomere with the compound, or the tautomer thereof, or the pharma- ceutically acceptable salt thereof.

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