2-substituted thiazole and benzothiazole compositions and methods as DUX4 inhibitors

Abstract

The present application provides compounds, compositions and methods for treating DUX4 misexpression diseases. In certain embodiments, the compounds and compositions of the present invention can be administered alone, and can also be administered in combination with other drugs for the treatment of DUX4 misexpression diseases.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 436,362, filed Dec. 30, 2022, and 63 / 616,498, filed Dec. 29, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application provides 2-substituted thiazole and benzothiazole compounds, methods, and pharmaceutical compositions for treating diseases such as neuromuscular diseases, inflammatory diseases, facioscapulohumeral muscular dystrophy, acute lymphoblastic leukemia, B-cell leukemia, sarcoma (e.g., small round cell sarcoma), prostate cancer, multiple myeloma, lung cancer, cancer, solid cancer, rheumatoid arthritis, axial spondyloarthritis, viral infections, mononucleosis, encephalitis, and chickenpox. In certain embodiments, 2-arylthiazole or benzothiazole compounds are provided for treating diseases such as those characterized by misexpression of double homeobox 4 (DUX4) in humans, such as cancer. Background Art

[0004] Double homeobox gene 4 (DUX4) is a gene of unknown function, the dysregulation of which leads to, for example, facioscapulohumeral muscular dystrophy. Lemmers, Richard J. L. F. et al., Science 2010, 329(5999): 1650-3; doi:10.1126 / science.1189044. There is currently no effective pharmaceutical method for controlling facioscapulohumeral muscular dystrophy.

[0005] Accordingly, there is a great need for compositions for effectively treating diseases and other disorders characterized by DUX4 misexpression. Summary of the Invention

[0006] This application provides compounds that can be used to treat diseases characterized by DUX4 misexpression (such as facioscapulohumeral muscular dystrophy; sarcoma; B-cell leukemia). In certain embodiments, 2-arylthiazole or benzothiazole compounds exhibit significant efficacy or bioavailability, or both, in humans.

[0007] In certain embodiments, this application provides a compound of formula I or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Wherein:

[0010] Each R 1 is independently selected from H and R 2 ;

[0011] m is an integer from 0 to 4;

[0012] The wavy bond represents a single-bond connection from L to the free site on the thiazole or benzothiazole ring;

[0013] L 1 is selected from a single bond, C 1-6 alkyl, and -(C=O)-;

[0014] L 2 is selected from -(C=O)(NR 3 )-, –(C=O)-alkyl-, and -(NR 3 )(C=O)-;

[0015] C y is selected from C 3-9 cycloalkyl, C 3-9 heterocyclic group, C3-C9 heteroaryl, and C 6-10 aryl;

[0016] Each R 2 is independently selected from halogen, C 1-3 alkoxy, C 1-3 alkyl, cyano, and R 5 ;

[0017] n is an integer from 0 to 2;

[0018] Each R 3 is independently selected from H and C 1-3 alkyl;

[0019] R 4 is C 1-6 alkylene, and R 4 is substituted with 0 to 4 R 7 groups;

[0020] R 5 is selected from -O(CO)R 6 , -N(R 3 )(CO)R 3 , -N(R 3 )(CO)R 6 , -OR 6 , -(CO)R 6 , -(CO)N(R 3 )R 3 , -(CO)N(R 3 )R 6 , C 3-7 cycloalkyl, C 3-9 heterocyclic group, C 6-10 aryl, and C3-C9 heteroaryl;

[0021] Or R 4 and R5 are joined together to form a 5- to 8-membered cycloalkyl, aryl, heterocyclic or heteroaryl ring, which may be substituted with 0 to 4 R 7 groups;

[0022] Each R 6 is independently selected from C 1-6 alkyl, C 3-7 cycloalkyl, C 3-9 heterocyclic, C 6-10 aryl and C 3- C9 heteroaryl, and R 6 is substituted with 0 to 4 R 7 groups;

[0023] Each R 7 is independently selected from halogen, C 1-3 alkoxy and C 1-3 alkyl.

[0024] In certain embodiments, the present application provides a compound of formula II or a pharmaceutically acceptable salt thereof:

[0025]

[0026] Wherein:

[0027] Y is selected from CH, CR 2 , -N=CH-, -N=CR 2 -, O, S and N; wherein Y and Z 1 are not both N;

[0028] Z 1 is selected from CH, CR 2 , -N=CH-, -N=CR 2 - and N; wherein Y and Z 1 are not both N.

[0029] In certain embodiments, the present application provides a compound of formula III or a pharmaceutically acceptable salt thereof:

[0030]

[0031] Wherein: Z 1 and Z 2 are each independently selected from CH, CR 2 and N; Z 1 and Z 2 are not both N.

[0032] In certain embodiments, the present application provides a compound of formula IV or a pharmaceutically acceptable salt thereof:

[0033]

[0034] Wherein:

[0035] R 1 is selected from H, halogen, and C 1-3 alkyl;

[0036] Each R 2 is independently selected from halogen, C 1-3 alkoxy, and C 1-3 alkyl;

[0037] R 4 is C 1-6 alkylene;

[0038] R 5 is selected from -O(CO)R 6 、-NH(CO)R 6 、-OR 6 、-(CO)R 6 、C 3-7 cycloalkyl, and C 3-9 heterocyclic group;

[0039] R 6 is selected from C 1-6 alkyl, C 3-7 cycloalkyl, C 3-9 heterocyclic group, and C 3-9 heteroaryl.

[0040] In certain embodiments, Z 1 and Z 2 are each independently selected from CH, CR 2 and N. In certain embodiments, Z 1 and Z 2 are each independently selected from CH and N.

[0041] In certain embodiments, R 1 is H or methyl.

[0042] In certain embodiments, n is 0.

[0043] In certain embodiments, R 4 is substituted with 0 R 2 groups.

[0044] In certain embodiments, R 5 is selected from -NH(CO)CH3, -O(CO)CH3, -(CO)CH3, and -OCH2CH3.

[0045] In certain embodiments, the compound is selected from the compounds in Table 6-1.

[0046] In certain embodiments, the compound inhibits the production of MBD3L2 RNA at 11 mM (e.g., according to the procedure of Example 7).

[0047] In certain embodiments, the compound DUX4 EC 50 is less than 10 mM. In certain embodiments, the compound DUX4 EC 50 is less than 1 mM.

[0048] In certain embodiments, the present application provides a pharmaceutical composition comprising: a compound disclosed in the present application, and a pharmaceutically acceptable excipient, carrier or diluent.

[0049] In certain embodiments, the composition is an oral formulation.

[0050] In certain embodiments, the present application provides a method of treating a patient, comprising administering an effective amount of a compound or composition disclosed in the present application. In certain embodiments, the patient is a human.

[0051] Detailed Description

[0052] Description of Exemplary Embodiments

[0053] The present application provides compounds, compositions and methods for treating cancer in a subject. Further provided are dosage forms that can be used in such methods.

[0054] Definitions

[0055] When referring to the compounds provided by the present application, unless otherwise specified, the following terms have the following meanings. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art. If there are multiple definitions for a term in the present application, unless otherwise specified, the definitions in this section shall prevail.

[0056] All publications, patent applications, patents and other references mentioned in the present application are hereby incorporated by reference in their entirety. In case of conflict, the content of the present application shall prevail.

[0057] The articles "a", "an" and "the" used in the present application include not only certain embodiments having a single element, but also embodiments having multiple elements. For example, an aspect "comprising a compound of formula Ib and an excipient" should be understood to provide, in certain embodiments, at least one additional compound of formula Ib, at least one additional excipient, or both.

[0058] Similarly, the term "or" as used in this application is the Boolean "or", unless the alternative options cannot be combined without logical incompatibility. For example, an aspect "comprising an excipient selected from A, B or C" should be understood to apply to embodiments comprising A and B; B and C; A and C; or A, B and C.

[0059] The term "about" as used in this application to modify a numerical value means a defined range near that numerical value. If "X" is a numerical value, "about X" generally means a value between 0.95X and 1.05X. Any reference to "about X" specifically represents at least the values of X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X and 1.05X. Thus, "about X" is intended to teach and provide written description support for claim features such as "0.98X". When "about" is applied to the beginning of a numerical range, it applies to both ends of the range. Thus, "from about 5% to 20%" is equivalent to "from about 5% to about 20%". When "about" is applied to the first numerical value of a set of numerical values, it applies to all the numerical values in the set. Thus, "about 7%, 9% or 11%" is equivalent to "about 7%, about 9% or about 11%".

[0060] Unless otherwise specified, the term "alkyl" as used in this application refers to saturated straight-chain or branched-chain hydrocarbons. In certain embodiments, the alkyl is a primary, secondary or tertiary hydrocarbon. In certain embodiments, the alkyl includes 1 to 10 carbon atoms, i.e., C 1-10 alkyl. In certain embodiments, the alkyl is C 1-12 alkyl, C 1-8 alkyl or C 1-6 alkyl. In certain embodiments, the alkyl is selected from methyl, CF3, CCl3, CFCl2, CF2Cl, ethyl, CH2CF3, CF2CF3, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl and 2,3-dimethylbutyl. The term includes substituted and unsubstituted alkyls, including haloalkyls. In certain embodiments, the alkyl is a fluorinated alkyl. In certain embodiments, the alkyl is unsubstituted. Non-limiting examples of groups that can substitute alkyl are halogen (fluorine, chlorine, bromine or iodine), hydroxy, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate or phosphonate; which can be unprotected or protected as needed as known to those skilled in the art, for example, as taught by Greene et al. in "Protective Groups in Organic Synthesis" (John Wiley and Sons, Second Edition, 1991), which is hereby incorporated by reference.

[0061] Unless otherwise specified, the term "lower alkyl" as used in this application refers to a saturated straight-chain or branched hydrocarbon having 1 to 6 carbon atoms, i.e., C1 to C6 alkyl. In certain embodiments, the lower alkyl is a primary, secondary, or tertiary hydrocarbon. The term includes substituted and unsubstituted groups. In certain embodiments, the lower alkyl is unsubstituted.

[0062] Unless otherwise specified, the term "alkylene" as used in this application refers to a straight-chain or branched divalent saturated aliphatic hydrocarbon group (especially having 1-11 carbon atoms). In certain embodiments, the alkylene contains 1 to 6 carbon atoms. The term includes substituted and unsubstituted groups. In certain embodiments, the alkylene is unsubstituted, such as methylene (-CH2-), ethylene (-CH2CH2-), propylene isomers (e.g., -CH2CH2CH2- and -CH(CH3)CH2-), etc.

[0063] Unless otherwise specified, the term "alkenyl" as used in this application refers to a monovalent olefinically unsaturated hydrocarbon group which, in certain embodiments, has up to about 11 carbon atoms, 2 to 8 carbon atoms, or 2 to 6 carbon atoms, can be straight-chain or branched, and has at least 1 or 1 to 2 olefinic unsaturation sites. The term includes substituted and unsubstituted groups. In certain embodiments, the alkenyl is unsubstituted. Exemplary alkenyls include vinyl (i.e., -CH=CH2), n-propenyl (-CH2CH=CH2), and isopropenyl (-C(CH3)=CH2), etc.

[0064] Unless otherwise specified, the term "alkenylene" as used in this application refers to a divalent olefinically unsaturated hydrocarbon group which, in certain embodiments, has up to about 11 carbon atoms or 2 to 6 carbon atoms, can be straight-chain or branched, and has at least 1 or 1 to 2 olefinic unsaturation sites. The term includes substituted and unsubstituted groups. In certain embodiments, the alkenylene is unsubstituted. Examples of the term include vinylene (-CH=CH-), propenylene isomers (e.g., -CH=CHCH2-; -C(CH3)=CH-; -CH=C(CH3)-), etc.

[0065] Unless otherwise specified, the term "alkynyl" as used in this application refers to an alkynically unsaturated hydrocarbon group which, in certain embodiments, has up to about 11 carbon atoms or 2 to 6 carbon atoms, can be straight-chain or branched, and has at least 1 or 1 to 2 alkynyl unsaturation sites. The term includes substituted and unsubstituted groups. In certain embodiments, the alkynyl is unsubstituted. Non-limiting examples of alkynyl include ethynyl (-C≡CH), propynyl (-CH2C≡CH), etc.

[0066] Unless otherwise specified, the term "alkoxy" as used in this application refers to an -OR' group, where R' is an alkyl or cycloalkyl group. Alkoxy groups include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, and n-butoxy, tert-butoxy, sec-butoxy, and n-pentyloxy, n-hexyloxy, and 1,2-dimethylbutoxy, etc.

[0067] Unless otherwise specified, the term "alkoxycarbonyl" as used in this application refers to -C(O)-alkoxy. Alkoxycarbonyl groups include, for example, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, etc.

[0068] Unless otherwise specified, the term "aryl" as used in this application refers to phenyl, biphenyl, or naphthyl. This term includes both substituted and unsubstituted groups. In certain embodiments, the aryl group can be substituted with any of the groups described, including but not limited to one or more selected from halogen (fluorine, chlorine, bromine, or iodine), alkyl, haloalkyl, hydroxy, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate groups; it can be unprotected or protected as needed as known to those skilled in the art, for example, as taught by Greene et al. in "Protective Groups in Organic Synthesis" (John Wiley and Sons, Second Edition, 1991), which is hereby incorporated by reference.

[0069] In certain embodiments, aryl refers to phenyl or naphthyl, which can be mono- or di-substituted with fluorine, chlorine, bromine, iodine, cyano, trifluoromethyl, nitro, carboxyl, aminocarbonyl, C 1-3 -alkyl (i.e., alkyl of one to three carbons) or C 1-3 -alkoxy. In certain embodiments, the aryl group is unsubstituted.

[0070] Unless otherwise specified, the term "amino" as used in this application refers to the -NH2 group.

[0071] Unless otherwise specified, the term "monoalkylamino" as used in this application refers to an alkylNR'- group, where R' is selected from hydrogen, alkyl, or cycloalkyl.

[0072] Unless otherwise specified, the term "alkylamino" or "arylamino" as used in this application refers to an amino group having one or two alkyl or aryl substituents, respectively. In certain embodiments, the alkyl substituent is a lower alkyl. In certain embodiments, the alkyl or lower alkyl is unsubstituted.

[0073] Unless otherwise specified, the term "acyl" as used in this application refers to a C(O)R' group, where R' is an alkyl or cycloalkyl (including lower alkyl) (i.e., "alkanoyl"), aryl (including phenyl), alkaryl (including benzyl), alkoxyalkyl (including methoxymethyl), aryloxyalkyl (such as phenoxymethyl); or a substituted alkyl (including lower alkyl) and aryl (including phenyl), optionally substituted by chlorine, bromine, fluorine, iodine, C1 to C4 alkyl or C1 to C4 alkoxy, sulfonate (including alkyl or arylalkylsulfonyl, including methanesulfonyl), mono / di / triphosphate, trityl or monomethoxytrityl, substituted benzyl, alkaryl, arylalkyl, including benzyl, alkoxyalkyl, including methoxymethyl, aryloxyalkyl (such as phenoxymethyl). The aryl in esters includes phenyl. In particular, acyl includes acetyl, trifluoroacetyl, methylacetyl, cyclopropylacetyl, propionyl, butyryl, hexanoyl, heptanoyl, octanoyl, neoheptanoyl, phenylacetyl, 2-acetoxy-2-phenylacetyl, diphenylacetyl, α-methoxy-α-trifluoromethylphenylacetyl, chloroacetyl, dichloroacetyl, 7H-dodecafluorheptanoyl, perfluoroheptanoyl, 7H-dodecafluorheptanoyl, 7-chlorododecafluorheptanoyl, 7-chlorotridecafluorheptanoyl, 4-(1,1,2,2-tetrafluoroethoxy)-benzoyl, 2-bromopropionyl, ω-aminooctanoyl, decanoyl, n-pentadecanoyl, stearoyl, 3-cyclopentyl-propionyl, 1-benzyl-carboxyl, O-acetylmandeloyl, pivaloyl-acetyl, 1-adamantane-carboxyl, cyclohexanecarboxyl, 2,6-pyridinedicarboxyl, cyclopropanecarboxyl, cyclobutanecarboxyl, perfluorocyclohexanecarboxyl, 4-methylbenzoyl, chloromethylisoxazolylcarbonyl, perfluoroisocyclohexanecarboxyl, crotonoyl, 1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl, 1-pyrrolidinylcarbonyl and 4-benzoyl.

[0074] Unless otherwise specified, the term "carboxyl" as used in this application refers to a -C(O)OH group.

[0075] Unless otherwise specified, the term "cycloalkyl" as used in this application refers to a saturated cycloalkane. In certain embodiments, the cycloalkyl can be saturated, bridged or unbridged, and / or a fused bicyclic group. In certain embodiments, the cycloalkyl includes three to ten carbon atoms, i.e., C3 to C 10 cycloalkyl. In some embodiments, the cycloalkyl has 3 to 15 (C 3-15 ), 3 to 10 (C 3-10 ) or 3 to 7 (C 3-7) carbon atoms. In certain embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, cycloheptyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, decahydronaphthyl, or adamantyl. The term includes both substituted and unsubstituted groups. In certain embodiments, the cycloalkyl group is unsubstituted.

[0076] Unless otherwise indicated, the term "cycloalkenyl" as used in this application refers to an unsaturated cyclic hydrocarbon. In certain embodiments, the cycloalkenyl group refers to a monocyclic or polycyclic system that includes at least one double bond. In certain embodiments, the cycloalkenyl group can be a bridged, unbridged, and / or fused bicyclic group. In certain embodiments, the cycloalkyl group includes from three to ten carbon atoms, i.e., C3 to C 10 cycloalkyl. In some embodiments, the cycloalkenyl group has 3 to 7 (C 3-7 ) or 4 to 7 (C 4-7 ) carbon atoms. The term includes both substituted and unsubstituted groups. In certain embodiments, the cycloalkenyl group is unsubstituted.

[0077] Unless otherwise indicated, the term "halogen" or "hal" as used in this application refers to chlorine, bromine, fluorine, or iodine.

[0078] Unless otherwise specified, the terms "heterocyclic group" or "heterocycle" as used in this application refer to a monovalent monocyclic non-aromatic ring system or a polycyclic system containing at least one non-aromatic ring; wherein one or more non-aromatic ring atoms are heteroatoms independently selected from O, S, or N, and the remaining ring atoms are carbon atoms. In certain embodiments, the heterocyclic group or heterocyclic moiety has 3 to 20, 3 to 15, 3 to 10, 3 to 8, 4 to 7, or 5 to 6 ring atoms. The heterocyclic group is attached to the remainder of the molecule through the non-aromatic ring. In certain embodiments, the heterocyclic group is a monocyclic, bicyclic, tricyclic, or tetracyclic system, which may include fused or bridged ring systems, wherein nitrogen or sulfur atoms may be oxidized, nitrogen atoms may be quaternized, and some rings may be partially or fully saturated or aromatic. The heterocyclic group may be attached to the main structure at any heteroatom or carbon atom, resulting in a stable compound. Examples of such heterocyclic groups include, but are not limited to, azolyl, benzodioxolyl, benzodioxinyl, benzopyrenyl, benzopyranyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, benzothiopyranyl, benzoxazinyl, β-carbonyl, tryptophanyl, tryptophan, cinnolinyl, coumarinyl, decahydroisoquinolinyl, dihydrobenzisothiazolyl, dihydroxybenzisoxazinyl, dihydrofuranyl, dihydroxyisoindolyl, dihydropyranyl, dihydropyrazolyl, dihydrochloropyrazinyl, dihydropyridyl, dihydropyrimidinyl, dialkylpyrrolyl, dioxolanyl, 1,4-dithianyl, furanonyl, imidazolidinyl, imidazolinyl, indolyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl, isocoumarinyl, isoindolyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinyl, oxazolidinyl, oxiranyl, piperazinyl, piperidinyl, 4-piperidinyl, pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydroxythienyl, thiamorpholinyl, thiazolidinyl, tetrahydroquinolinyl, and 1,3,5-trithianyl. The term includes substituted and unsubstituted groups. In certain embodiments, the heterocyclic group is unsubstituted.

[0079] Unless otherwise specified, the term "heteroaryl" as used in this application refers to a monovalent monocyclic aryl group and / or a polycyclic aryl group containing at least one aromatic ring, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S, and N. The heteroaryl group is attached to the rest of the molecule through the aromatic ring. The heteroaryl group may contain at most one or two O atoms, one or two S atoms, or one to four N atoms, provided that the total number of heteroatoms on each ring is four or less and each ring contains at least one carbon atom. In certain embodiments, the heteroaryl group has 5 to 20, 5 to 15, or 5 to 10 ring atoms. Examples of monocyclic heteroaryl groups include, but are not limited to, furyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl, and triazolyl. Examples of bicyclic heteroaryl groups include, but are not limited to, benzofuryl, benzimidazolyl, benzisoxazolyl, benzopyranyl, benzothiadiazolyl, benzodithiazolyl, benzotriazolyl, phenyloxazolyl, furylpyridyl, imidazolylpyridyl, imidazothiazolyl, mesitylenediyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolyl, isothiazolyl, naphthyridinyl, oxazolylpyridyl, phthalazinyl, pteridinyl, purinyl, pyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolylpyridyl, and thienylpyridyl. Examples of tricyclic heteroaryl groups include, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuryl, pyrimidinyl, phenanthrolinyl, phenanthridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and xanthenyl. The term includes substituted and unsubstituted groups. In certain embodiments, the heteroaryl group is unsubstituted.

[0080] Unless otherwise specified, the term "protecting group" as used in this application refers to a group added to an oxygen, nitrogen, or phosphorus atom to prevent further reaction or for other purposes. A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis.

[0081] Unless otherwise indicated, the term "pharmaceutically acceptable salt" as used in this application refers to any salt of a compound provided in this application, which retains its biological properties and has no toxic or other properties unsuitable for pharmaceutical use. Such salts can be derived from a variety of organic and inorganic counterions well known in the art. Such salts include, but are not limited to: (1) acid addition salts formed with organic or inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, glutaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, picric acid, cinnamic acid, mandelic acid, phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, dodecylsulfuric acid, gluconic acid, benzenesulfonic acid, glutamic acid, hydroxy- naphthoic acid, salicylic acid, stearic acid, cyclohexylaminosulfonic acid, quinic acid, mucic acid, and other acids; (2) salts formed when an acidic proton is present in the parent compound under the following two circumstances: (a) being replaced by a metal ion (such as an alkali metal ion, alkaline earth ion, or aluminum ion) or an alkali metal or alkaline earth metal hydroxide (such as the hydroxides of sodium, potassium, calcium, magnesium, aluminum, lithium, zinc, and barium, as well as ammonia); (b) coordinating with an organic base (such as an aliphatic, alicyclic, or aromatic organic amine, such as ammonia, methylamine, dimethylamine, diethylamine, pyridine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, procaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucosamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, etc.).

[0082] Pharmaceutically acceptable salts also include (by way of example only and not limitation): sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc.; when the compound contains a basic functional group, salts of non-toxic organic or inorganic acids, such as hydrohalides, such as hydrochloride and hydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentanepropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate, tartrate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, mesylate, esylate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, dodecylsulfate, gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate, mucate, etc.

[0083] As used in this application, the terms "substantially free of" or "substantially absent", unless otherwise specified, in the context of a composition, refer to a composition that contains at least 85 or 90% by weight, and in certain embodiments 95, 98, 99 or 100% by weight, of the designated enantiomer of the compound. In certain embodiments, in the methods and compounds provided in this application, the compound is substantially free of other enantiomers or diastereomers.

[0084] Similarly, unless otherwise specified, the term "isolated" as used in this application, in the context of a composition, refers to a composition that contains at least 85%, 90%, 95%, 98%, 99% to 100% by weight of the said compound, with the remainder containing other chemical substances or enantiomers.

[0085] Unless otherwise specified, the term "solvate" as used in this application refers to a compound or its salt provided in this application, and also includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0086] "Isotope composition" refers to the content of each isotope in a given atom, and "natural isotope composition" refers to the natural isotope composition or abundance of a given atom. Atoms containing the natural isotope composition may also be referred to as "non-enriched" atoms in this application. Unless otherwise specified, the atoms of the compounds described in this application refer to any stable isotope representing the atom. For example, unless otherwise specified, when a position is specifically designated as "H" or "hydrogen", that position is understood to have hydrogen with the natural isotope composition.

[0087] "Isotope enrichment" refers to the percentage of incorporation of a specific isotope on a given atom in a molecule in place of the natural isotope abundance of that atom. For example, 1% deuterium enrichment at a given position means that 1% of the molecules in the sample contain deuterium at the designated position. Since the natural distribution of deuterium is approximately 0.0156%, the deuterium enrichment at any position in a compound synthesized using unenriched starting materials is approximately 0.0156%. The isotope enrichment of the compounds provided in this application can be determined using conventional analytical methods known to those of ordinary skill in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy.

[0088] "Isotope-enriched" refers to an atom having an isotope composition different from the natural isotope composition of that atom. "Isotope-enriched" can also refer to a compound containing at least one atom whose isotope composition is different from the natural isotope composition of that atom.

[0089] The "alkyl", "cycloalkyl", "alkenyl", "cycloalkenyl", "alkynyl", "aryl", "alkoxy", "alkoxycarbonyl", "carboxyl", "alkylamino", "arylamino", "heterocyclic", "heteroaryl", "acyl" and "carboxyl" groups used in this application may contain deuterium at one or more positions where a hydrogen atom is present, and the deuterium composition of the atoms is different from the natural isotope composition.

[0090] Similarly, the "alkyl", "cycloalkyl", "alkenyl", "cycloalkenyl", "alkynyl", "aryl", "alkoxy", "alkoxycarbonyl", "carboxyl", "alkylamino", "arylamino", "heterocyclic", "heteroaryl", "acyl" and "carboxyl" groups used in this application may contain carbon-13, the content of which is different from the natural isotope composition.

[0091] As used in this application, the term "EC 50 " refers to the dose, concentration or amount that can elicit the expression of a dose-dependent response of 50% of the maximum response that a test compound can induce, initiate or enhance.

[0092] As used in this application, the term "IC 50 " refers to the amount, concentration or dose that can achieve 50% of the maximum inhibitory response of the compound in an assay. <s

[0093] The terms "subject" and "patient" are used interchangeably in this application. The term "subject" refers to an animal, such as a mammal, including non - primates (such as cows, pigs, horses, cats, dogs, rats, and mice) and primates, such as monkeys like cynomolgus monkeys, chimpanzees, and humans, and for example, humans. In certain embodiments, the subject is refractory or non - responsive to current treatments for a proliferative disease. In another embodiment, the subject is a farm animal (such as a horse, cow, pig, etc.) or a pet (such as a dog or a cat). In certain embodiments, the subject is a human.

[0094] Unless otherwise indicated, the terms "therapeutic agent" and "therapeutic drug" as used in this application refer to any drug that can be used to treat a disease or one or more of its symptoms. In certain embodiments, the term "therapeutic agent" includes the compounds provided in this application. In certain embodiments, a therapeutic agent is a reagent known to be, having been, or currently being used to treat a disease or one or more of its symptoms.

[0095] The term "therapeutically effective amount" or "effective amount" as used in this application, unless otherwise indicated, refers to a dose of a compound or composition that, when administered to a subject to treat a disease, is sufficient to effect treatment of the disease. A "therapeutically effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject.

[0096] In certain embodiments, "treatment" of any disease or disorder refers to ameliorating the disease or disorder present in a subject. In another embodiment, "treatment" includes ameliorating at least one physical parameter that may not be perceptible to the subject. In certain embodiments, "treatment" includes modulating the disease or disorder, either physically (such as stabilization of discernible symptoms) or physiologically (such as stabilization of physical parameters) or both. In certain embodiments, "treatment" includes delaying the progression of the disease or disorder.

[0097] As used in this application, the terms "preventive agent" and "preventive drug" refer to any drug that can be used to prevent a disease or one or more of its symptoms. In certain embodiments, the term "preventive agent" includes the compounds provided in this application. In certain other embodiments, the term "preventive agent" does not refer to the compounds provided in this application. For example, a preventive agent is a drug known to be, having been, or currently being used to prevent or arrest the onset, development, progression, and / or severity of a disease.

[0098] As used in this application, the term "preventive effective amount" refers to a dose of a treatment (such as a preventive agent) that is sufficient to prevent or reduce the development, recurrence, or onset of one or more symptoms associated with a disease, or enhance or improve the preventive effect of another treatment (such as another preventive agent).

[0099] Compound

[0100] In certain embodiments, the present application provides a compound of formula I or a pharmaceutically acceptable salt thereof:

[0101]

[0102] Wherein:

[0103] Each R 1 is independently selected from H and R 2 ;

[0104] m is an integer from 0 to 4;

[0105] The wavy bond represents a single bond connection from L to the free site on the thiazole or benzothiazole ring,

[0106] L 1 is selected from a single bond, C 1-6 alkyl, and -(C=O)-;

[0107] L 2 is selected from -(C=O)(NR 3 )-, -(C=O)-alkyl-, and -(NR 3 )(C=O)-;

[0108] C y is selected from C 3-9 cycloalkyl, C 3-9 heterocyclic group, C3-C9 heteroaryl, and C 6-10 aryl;

[0109] Each R 2 is independently selected from halogen, C 1-3 alkoxy, C 1-3 [[ID=5 six]]alkyl, cyano, and R 5 ;

[0110] n is an integer from 0 to 2;

[0111] Each R 3 is independently selected from H and C 1-3 alkyl;

[0112] R 4 is C 1-6 alkylene, and R 4 is substituted with 0 to 4 R 7 groups;

[0113] R 5 is selected from -O(CO)R 6 , -N(R 3 )(CO)R 3 , -N(R 3 )(CO)R 6 , -OR 6 , -(CO)R6 、 -(CO)N(R 3 )R 3 、 -(CO)N(R 3 )R 6 、 C 3-7 cycloalkyl, C 3-9 heterocyclic group, C 6-10 aryl and C3-C9 heteroaryl;

[0114] Alternatively, R 4 and R 5 are joined together to form a 5- to 8-membered cycloalkyl, aryl, heterocyclic or heteroaryl ring, which may be substituted by 0 to 4 R 7 groups;

[0115] Each R 6 is independently selected from C 1-6 alkyl, C 3-7 cycloalkyl, C 3-9 heterocyclic group, C 6-10 aryl and C 3- C9 heteroaryl, and R 6 is substituted by 0 to 4 R 7 groups;

[0116] Each R 7 is independently selected from halogen, C 1-3 alkoxy and C 1-3 alkyl.

[0117] In certain embodiments, the present application provides a compound of formula I-B or a pharmaceutically acceptable salt thereof:

[0118]

[0119] Wherein:

[0120] Each R 1 is independently selected from H and R 2 ;

[0121] m is an integer from 0 to 4;

[0122] C y is selected from C 3-9 heterocyclic group, C3-C9 heteroaryl and C 6-10 aryl;

[0123] Each R 2 is independently selected from halogen, C 1-3 alkoxy, C 1-3 alkyl and R 5 ;

[0124] n is an integer from 0 to 2;

[0125] R3 Selected from H and C 1-3 alkyl;

[0126] R 4 is C 1-6 alkylene, and R 4 is substituted with 0 to 4 R 7 groups;

[0127] Each R 5 is independently selected from -O(CO)R 6 、-NH(CO)R 6 、-OR 6 、-(CO)R 6 、C 3-7 cycloalkyl, C 3-9 heterocyclic group, C 6-10 aryl, and C 3- C9 heteroaryl;

[0128] Each R 6 is independently selected from C 1-6 alkyl, C 3-7 [[ID=4!]]cycloalkyl, C 3-9 heterocyclic group, C 6-10 aryl, and C 3- C9 heteroaryl, and R 6 is substituted with 0 to 4 R 7 groups;

[0129] Each R 7 is independently selected from halogen, C 1-3 alkoxy, and C 1-3 alkyl.

[0130] In certain embodiments, the present application provides a compound of formula II or a pharmaceutically acceptable salt thereof:

[0131]

[0132] Wherein:

[0133] Y is selected from CH, CR 2 、-N=CH-, -N=CR 2- 、O, S, and N; wherein Y and Z 1 are not both N;

[0134] Z 1 is selected from CH, CR 2 、-N=CH-, -N=CR 2 -, and N; wherein Y and Z 1 are not both N.

[0135] In certain embodiments, the present application provides a compound of formula II-B or a pharmaceutically acceptable salt thereof:

[0136]

[0137] Wherein:

[0138] Y is selected from CH, CR 2 , O, S, and N; wherein Y and Z 1 are not both N;

[0139] Z 1 is selected from CH, CR 2 and N; wherein Y and Z 1 are not both N.

[0140] In certain embodiments, Y is O, and Z 1 is N, CH, or CR 2 . In certain embodiments, Y is O, and Z 1 is CH or CR 2 (e.g., CH). In certain embodiments, Y is O, and Z 1 is N.

[0141] In certain embodiments, the present application provides a compound of Formula III or a pharmaceutically acceptable salt thereof:

[0142]

[0143] Wherein: Z 1 and Z 2 are each independently selected from CH, CR 2 and N; wherein Z 1 and Z 2 are not both N.

[0144] In certain embodiments, Z 1 is N, and Z 2 is CH or CR 2 (e.g., CH). In certain embodiments, Z 1 is CH or CR 2 (e.g., CH), and Z 2 is N. In certain embodiments, Z 1 and Z 2 are both independently selected from CH or CR 2 . In certain embodiments, Z 1 and Z 2 are both CH. In certain embodiments, Z 1 and Z 2 are each independently selected from CH or N.

[0145] In certain embodiments, the present application provides a compound of Formula IV or a pharmaceutically acceptable salt thereof:

[0146]

[0147] Wherein:

[0148] R 1 is selected from H, halogen, and C 1-3 alkyl;

[0149] Each R 2 is independently selected from halogen, C 1-3 alkoxy, and C 1-3 alkyl;

[0150] R 4 is C 1-6 alkylene;

[0151] R 5 is selected from -O(CO)R 6 , -NH(CO)R 6 , -OR 6 , -(CO)R 6 , C 3-7 cycloalkyl, and C 3-9 heterocyclic group;

[0152] R 6 is selected from C 1-6 alkyl, C 3-7 cycloalkyl, C 3-9 heterocyclic group, and C 3-9 heteroaryl.

[0153] In certain embodiments, R 1 is independently selected from H, F, Cl, methoxy, methyl, ethyl, and acetoxy. In certain embodiments, R 1 is independently selected from H and methyl.

[0154] In certain embodiments, R 1 is H or methyl.

[0155] In certain embodiments, R 1 is 6-substituted. In certain embodiments, R 1 is 5-substituted. In certain embodiments, R 1 is 7-substituted. In certain embodiments, R 1 is 4-substituted.

[0156] In certain embodiments, m is an integer from 0 to 4 (e.g., 0, 1, 2, 3, or 4). In certain embodiments, m is 0, 1, 2, or 3. In certain embodiments, m is 0, 1, or 2. In certain embodiments, m is 0 or 1. In certain embodiments, m is 0.

[0157] In certain embodiments, C y is selected from C3-9 Heterocyclic group, C3-C9 heteroaryl and C 6-10 aryl. In certain embodiments, C y is selected from C3-C9 heteroaryl and C 6-10 aryl. In certain embodiments, C y is C 6-10 aryl (e.g., benzene ring). In certain embodiments, C y is C3-C9 heteroaryl (e.g., furyl, pyridyl, pyrimidinyl).

[0158] In certain embodiments, each R 2 is independently selected from halogen, C 1-3 alkoxy, C 1-3 alkyl. R 5 is independently selected from H, F, Cl, methoxy, methyl, ethyl and acetoxy. In certain embodiments, R 1 is independently selected from H and methyl.

[0159] In certain embodiments, n is an integer from 0 to 2 (e.g., 0, 1 or 2). In certain embodiments, n is 0 or 1. In certain embodiments, n is 0.

[0160] In certain embodiments, each R 3 is independently selected from H and C 1-3 alkyl. In certain embodiments, R 3 is H. In certain embodiments, R 3 is methyl. In certain embodiments, R 3 is ethyl.

[0161] In certain embodiments, R 4 is C 1-6 alkylene, and R 4 is substituted with 0 to 4 R 7 groups. In certain embodiments, R 4 is methylene. In certain embodiments, R 4 is vinylene, propylene or butylene. In certain embodiments, R 4 is substituted with 0 R 7 groups. In certain embodiments, R 4 is substituted with 1 or 2 0 R 7 groups (e.g., methyl).

[0162] In certain embodiments, R 4 is substituted with 0 R 2 groups.

[0163] In certain embodiments, R 5 is selected from -O(CO)R 6, -N(R 3 )(CO)R 3 , -N(R 3 )(CO)R 6 , -OR 6 , -(CO)R 6 , -(CO)N(R 3 )R 3 , -(CO)N(R 3 )R 6 , C 3-7 cycloalkyl, C 3-9 heterocyclic group, C 6-10 aryl and C3-C9 heteroaryl. In certain embodiments, R 5 is selected from -O(CO)R 6 , -N(R 3 )(CO)R 6 , -OR 6 , -(CO)R 6 , -(CO)N(R 3 )R 6 , C 3-7 cycloalkyl, C 3-9 heterocyclic group, C 6-10 aryl and C3-C9 heteroaryl.

[0164] In certain embodiments, R 5 is independently selected from -O(CO)R 6 , -NH(CO)R 6 , -OR 6 , -(CO)R 6 , C 3-7 cycloalkyl, C 3-9 heterocyclic group, C 6-10 aryl and C3-C9 heteroaryl. In certain embodiments, R 5 is -O(CO)R 6 (such as acetoxy). In certain embodiments, R 5 is -NH(CO)R 6 (such as acetamido). In certain embodiments, R 5 is -OR 6 (such as methoxy, ethoxy or isopropoxy). In certain embodiments, R 5 is -(CO)R 6 (such as -(CO)methyl). In certain embodiments, R 5 is C 3-7 cycloalkyl (such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl). In certain embodiments, R 5 is C 3-9Heterocyclic group (such as 1-substituted pyrrolidine-2,5-dione; 1-substituted pyrrolidinyl-2-one; 5-substituted pyrrolidin-2-one). In certain embodiments, R 5 is C 6-10 aryl (such as phenyl, naphthyl). In certain embodiments, R 5 is a C3-C9 heteroaryl (such as, 1-, 2-, 4- or 5-imidazolyl; 1- or 4-triazolyl; 1-, 3-, 4- or -5-pyrazolyl; 2-, 4-, or 5-oxazolyl; 2-, 3- or 4-pyridyl; 1-, 3-, 4-, 5- or 6-substituted pyridin-2-one; 2-, 4-, 5- or 6-pyrimidinyl).

[0165] In certain embodiments, R 5 is selected from -NH(CO)CH3, -O(CO)CH3, -(CO)CH3 and -OCH2CH3.

[0166] In certain embodiments, each R 6 is independently selected from C 1-6 alkyl (such as methyl, ethyl or isopropyl), C 3-7 cycloalkyl, C 3-9 heterocyclic group, C 6-10 aryl and C 3- C9 heteroaryl, and R 6 is substituted with 0 to 4 R 7 groups. In certain embodiments, R 6 is C 1-6 alkyl (such as methyl, ethyl or isopropyl). In certain embodiments, R 6 is C 3-7 cycloalkyl (such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl). In certain embodiments, R 6 is C 3-9 heterocyclic group (such as 1-substituted pyrrolidine-2,5-dione; 1-substituted pyrrolidinyl-2-one; 5-substituted pyrrolidin-2-one). In certain embodiments, R 6 is C 6-10 aryl (such as phenyl, naphthyl). In certain embodiments, R 6 is a C3-C9 heteroaryl (such as, 1-, 2-, 4- or 5-imidazolyl; 1- or 4-triazolyl; 1-, 3-, 4- or -5-pyrazolyl; 2-, 4-, or 5-oxazolyl; 2-, 3- or 4-pyridyl; 1-, 3-, 4-, 5- or 6-substituted pyridin-2-one; 2-, 4-, 5- or 6-pyrimidinyl).

[0167] In certain embodiments, R 6 is substituted with 0 to 4 independently selected R 7Group substitution. In certain embodiments, R 6 is substituted with 0 to 3 independently selected R 7 groups. In certain embodiments, R 6 is substituted with 0 to 2 independently selected R 7 groups. In certain embodiments, R 6 is substituted with 0 to 1 R 7 group. In certain embodiments, R 6 is not substituted with R 7 groups.

[0168] In certain embodiments, each R 7 is independently selected from halogen, C 1-3 alkoxy, and C 1-3 alkyl. In certain embodiments, each R 7 is independently selected from halogen and C 1-3 alkyl. In certain embodiments, each R 7 is halogen (e.g., F). In certain embodiments, each R 7 is C 1-3 alkyl (e.g., methyl). In certain embodiments, each R 7 is C 1-3 alkoxy (e.g., methoxy, ethoxy).

[0169] In certain embodiments, the present application provides a compound selected from the following:

[0170]

[0171]

[0172]

[0173]

[0174] In certain embodiments, the present application provides a compound selected from the following:

[0175]

[0176]

[0177]

[0178] In certain embodiments, the compound is selected from the compounds of Table 6-1. In certain embodiments, the compound is any one of the compounds in Table 6-1.

[0179] In certain embodiments, one or more variables of formula (I) or formula (I-B) (i.e., R1 , m, C y , R 2 , n, R 3 , R 4 , R 5 , R 6 or R 7 ) variables corresponding to the compounds in Table 6-1.

[0180] In certain embodiments, one or more variables of formula (II) or formula (II-B) (i.e., R 1 , m, Y, Z 1 , R 2 , n, R 3 , R 4 , R 5 , R 6 or R 7 ) variables corresponding to the compounds in Table 6-1.

[0181] In certain embodiments, one or more variables of formula (III) (i.e., R 1 , m, Z 1 , Z 2 , R 2 , n, R 3 , R 4 , R 5 , R 6 or R 7 ) variables corresponding to the compounds in Table 6-1.

[0182] In certain embodiments, one or more variables of formula (IV) (i.e., R 1 , Z 1 , Z 2 , R 2 , n, R 3 , R 4 , R 5 , R 6 or R 7 ) variables corresponding to the compounds in Table 6-1.

[0183] In certain embodiments, the compound inhibits the production of MBD3L2 RNA at 11 mM (e.g., according to the procedure of Example 7).

[0184] In certain embodiments, the DUX4 EC of the compound 50 is less than 10 mM (e.g., codes "B" or "C" in Table 6-1). In certain embodiments, the DUX4 EC of the compound 50 is less than 1 mM (e.g., code "C" in Table 6-1).

[0185] In some embodiments, the present application provides:

[0186] Compounds as described in the present application, such as compounds of formula I, II, III or IV, and their pharmaceutically acceptable salts and compositions;

[0187] Compounds as described in the present application, such as compounds of formula I, II, III or IV, and their pharmaceutically acceptable salts and compositions, for the treatment and / or prevention of diseases characterized by DUX4 misexpression;

[0188] Methods for preparing the compounds as described in the present application, such as compounds of formula I, II, III or IV, will be described in more detail below;

[0189] Pharmaceutical formulations comprising the compounds as described in the present application, such as compounds of formula I, II, III or IV or their pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier or diluent;

[0190] Pharmaceutical formulations comprising the compounds as described in the present application, such as compounds of formula I, II, III or IV or their pharmaceutically acceptable salts, and one or more other effective pharmaceutical formulations (which may be in a pharmaceutically acceptable carrier or diluent), for the treatment of diseases characterized by DUX4 misexpression.

[0191] Optically active substances

[0192] The compounds provided in the present application may have several chiral centers and may exist and be separated in optically active and racemic forms. Some compounds may exhibit polymorphism. Any racemic, optically active, diastereoisomeric, polymorphic or stereoisomeric form or mixture thereof of the compounds provided in the present application having the useful properties described in the present application is within the scope of the present invention. Optically active forms can be prepared by any method known to those skilled in the art, for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase.

[0193] Examples of methods for obtaining optically active materials are known in the art and include at least the following methods.

[0194] Physical separation of crystals - a technique for manually separating macroscopic crystals of individual enantiomers. This technique can be used if there are crystals of the individual enantiomers, i.e., the material is a large crystal and the crystals are visually different;

[0195] Simultaneous crystallization - a technique for separately crystallizing individual enantiomers from a solution of a racemate, which is only possible when the latter is a large solid crystal;

[0196] Enzymatic resolution—a technique for the partial or complete separation of a racemate by the different reaction rates of enantiomers with an enzyme;

[0197] Enzymatic asymmetric synthesis—a synthetic technique in which at least one step of the synthesis uses an enzyme reaction to obtain an enantiopure or enantiomer-enriched synthetic precursor of the desired enantiomer;

[0198] Chemical asymmetric synthesis—a synthetic technique in which the desired enantiomer is synthesized from an achiral precursor under conditions that generate asymmetry (i.e., chirality) in the product, which can be achieved by using a chiral catalyst or a chiral auxiliary;

[0199] Diastereomeric resolution—a technique in which a racemic compound reacts with an enantiopure reagent (chiral auxiliary) that converts the individual enantiomers into diastereoisomers. The resulting diastereoisomers are then separated by chromatography or crystallization due to their now more distinct structural differences, and the chiral auxiliary is subsequently removed to obtain the desired enantiomer;

[0200] First- and second-order asymmetric transformation—a technique by which the diastereomeric equilibrium in a racemate is shifted in solution to create a preponderance of diastereoisomers from the desired enantiomer, or the equilibrium is disrupted by the preferential crystallization of diastereoisomers from the desired enantiomer, ultimately converting in principle all of the material to crystalline diastereoisomers. The desired enantiomer is then released from the diastereoisomers;

[0201] Kinetic resolution—a technique in which, under kinetic conditions, partial or complete resolution of a racemate (or further resolution of a partially resolved compound) is achieved due to the unequal reaction rates of enantiomers with a chiral non-racemic reagent or catalyst;

[0202] Enantioselective synthesis from a non-racemic precursor—a technique by which the desired enantiomer is obtained from achiral starting materials and the stereochemical integrity is either not impaired or only minimally impaired during the synthesis;

[0203] Chiral liquid chromatography—a technique for separating the enantiomers of a racemate in a liquid mobile phase by their different interactions with a stationary phase. The stationary phase can be made of a chiral material, or the mobile phase can contain additional chiral material to induce different interactions;

[0204] Chiral gas chromatography—a technique for the volatilization and enantiomeric separation of a racemate due to their different interactions with a column containing a stationary non-racemic chiral adsorbent phase in a gas mobile phase;

[0205] Extraction with a chiral solvent—a technique for separating enantiomers by preferentially dissolving one enantiomer into a specific chiral solvent;

[0206] Transport of chiral membranes - a technique in which a racemate is brought into contact with a thin film barrier. The barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as a concentration or pressure difference causes preferential passage through the membrane barrier. The separation is due to the non-racemic chiral nature of the membrane, which allows only one enantiomer of the racemate to pass through.

[0207] In some embodiments, the composition of the compounds of the present invention is substantially free of the designated enantiomer of the compound. In certain embodiments, in the methods and compounds of the present invention, the compound is substantially free of enantiomers. In some embodiments, the composition comprises a compound that comprises at least 85 wt%, 90 wt%, 95 wt%, 98 wt%, 99 wt% to 100 wt% of the composition, and the remainder comprises other chemical substances or enantiomers.

[0208] Isotope-enriched compounds

[0209] This application also provides isotope-enriched compounds.

[0210] Isotope enrichment (e.g., deuteration) of drugs has previously been shown to improve pharmacokinetic ("PK"), pharmacodynamic ("PD"), and toxicity profiles for certain classes of drugs. See, e.g., Lijinsky et al., Food Cosmet. Toxicol., 20:393 (1982); Lijinsky et al., J. Nat. Cancer Inst., 69:1127 (1982); Mangold et al., Mutation Res. 308:33 (1994); Gordon et al., Drug Metab. Dispos., 15:589 (1987); Zello et al., Metabolism, 43:487 (1994); Gately et al., J. Nucl. Med., 27:388 (1986); Wade D, Chem. Biol. Interact. 117:191 (1999).

[0211] Isotope enrichment of drugs can be used, for example, (1) to reduce or eliminate unwanted metabolites, (2) to increase the half-life of the parent drug, (3) to reduce the dose required to achieve the desired effect, (4) to reduce the dose required to achieve the intended effect, (5) to increase the formation of active metabolites (if any), or (6) to reduce the production of harmful metabolites in specific tissues, or to create a more effective or safer drug for combination therapy (whether the combination therapy is intentional or unintentional).

[0212] Replacing an atom with an isotope of that atom generally results in a change in the reaction rate of a chemical reaction. This phenomenon is known as the kinetic isotope effect (“KIE”). For example, if a C-H bond breaks in the rate-determining step of a chemical reaction (i.e., the step with the highest transition state energy), replacing hydrogen with deuterium will result in a decrease in the reaction rate and the process will slow down. This phenomenon is known as the deuterium kinetic isotope effect (“DKIE”). (See Foster et al., Adv. Drug Res., vol. 14, pp. 1-36 (1985); Kushner et al., Can. J. Physiol. Pharmacol., vol. 77, pp. 79-88 (1999)).

[0213] The magnitude of the DKIE can be expressed as the ratio of the specific reaction rate for C-H bond breakage to the same reaction rate with deuterium substitution for hydrogen. The DKIE can range from approximately 1 (no isotope effect) to very large numbers such as 50 or more, meaning that when deuterium replaces hydrogen, the reaction can be 50 times slower or more. High DKIE values may be due in part to a phenomenon called tunneling, which is a result of the uncertainty principle. Tunneling is attributed to the small mass of the hydrogen atom and occurs because, in the absence of the required activation energy, a transition state involving the proton sometimes forms. Since deuterium has a larger mass than hydrogen, statistically it has a much lower probability of this phenomenon occurring.

[0214] Tritium (“T”) is a radioactive isotope of hydrogen used in research, fusion reactors, neutron generators, and radiopharmaceuticals. Tritium is a hydrogen atom with 2 neutrons in its nucleus and an atomic weight close to 3. It occurs naturally in the environment at very low concentrations, most commonly as T2O. Tritium decays slowly (half-life = 12.3 years) and emits a low-energy beta particle that cannot penetrate the outer layer of human skin. Internal exposure is the main hazard associated with this isotope, but large amounts must be ingested to pose a significant risk to health. Compared to deuterium, less tritium must be consumed before reaching dangerous levels. Replacing hydrogen with tritium (“T”) produces a stronger bond than deuterium and a numerically larger isotope effect. Similarly, replacing other elements with isotopes, including but not limited to 13 C or 14 C, 33 S, 34 S or 36 S, 15 N and 17 O or 18 O, may result in similar kinetic isotope effects.

[0215] For example, DKIE is used to reduce the hepatotoxicity of halothane, possibly by limiting the production of reactive species such as trifluoroacetyl chloride. However, this approach may not be applicable to all drug classes. For example, the incorporation of deuterium can lead to metabolic switching. The concept of metabolic switching holds that when a foreign substance is sequestered by phase I enzymes, it can transiently bind and rebind in various conformations prior to a chemical reaction such as oxidation. This hypothesis is supported by the relatively large binding pockets in many phase I enzymes and the promiscuity of many metabolic reactions. Metabolic switching can result in different proportions of known metabolites as well as entirely new metabolites. This new metabolic profile may produce more or less toxicity.

[0216] Animal bodies express various enzymes to clear foreign substances such as therapeutic agents from their circulatory systems. Examples of such enzymes include cytochrome P450 enzymes ("CYPs"), esterases, proteases, reductases, dehydrogenases, and monoamine oxidases, which react with these foreign substances and convert them into more polar intermediates or metabolites for renal excretion. Some of the most common metabolic reactions of drug compounds involve the oxidation of carbon-hydrogen (C-H) bonds to carbon-oxygen (C-O) or carbon-carbon (C-C) π bonds. The resulting metabolites may be stable or unstable under physiological conditions, and their pharmacokinetic, pharmacodynamic, and acute and long-term toxicity profiles may be very different from those of the parent compound. For many drugs, this oxidation reaction is rapid. Therefore, these drugs typically require multiple or high-dose daily administrations.

[0217] Thus, compared to similar compounds with a natural isotope composition, the isotope enrichment of the compounds provided in the present application at certain positions will produce a detectable KIE, which will affect the pharmacokinetic, pharmacological, and / or toxicological characteristics of the compounds provided in the present application.

[0218] Preparation of Compounds

[0219] The compounds provided in the present application can be prepared, isolated, or obtained by any method obvious to those skilled in the art. Exemplary preparation methods are described in detail in the following examples.

[0220] Pharmaceutical Compositions and Methods of Administration

[0221] In certain embodiments, the present application provides pharmaceutical compositions comprising: a compound disclosed in the present application; and a pharmaceutically acceptable excipient, carrier, or diluent.

[0222] In certain embodiments, the composition is an oral formulation.

[0223] In certain embodiments, the compounds can be formulated into pharmaceutical compositions using methods available in the art and the methods disclosed in the present application. Any compound disclosed in the present application can be provided in a suitable pharmaceutical composition and administered via a suitable route of administration.

[0224] The methods provided by the present application include administering a pharmaceutical composition comprising at least one compound described herein, including compounds of general formula I, II, III or IV and their suitable salts, which can be used alone or in combination with one or more compatible and pharmaceutically acceptable carriers (such as diluents or adjuvants), or in combination with another pharmaceutical preparation for treating diseases characterized by DUX4 misexpression.

[0225] In certain embodiments, another agent can be formulated or packaged together with the compounds provided by the present application. It is only possible to formulate another agent together with the compounds provided by the present application when, in the judgment of those skilled in the art, such a combined preparation does not interfere with the activity or method of administration of either agent. In certain embodiments, the compounds provided by the present application and another agent are formulated separately. For the convenience of practitioners in the art, they can be packaged together or separately packaged.

[0226] In clinical practice, the active agents provided by the present application can be administered by any conventional route, such as orally, parenterally, rectally or by inhalation (e.g., in the form of an aerosol). In certain embodiments, the compounds provided by the present application are administered orally.

[0227] Tablets, pills, hard gelatin capsules, powders or granules can be used as oral solid compositions. In these compositions, the active ingredient is mixed with one or more inert diluents or adjuvants, such as sucrose, lactose or starch.

[0228] These compositions can contain substances other than diluents, such as lubricants like magnesium stearate, or coatings for controlled release.

[0229] As liquid compositions for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents (such as water or liquid paraffin) can be used. These compositions can also contain substances other than diluents, such as wetting, sweetening or flavoring agents.

[0230] Compositions for parenteral administration can be emulsions or sterile solutions. Propylene glycol, polyethylene glycol, vegetable oils especially olive oil, or injectable organic esters such as ethyl oleate can be used as solvents or carriers. These compositions can also contain adjuvants, especially wetting agents, isotonic agents, emulsifying agents, dispersing agents and stabilizers. Sterilization can be carried out in various ways, such as using a bacteriological filter, radiation or heating. They can also be prepared in the form of sterile solid compositions, which can be dissolved in sterile water or any other injectable sterile medium when in use.

[0231] The composition for rectal administration is a suppository or a rectal capsule, which contains excipients such as cocoa butter, semi-synthetic glycerides or polyethylene glycols in addition to the active ingredient.

[0232] The composition can also be an aerosol. For use in the form of a liquid aerosol, the composition can be a stable sterile solution or a solid composition that is dissolved in pyrogen-free sterile water, saline or any other pharmaceutically acceptable carrier at the time of use. For use in the form of a dry aerosol for direct inhalation, the active ingredient is pulverized and combined with a water-soluble solid diluent or carrier, such as dextran, mannitol or lactose.

[0233] In certain embodiments, the composition provided by the present application is a pharmaceutical composition or a single-unit dosage form. The pharmaceutical compositions and single-unit dosage forms provided by the present application contain a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (such as the compounds provided by the present application or other prophylactic or therapeutic agents), and usually one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment, in this case, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency, or listed in the United States Pharmacopeia or other recognized pharmacopeias, for use in animals, and more particularly in humans. The term "carrier" includes diluents, excipients or vehicles for administering therapeutic agents. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water can be used as a carrier. Aqueous solutions of saline, glucose and glycerol can also be used as liquid carriers, especially for injection solutions. Examples of suitable pharmaceutical carriers are described by E.W. Martin in Remington's Pharmaceutical Sciences, the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0234] Typical pharmaceutical compositions and dosage forms contain one or more excipients. Suitable excipients are well known to those skilled in the art of pharmacy, and non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art, including but not limited to the manner in which the dosage form is to be administered to the subject and the particular active ingredient in the dosage form. If desired, the composition or single-unit dosage form may also contain small amounts of wetting agents or emulsifying agents, or pH buffering agents.

[0235] The lactose-free compositions provided by the present application may contain excipients well known in the art, such as those listed in the United States Pharmacopia (USP) SP (XXI) / NF (XVI). Generally, the lactose-free compositions contain a pharmaceutically compatible and pharmaceutically acceptable amount of an active ingredient, a binder / filler, and a lubricant. Exemplary lactose-free dosage forms include an active ingredient, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

[0236] Since water can promote the degradation of certain compounds, the present application also includes anhydrous pharmaceutical compositions and dosage forms containing an active ingredient. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical field as a means of simulating long-term storage to determine characteristics such as the shelf life of a formulation or its stability over time. See, for example, Jens T. Carstensen, Drug Stability: Principles and Practice, 2d. Edited by Marcel Dekker, New York, N.Y., 1995, pp. 379-80. In fact, water and heat can accelerate the decomposition of certain compounds. Thus, the effect of water on a formulation can be significant because moisture and / or humidity are typically encountered during the manufacture, handling, packaging, storage, shipping, and use of a formulation.

[0237] The anhydrous pharmaceutical compositions and dosage forms provided by the present application can be prepared using anhydrous or low-moisture ingredients and low-moisture or low-humidity conditions. If it is estimated that there will be significant exposure to moisture and / or humidity during manufacture, packaging, and / or storage, pharmaceutical compositions and dosage forms containing lactose, at least one active ingredient, and a primary or secondary amine can be anhydrous.

[0238] By preparing and storing anhydrous pharmaceutical compositions to maintain their anhydrous nature. Thus, anhydrous compositions can be packaged using materials known to prevent exposure to water in order to be included in a suitable prescription kit. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit-dose containers (such as vials), blister packs, and strip packs.

[0239] The present application further provides pharmaceutical compositions and dosage forms that contain one or more compounds that reduce the rate of decomposition of an active ingredient. Such compounds, referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[0240] The pharmaceutical compositions and single unit dosage forms can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions and dosage forms will contain a prophylactically or therapeutically effective amount of the prophylactic or therapeutic agent in purified form, as well as a suitable amount of carrier to provide a suitable dosage form for the subject. The formulation should be adapted to the mode of administration. In one embodiment, the pharmaceutical composition or single unit dosage form is sterile and in a form suitable for administration to a subject, such as an animal subject, such as a mammalian subject (e.g., a human subject).

[0241] The pharmaceutical composition formulation should match the intended route of administration. Examples of routes of administration include but are not limited to parenteral administration, such as intravenous, intradermal, subcutaneous, intramuscular, subcutaneous, oral, buccal, sublingual, inhalation, intranasal, transdermal, topical, intra mucosal, intratumoral, intra synovial, and rectal administration. In a specific embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration. In one embodiment, the pharmaceutical composition is formulated according to conventional procedures for subcutaneous administration to a human. Generally, a composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. When necessary, the composition may also include solubilizing agents and local anesthetics, such as lidocaine, to relieve the pain at the injection site.

[0242] Examples of dosage forms include but are not limited to: tablets; cachets; capsules (such as soft elastic gelatin capsules); lozenges; pills; troches; dispersions; suppositories; ointments; cataplasms (plasters); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (such as nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a subject, including suspensions (such as aqueous or non aqueous liquid suspensions, oil in water emulsions or water in oil liquid emulsions); solutions and elixirs; liquid dosage forms suitable for parenteral administration to a subject; and sterile solids (such as crystalline or amorphous solids) that can be reconstituted to provide a liquid dosage form suitable for parenteral administration to a subject.

[0243] The composition, shape, and type of the dosage forms provided in this application will generally vary according to their use. For example, a dosage form for the initial treatment of a viral infection may contain a greater amount of one or more active ingredients than a dosage form for maintenance treatment of the same infection. Similarly, a parenteral dosage form may contain a lesser amount of one or more active ingredients compared to an oral dosage form for treating the same disease or condition. Those skilled in the art will readily understand the various different dosage forms covered by this application, for example, see Remington's Pharmaceutical Sciences, 20th Edition, Mack Publishing, Easton PA (2000).

[0244] Typically, the components of the composition can be supplied separately or mixed together in unit dosage forms, for example, as dry freeze-dried powders or anhydrous concentrates, packed in sealed containers such as ampoules or sachets, indicating the amount of the active ingredient. When the composition is administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, sterile water for injection or saline can be provided in an ampoule for mixing the components before administration.

[0245] Typical dosage forms include the compounds provided in this application or their pharmaceutically acceptable salts, solvates or hydrates, administered once daily in the morning at a dose in the range of about 0.1 mg to about 1000 mg, or taken in divided doses throughout the day with food. In certain embodiments, the dosage form can contain about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100, 200, 250, 500 or 1000 mg of the active compound.

[0246] Oral dosage forms

[0247] Pharmaceutical compositions suitable for oral administration can be in discrete dosage forms, such as but not limited to tablets (such as chewable tablets), capsules, cachets and solutions (such as flavored syrups). Such dosage forms contain a predetermined amount of the active ingredient and can be prepared by pharmaceutical methods well known to those skilled in the art. See Remington's Pharmaceutical Sciences, 20th Edition, Mack Publishing, Easton PA (2000).

[0248] In certain embodiments, the oral dosage form is solid and prepared with anhydrous components under anhydrous conditions as described in detail above. However, the scope of the compositions provided in this application extends beyond anhydrous solid oral dosage forms. Accordingly, other dosage forms are described in this application.

[0249] Typical oral dosage forms are prepared by intimately mixing the active ingredient with at least one excipient according to conventional pharmaceutical formulation techniques. Depending on the form of the preparation required for administration, the excipient can take various forms. For example, excipients suitable for oral liquid or aerosol dosage forms include but are not limited to water, glycols, oils, alcohols, flavoring agents, preservatives and coloring agents. Examples of excipients suitable for solid oral dosage forms (such as powders, tablets, capsules and cachets) include but are not limited to starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrants.

[0250] Due to their ease of administration, tablets and capsules are the most advantageous oral dosage unit forms and solid excipients are used in this case. If desired, tablets can be coated by standard aqueous or non-aqueous techniques. This dosage form can be prepared by any pharmaceutical method. Generally, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately mixing the active ingredient with a liquid carrier, a comminuted solid carrier, or both, and then shaping the product into the desired form, if necessary.

[0251] For example, tablets can be prepared by compression or molding. Tablets can be compressed by compressing the active ingredient in free-flowing form, such as a powder or granule (which can be mixed with excipients), in a suitable machine. A mixture of powdered compounds moistened with an inert liquid diluent is molded in a suitable machine to produce molded tablets.

[0252] Examples of excipients suitable for oral dosage forms include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as gum arabic, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (such as ethyl cellulose, cellulose acetate, calcium carboxymethyl cellulose, sodium carboxymethylcellulose), polyvinylpyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose (such as grades 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

[0253] Examples of fillers suitable for the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to: talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrin, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof. Binders or fillers in pharmaceutical compositions typically comprise from about 50 to about 99 weight % of the pharmaceutical composition or dosage form.

[0254] Suitable forms of microcrystalline cellulose include, but are not limited to, materials sold under the trade names AVICEL PH 101, AVICEL PH103, AVICEL RC 581, AVICEL PH 105 (available from FMC Corporation, American Viscose Division, AVICEL Sales, Marcus Hook, PA) and mixtures thereof. A particular binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose (sold under the trade name AVICEL RC 581). Suitable anhydrous or low-moisture excipients or additives include AVICEL PH 103 TM and Starch 1500LM.

[0255] Disintegrants can be used in the composition to provide tablets that disintegrate upon exposure to an aqueous environment. Tablets containing too much disintegrant may disintegrate during storage, while tablets containing too little disintegrant may not disintegrate at the desired rate or under the desired conditions. Therefore, an amount of disintegrant sufficient to form a solid oral dosage form, i.e., neither too much nor too little so as not to adversely affect the release of the active ingredient, should be used. The amount of disintegrant used varies according to the type of formulation and can be readily determined by one of ordinary skill in the art. Typical pharmaceutical compositions contain from about 0.5 to about 15% by weight of disintegrant, particularly from about 1 to about 5% by weight of disintegrant.

[0256] Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to: agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pregelatinized starch, other starches, clays, other alginates, other celluloses, gums, and mixtures thereof.

[0257] Lubricants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to: calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other diols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and mixtures thereof. Other lubricants include, for example, syloid silica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore, Maryland), coagulated aerosol of synthetic silica (sold by Degussa Co. of Plano, Texas), CAB-O-SIL (a pyrogenic silica product sold by Cabot Co. of Boston, Massachusetts), and mixtures thereof. If a lubricant is used, the amount thereof is generally less than about 1% by weight of the pharmaceutical composition or dosage form into which it is incorporated.

[0258] Delayed release dosage form

[0259] The compounds (active ingredients) provided by the present application can be administered by controlled release means or delivery devices well-known to those of ordinary skill in the art. Examples include, but are not limited to, those described in the following U.S. patents: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566; 5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945; 5,993,855; 6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363; 6,264,970; 6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; 6,699,500; each of the above patents is hereby incorporated by reference in its entirety into the present application. Such dosage forms can be used to provide slow or controlled release of one or more active ingredients, for example, using hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multi-layer coatings, microparticles, liposomes, microspheres or combinations thereof, in different proportions to provide the desired release profile. Those of ordinary skill in the art can easily select known suitable controlled release formulations, including those described in the present application, for use with the active ingredients provided by the present application. Accordingly, the present application encompasses single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel caps and cachets suitable for controlled release.

[0260] All controlled release drug products have a common goal of improving drug therapy to make it superior to non-controlled release drugs. Ideally, the characteristics of the best designed controlled release formulations used in medicine are to cure or control the condition with the least amount of drug in the shortest time. The advantages of controlled release formulations include prolonging the activity of the drug, reducing the dosing frequency and improving the compliance of the subject. In addition, controlled release formulations can be used to affect the onset time or other characteristics, such as the blood level of the drug, thereby affecting the occurrence of side effects (such as adverse reactions).

[0261] Most controlled release formulations are designed to initially release a certain amount of drug (active ingredient) to rapidly produce the desired therapeutic effect and gradually and continuously release other amounts of drug to maintain this therapeutic or prophylactic effect over a longer period of time. To maintain a constant level of drug in the body, the drug must be released from the dosage form at a certain rate to replace the amount of drug metabolized and excreted in the body. The controlled release of the active ingredient can be stimulated by various conditions, including but not limited to pH, temperature, enzymes, water or other physiological conditions or compounds.

[0262] In certain embodiments, the drug can be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other modes of administration. In certain embodiments, a pump can be used (see, e.g., Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, a polymeric material can be used. In another embodiment, the controlled release system can be placed at an appropriate site within the subject determined by one of ordinary skill in the art, such that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)). The active ingredient can be dispersed in an inner solid matrix surrounded by an outer polymeric membrane. The inner matrix can be, for example, polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, silicone carbonate copolymer, hydrophilic polymers such as hydrogels of acrylates and methacrylates, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate. The outer polymeric membrane is insoluble in body fluids and can be, for example, polyethylene, polypropylene, ethylene / propylene copolymer, ethylene / ethyl acrylate copolymer, ethylene-vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, chloroprene rubber, chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride with vinyl acetate, vinylidene chloride, ethylene, and propylene, ionomers, polyethylene terephthalate, butyl rubber, epichlorohydrin rubber, ethylene / vinyl alcohol copolymer, ethylene / vinyl acetate / vinyl alcohol terpolymer, and ethylene / ethylene oxyethanol copolymer. In the release rate controlling step, the active ingredient diffuses through the outer polymeric membrane. The percentage of the active ingredient in such parenteral compositions depends largely on its specific properties and the needs of the subject.

[0263] Parenteral dosage forms

[0264] In certain embodiments, parenteral dosage forms are provided. Parenteral dosage forms can be administered by various routes, including but not limited to subcutaneous, intravenous (including bolus), intramuscular, and intraarterial. Because their administration typically bypasses the subject's natural defenses against contaminants, parenteral dosage forms are generally sterile or capable of being sterilized in the subject prior to administration. Examples of parenteral dosage forms include but are not limited to injection solutions, dry products that are soluble or suspendable in a pharmaceutically acceptable injection vehicle, injectable suspensions, and emulsions.

[0265] Suitable carriers for providing parenteral dosage forms are well known to those skilled in the art. Examples include but are not limited to: Water for Injection USP; aqueous carriers, including but not limited to Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-soluble carriers, such as but not limited to ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous carriers, such as but not limited to corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0266] Compounds that increase the solubility of one or more of the active ingredients disclosed in this application can also be incorporated into parenteral dosage forms.

[0267] Transdermal, topical, and mucosal dosage forms

[0268] This application also provides transdermal, topical, and mucosal dosage forms. Transdermal, topical, and mucosal dosage forms include but are not limited to: ophthalmic solutions, sprays, aerosols, creams, emulsions, ointments, gels, solutions, emulsions, suspensions, or other forms known to those skilled in the art. For example, see Remington′s Pharmaceutical Sciences, 16th, 18th and 20th eds., Mack Publishing, Easton PA (1980, 1990 & 2000), and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treating intraoral mucosal tissues can be formulated as mouthwashes or oral gels. In addition, transdermal dosage forms include "reservoir-type" or "matrix-type" patches that can be applied to the skin and worn for a specific time to allow the desired amount of the active ingredient to penetrate.

[0269] Suitable excipients (e.g., carriers and diluents) and other materials for providing the transdermal, topical, and mucosal dosage forms covered by the present application are well known to those skilled in the pharmaceutical art and depend on the particular tissue to which the given pharmaceutical composition or dosage form is applied. Given this fact, typical excipients include, but are not limited to: water, acetone, ethanol, ethylene glycol, propylene glycol, 1,3 - butanediol, isopropyl myristate, isopropyl palmitate, and mineral oil and mixtures thereof to form non - toxic and pharmaceutically acceptable lotions, tinctures, creams, emulsions, gels, or ointments. If desired, humectants or wetting agents can also be added to the pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art, see, for example, Remington′s Pharmaceutical Sciences, 16th, 18th and 20th eds., Mack Publishing, Easton PA (1980, 1990 & 2000).

[0270] Depending on the particular tissue to be treated, additional ingredients can be used before, during, or after the application of the provided active ingredient for treatment. For example, penetration enhancers can be used to assist in delivering the active ingredient to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl alcohol, and tetrahydrofurfuryl alcohol; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethylformamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (povidone, polyvinylpyrrolidone); urea; and various water - soluble or water - insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

[0271] The pH value of the pharmaceutical composition or dosage form, or the pH value of the tissue to which the pharmaceutical composition or formulation is applied, can also be adjusted to improve the delivery of one or more active ingredients. Similarly, the polarity, ionic strength, or tonicity of the solvent carrier can be adjusted to improve delivery. Compounds such as stearates can also be added to the pharmaceutical composition or dosage form to improve the hydrophilicity or lipophilicity of one or more active ingredients, thereby improving delivery. In this regard, stearates can act as lipid carriers, emulsifiers, or surfactants of the formulation, as well as delivery enhancers or penetration enhancers. Different salts, hydrates, or solvates of the active ingredient can be used to further adjust the properties of the resulting composition.

[0272] Dosage and Unit Dosage Forms

[0273] In certain embodiments, the present application provides methods of treating a patient, including administering an effective therapeutically - active amount of a compound or composition disclosed herein. In certain examples, the patient is a human.

[0274] In the treatment of human cases, the doctor will determine the cause he believes to be most appropriate based on prophylactic or therapeutic purposes, as well as the age, weight, stage of infection, and other specific factors of the subject. In certain embodiments, the dosage for an adult is about 1 to about 1000 mg per day, or about 5 to about 250 mg per day or about 10 to 50 mg per day. In certain embodiments, the dosage for each adult is about 5 to about 400 mg per day or 25 to 200 mg per day. In certain embodiments, a dosage of about 50 to about 500 mg per day may also be considered.

[0275] In another aspect, a method for treating or preventing a disease characterized by DUX4 misexpression in a subject is provided by administering to the subject in need thereof an effective amount of a compound provided by the present application or a pharmaceutically acceptable salt thereof. The amount of the compound or composition effective to prevent or treat a disease or one or more of its symptoms will vary depending on the nature and severity of the disease or condition and the route of administration of the active ingredient. The frequency and dosage will also vary according to the specific factors of each subject, depending on the specific therapy administered (e.g., therapeutic agent or prophylactic agent), the severity of the disease, disorder or condition, the route of administration, and the age, body, weight, response, and past medical history of the subject. The effective dose can be inferred from a dose-response curve obtained from in vitro or animal model test systems.

[0276] In certain embodiments, exemplary dosages of the composition include milligram or microgram amounts of the active compound per kilogram of subject or sample weight (e.g., about 10 micrograms / kg to about 50 mg / kg, about 100 micrograms / kg to about 25 mg / kg or about 100 micrograms / kg to about 10 mg / kg). For the composition provided by the present application, in certain embodiments, based on the weight of the active compound, the dosage administered to the subject is 0.140 mg / kg to 3 mg / kg of the subject's body weight. In certain embodiments, the dosage administered to the subject is 0.20 mg / kg to 2.00 mg / kg or 0.30 mg / kg to 1.50 mg / kg of the subject's body weight.

[0277] In certain embodiments, for the conditions described in the present application, the recommended daily dose range of the compositions provided by the present application is in the range of from about 0.1 mg to about 1000 mg per day, administered as a once-daily dose or in divided doses throughout the day. In certain embodiments, the daily dose is administered twice daily in equal divided doses. In certain embodiments, the daily dose range should be from about 10 mg to about 200 mg per day, from about 10 mg to about 150 mg per day in other embodiments, and can be from about 25 to about 100 mg per day in further embodiments. In some cases, it may be necessary to use an active ingredient dose outside the scope of the present application, which will be apparent to those of ordinary skill in the art. In addition, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate the treatment in combination with the response of the subject.

[0278] As is well known to those of ordinary skill in the art, different therapeutically effective amounts may be applicable to different diseases and conditions. Similarly, the above-described dose and dose frequency regimens also include amounts sufficient to prevent, manage, treat, or improve such diseases, but not sufficient to cause or sufficient to reduce adverse reactions associated with the compositions provided by the present application. In addition, when multiple doses of the compositions of the present application are administered to a subject, not all doses need to be the same. For example, the dose administered to the subject can be increased to enhance the prophylactic or therapeutic effect of the composition, or the dose administered can be reduced to reduce one or more side effects that a particular subject is experiencing.

[0279] In certain embodiments, based on the weight of the active compound, the dose of the compositions provided by the present application for preventing, treating, managing, or improving a disease or one or more symptoms thereof in a subject is 0.1 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 10 mg / kg, or 15 mg / kg or more of the subject's body weight. In another embodiment, the dose of the composition for preventing, treating, managing, or improving a disease or one or more symptoms thereof in a subject or the compositions provided by the present application is 0.1 mg - 200 mg, 0.1 mg - 100 mg, 0.1 mg - 50 mg, 0.1 mg - 25 mg, 0.1 mg - 15 mg, 0.1 mg - 10 mg, 0.1 mg - 7.5 mg, 0.1 mg - 5 mg, 0.1 - 2.5 mg, 0.25 mg - 20 mg, 0.25 - 15 mg, 0.25 - 12 mg, 0.25 - 10 mg, 0.25 mg - 7.5 mg, 0.25 - 5 mg, 0.5 mg - 2.5 mg, 1 mg - 20 mg, 1 mg - 15 mg, 1 mg - 12 mg, 1 mg - 10 mg, 1 mg - 7.5 mg, 1 mg - 5 mg, or 1 mg - 2.5 mg.

[0280] In certain embodiments, treatment or prophylaxis can be initiated with one or more loading doses of a compound or composition provided by the present application, followed by one or more maintenance doses. In these embodiments, the loading dose can be, for example, from about 60 to about 400 mg per day, or from about 100 to about 200 mg per day, for one day to five weeks. One or more maintenance doses can be administered after the loading dose. In certain embodiments, each maintenance dose is independently from about 10 mg to about 200 mg per day, from about 25 mg to about 150 mg per day, or from about 25 to about 80 mg per day. The maintenance dose can be administered daily, or as a single dose or divided doses.

[0281] In certain embodiments, a dose of a compound or composition provided by the present application is administered to achieve a steady-state concentration of the active ingredient in the blood or serum of a subject. The steady-state concentration can be determined by measurement using techniques available to those skilled in the art, or can be based on the physical characteristics of the subject, such as height, weight, and age. In certain embodiments, an amount of a compound or composition provided by the present application is administered sufficient to achieve a steady-state concentration in the blood or serum of the subject of from about 300 to about 4000 ng / mL, from about 400 to about 1600 ng / mL, or from about 600 to about 1200 ng / mL. In certain embodiments, a maintenance dose can be administered to achieve a steady-state concentration in the blood or serum of the subject of from about 300 to about 4000 ng / mL, from about 400 to about 1600 ng / mL, or from about 600 to about 1200 ng / mL.

[0282] In certain embodiments, the same composition can be administered repeatedly, and can be administered at intervals of at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. In other embodiments, the same prophylactic or therapeutic agent can be administered repeatedly, and can be administered at intervals of at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.

[0283] In certain aspects, the present application provides unit doses comprising a compound or a pharmaceutically acceptable salt thereof in a suitable dosage form. These forms are described in detail above. In certain embodiments, the unit dose comprises from 1 to 1000 mg, from 5 to 250 mg, or from 10 to 50 mg of the active ingredient. In certain embodiments, the unit dose comprises about 1, 5, 10, 25, 50, 100, 125, 250, 500, or 1000 mg of the active ingredient. Such unit doses can be prepared using techniques familiar to those skilled in the art.

[0284] The dosage of the second agent will be used in the combination therapy provided by the present application. In certain embodiments, the dosage used in the combination therapy provided by the present application is lower than the dosage that has been or is currently used for preventing or treating diseases characterized by DUX4 misexpression. The recommended dosage of the second agent can be obtained from the knowledge of those skilled in the art. For second agents approved for clinical use, their recommended dosages are documented in the literature, such as Hardman et al., eds., 1996, Goodman & Gilman’s The Pharmacological Basis Of Therapeutics 9th Ed, Mc-Graw-Hill, New York; Physician’s Desk Reference (PDR) 57th Ed., 2003, Medical Economics Co., Inc., Montvale, NJ, which are hereby incorporated by reference in their entirety into the present application.

[0285] In various embodiments, the therapies (such as the compounds and the second agent provided by the present application) are administered at intervals of less than 5 minutes, less than 30 minutes, or 1-hour intervals, about 1-hour intervals, 1 to 2 hours, 2 to 3 hours, 3 to 4 hours, 4 to 5 hours, 5 to 6 hours, 6 to 7 hours, 7 to 8 hours, 8 to 9 hours, 9 to 10 hours, 10 to 11 hours, 11 to 12 hours, 12 hours to 18 hours, 18 hours to 24 hours, 24 hours to 36 hours, 36 hours to 48 hours, 48 hours to 52 hours, 52 hours to 60 hours, 60 hours to 72 hours, 72 hours to 84 hours, 84 hours to 96 hours, or 96 hours to 120 hours. In various embodiments, the treatment intervals do not exceed 24 hours or do not exceed 48 hours. In certain embodiments, two or more therapies are administered during the same patient visit. In other embodiments, the compounds and the second agent provided by the present application are administered simultaneously.

[0286] In other embodiments, the compounds and the second agent provided by the present application are administered at intervals of about 2 to 4 days, about 4 to 6 days, about 1 week, about 1 to 2 weeks, or more than 2 weeks.

[0287] In certain embodiments, the same agent can be repeatedly administered, and the intervals are at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. In other embodiments, the same agent can be repeatedly administered, and the intervals are at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.

[0288] In certain embodiments, the compounds provided by the present application and the second medicament are administered to a patient, such as a mammal like a human, in a certain order and at certain time intervals, such that the compounds provided by the present application can act together with other medicaments to provide greater benefits than when administered otherwise. For example, the second active medicament can be administered simultaneously or in any order at different time points; however, if not administered simultaneously, it should be administered in a timely manner to provide the desired therapeutic or prophylactic effect. In certain embodiments, the compounds provided by the present application and the second active medicament exert their effects during overlapping times. Each second active medicament can be administered alone in any suitable form and by any suitable route. In other embodiments, the compounds provided by the present application are administered before, simultaneously with, or after the administration of the second active medicament.

[0289] In certain embodiments, the compounds provided by the present application and the second medicament are administered to the patient periodically. Cyclic therapy includes administering a first drug (e.g., a first prophylactic or therapeutic drug) over a period of time, and then taking a second drug and / or a third drug (such as a second and / or a first prophylactic or therapeutic drug) over a second period of time, and repeating this order of administration. Cyclic therapy can reduce resistance to one or more therapies, avoid or reduce side effects of one of the therapies, and / or enhance the efficacy of the treatment.

[0290] In certain embodiments, the compounds provided by the present application and the second active medicament are administered in cycles of less than about 3 weeks, about once every two weeks, about once every 10 days, or about once a week. One cycle can include administering the compounds provided by the present application and the second medicament within about 90 minutes per cycle, about 1 hour per cycle, or about 45 minutes per cycle by infusion. Each cycle can include a rest of at least 1 week, at least 2 weeks, and at least 3 weeks. The number of cycles of administration is about 1 to about 12 cycles, more typically about 2 to about 10 cycles, and most typically about 2 to about 8 cycles.

[0291] In other embodiments, different treatment courses are administered to the patient simultaneously, that is, the individual doses of the second medicament are administered separately, but at certain time intervals such that the compounds provided by the present application can act together with the second active medicament. For example, one component can be administered once a week, and the other components can be administered once every two weeks or once every three weeks. In other words, even if the therapeutic drugs are not administered simultaneously or within the same day, the dosing regimens are carried out simultaneously.

[0292] The second agent can act additively or synergistically with the compounds provided by the present application. In certain embodiments, the compounds provided by the present application and one or more second agents form the same pharmaceutical composition and are administered simultaneously. In another embodiment, the compounds provided by the present application and one or more second agents form separate pharmaceutical compositions respectively, but are administered simultaneously. In another embodiment, the compounds provided by the present application are administered before or after the administration of the second agent. It is also contemplated to administer the compounds and the second agent provided by the present application by the same or different routes of administration, such as oral and parenteral administration. In certain embodiments, when the compounds provided by the present application are administered simultaneously with a second agent that may produce adverse side effects including but not limited to toxicity, the second active agent can be advantageously administered at a dose below the threshold for triggering adverse side effects.

[0293] Kit

[0294] The present application also provides a kit for a method of treating a disease characterized by DUX4 misexpression. The kit may include the compounds or compositions provided by the present application, second agents or compositions, and instructions for providing healthcare practitioners with information on the use of treating the disease. The instructions may be provided in printed form or in the form of electronic media such as floppy disks, CDs or DVDs, or in the form of a website address where such instructions can be obtained. When a subject administers the compounds or compositions or second agents or compositions provided by the present application, the unit dose is such that the therapeutically or prophylactically effective plasma level of the compound or composition in the subject can be maintained for at least 1 day. In some embodiments, it may contain the compound or composition as a sterile aqueous pharmaceutical composition or a dry powder (e.g., lyophilized) composition.

[0295] In some embodiments, suitable packaging is provided. The "packaging" used in the present application includes solid matrices or materials commonly used in the system, which can hold the compounds provided by the present application and / or second agents suitable for administration to a subject within a fixed range. These materials include glass and plastic (such as polyethylene, polypropylene and polycarbonate) bottles, vials, paper, plastic and plastic foil laminated envelopes, etc. If electron beam sterilization technology is used, the packaging should have a low enough density to allow sterilization of the contents.

[0296] Method of use

[0297] In certain embodiments, the present application provides a method of treating a patient, including administering an effective therapeutically amount of the compounds or compositions disclosed by the present application. In certain embodiments, the patient is a human. In certain embodiments, the patient is a subject in need of treatment (i.e., a patient in need of treatment). In certain embodiments, the patient is a subject previously treated with another chemotherapeutic compound or composition.

[0298] In certain embodiments, the present application provides methods for treating and / or preventing diseases characterized by DUX4 misexpression, including administering an effective amount of a compound provided by the present application or a pharmaceutically acceptable salt thereof. In certain embodiments, the present application provides methods for treating diseases characterized by DUX4 misexpression in a subject. In certain embodiments, the method includes the step of administering to a subject in need thereof an amount of a compound that is effective in treating or preventing a disease characterized by DUX4 misexpression and is used in combination with a second agent that is effective in treating or preventing the disease. The compound can be any compound as described in the present application, and the second agent can be any second agent described in the art or in the present application. In certain embodiments, as described in the present application, the compound is in the form of a pharmaceutical composition or dosage form.

[0299] In certain embodiments, the subject has never received treatment or prophylaxis for a disease characterized by DUX4 misexpression. In further embodiments, the subject has previously received treatment or prophylaxis for a disease characterized by DUX4 misexpression.

[0300] In certain embodiments, the subject is a subject who has discontinued treatment for a disease characterized by DUX4 misexpression due to one or more treatment-related adverse events. In certain embodiments, the subject is a subject who is not suitable for the current treatment regimen.

[0301] In certain embodiments, the subject has received treatment for a disease characterized by DUX4 misexpression and has discontinued such treatment prior to administration of the method provided by the present application. In further embodiments, the subject has received treatment and continues to receive such treatment as well as administration of the method provided by the present application. These methods can be co-administered with other therapies for the disease, as determined by those skilled in the art. In certain embodiments, the methods or compositions provided by the present application can be co-administered with other therapies for diseases characterized by DUX4 misexpression at reduced doses.

[0302] In certain embodiments, methods are provided for treating subjects who are unresponsive to treatment for diseases characterized by DUX4 misexpression. For example, in some embodiments, the subject can be a subject who does not respond to one or more drug treatments for a disease characterized by DUX4 misexpression. In some embodiments, the subject can be a subject who has a poor response to one or more drug treatments for a disease characterized by DUX4 misexpression.

[0303] Method

[0304] The activity of a compound against a disease characterized by DUX4 misexpression can be determined according to any assay method known to those skilled in the art.

[0305] Second therapeutic agent

[0306] In certain embodiments, the compounds and compositions provided by the present application can be used in methods for treating liver diseases, which methods comprise further administering to a subject in need thereof a second agent effective in treating the disease. The second agent can be any agent known to those skilled in the art to be effective in treating the disease, including those currently approved by the FDA.

[0307] In certain embodiments, the compounds provided by the present application are administered in combination with a second agent. In further embodiments, the second agent is administered in combination with two second agents. In further embodiments, the second agent is administered in combination with two or more second agents.

[0308] As used herein, the term "combination" includes the use of multiple therapies (e.g., one or more prophylactic and / or therapeutic agents). The use of the term "in combination" does not limit the order in which the therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject having a disease. The first therapy (e.g., a prophylactic or therapeutic agent, such as a compound provided by the present application) can be administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., a prophylactic agent or a therapeutic agent) to a subject having a disease.

[0309] As used herein, the term "synergistic" includes combinations of the compounds provided by the present application and another therapy (e.g., a prophylactic agent or a therapeutic agent) that has been or is currently used for preventing, managing, or treating a disease, which combination is more effective than the additive effect of the therapies. The synergistic effect of combinations of multiple therapies (e.g., combinations of prophylactic or therapeutic agents) makes it possible to use lower doses of one or more of the therapies and / or administer the therapies less frequently to a subject having a disease, thereby reducing the toxicity associated with administering the treatment to the subject without reducing the efficacy of the therapies in preventing or treating the disease. In addition, the synergistic effect can enhance the efficacy of the drugs in preventing or treating the disease. Finally, the synergistic effect of combinations of multiple therapies (e.g., combinations of prophylactic or therapeutic drugs) can avoid or reduce adverse or unwanted side effects associated with the use of any one of the therapies alone.

[0310] The active compounds provided by the present application can be administered in combination with or alternately with another therapeutic agent. In combination therapy, the effective doses of two or more drugs are administered together, while in alternate or sequential step therapy, the effective amounts of each drug are administered continuously or sequentially. The doses administered will depend on the rates of absorption, inactivation, and excretion of the drugs and other factors known to those skilled in the art. It should be noted that the dose values also vary with the severity of the condition to be alleviated. It should be further understood that for any particular subject, the specific dosage regimen and schedule should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition. In certain embodiments, the compound for treating a disease characterized by DUX4 misexpression has an EC of 1 to 15 μM 50 . In certain embodiments, an EC 50 less than 1 to 5 μM of the compound is desirable.

[0311] Examples of the second drug include losaprimo, vitamin C, vitamin E, zinc gluconate, and selenomethionine. Examples

[0312] The symbols and conventions used in the processes, protocols, and examples of the present application, whether or not specific abbreviations are specifically defined, are consistent with those used in contemporary scientific literature, such as the Journal of the American Chemical Society or the Journal of Biological Chemistry. Specifically, but not limited to, the following abbreviations may be used in the examples and throughout the specification: g (gram); mg (milligram); mL (milliliter); μL (microliter); mM (millimole); μM (micromole); Hz (hertz); MHz (megahertz); mmol (millimole); h, hr or hrs (hour); min (minute); thin layer chromatography; HPLC (high performance liquid chromatography); THF (tetrahydrofuran); CDCl3 (deuterated chloroform); DCM (dichloromethane); DMSO (dimethyl sulfoxide); DMSO-d6 (deuterated dimethyl sulfoxide); EtOAc (ethyl acetate).

[0313] For all the following examples, standardized work-up and purification methods known to those skilled in the art can be used. Unless otherwise stated, all temperatures are in °C (degrees Celsius). Unless otherwise stated, all reactions are carried out at room temperature. The synthetic methods shown in the present application are intended to illustrate the applicable chemical reactions by specific examples and do not limit the scope of the present application.

[0314] Example 1

[0315] Synthesis of N-(6-(benzothiazol-2-yl)pyridin-3-yl)-3-(2,5-dioxopyrrolidin-1-yl)acrylamide (AT003) (5a)

[0316]

[0317] Synthesis of 6-(benzo[d]thiazol-2-yl)pyridin-3-amine (3)

[0318]

[0319] Under a nitrogen atmosphere, polyphosphoric acid (30 g) was added to a mixture of 2-aminobenzenethiol (1a) (2 g, 15.98 mmol) and 5-aminopicolinic acid (2) (2.20 g, 15.98 mmol) at room temperature. The reaction mixture was heated at 130 °C for 4 h. TLC analysis showed consumption of the starting materials. The reaction mixture was quenched with water (50 mL), and the pH was slowly adjusted to neutral with saturated sodium hydroxide solution. The resulting precipitate was filtered and washed with water. The obtained solid was triturated with MTBE and dried in vacuo to give 6-(benzo[d]thiazol-2-yl)pyridin-3-amine (3a) as a pale yellow solid (1.9 g, 8.36 mmol, 52.3% yield). LCMS (ESI, +ve mode): 83.94%, observed: C 12 H9N3S (M+H) was 228.2, RT: 1.74 min.

[0320] Synthesis of N-(6-(benzo[d]thiazol-2-yl)pyridin-3-yl)-3-(2,5-dioxopyrrolidin-1-yl)propanamide (5a) (AT003)

[0321]

[0322] A solution of 6-(benzo[d]thiazol-2-yl)pyridin-3-amine (3a) (100 mg, 0.440 mmol) in dichloromethane (10 mL) was stirred, and 3-(2,5-dioxopyrrolidin-1-yl)propanoic acid (4a) (75 mg, 0.440 mmol) was added thereto under a nitrogen atmosphere. The reaction mixture was cooled to 0 °C, and then triethylamine (0.307 mL, 2.200 mmol) and a solution of T3P in EtOAc (0.786 mL, 1.320 mmol, 50% solution) were added. The reaction mixture was stirred at room temperature for 16 h. TLC analysis showed consumption of the starting materials. The reaction mixture was diluted with DCM (15 mL) and washed with 10% sodium bicarbonate solution (10 mL) and brine solution (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude residue. The obtained residue was purified by preparative HPLC (0.1% HCOOH in ACN) and lyophilized to give N-(6-(benzo[d]thiazol-2-yl)pyridin-3-yl)-3-(2,5-dioxopyrrolidin-1-yl)propanamide (5a) as a white solid (38 mg, 0.099 mmol, 22.50% yield). 11H-NMR (400 MHz, DMSO-d6): δ 10.57 (s, 1H), 8.81 (s, 1H), 8.29 (s, 2H), 8.15 (d, J = 8.00 Hz, 1H). LCMS (ESI, +ve mode): 99.61%, observed: C 21 H 19 The [M+H]+ of C 21 H 19 N3O3S is 381.1, RT: 2.07 min. HPLC: 99.09%, RT: 3.55 min.

[0323] Example 2

[0324] Synthesis of 4-acetamido-N-(4-(benzothiophen-2-yl)phenyl)-N-methylbutyramide (AT037) (5b)

[0325]

[0326] Synthesis of 4-(benzothiophen-2-yl)-N-methylaniline (3b)

[0327]

[0328] Dissolve benzothiophene-2-ylboronic acid (1b) (500 mg, 2.81 mmol) in a 25 mL sealed tube containing a mixed solution of dioxane (6 mL) and water (2 mL). At room temperature, add 4-bromo-N-methylaniline (2b) (523 mg, 2.81 mmol) and potassium carbonate (1165 mg, 8.43 mmol). Degas the reaction mixture with nitrogen for 5 minutes, then add dichlorobis(triphenylphosphine)palladium(II) (197 mg, 0.281 mmol). Stir the reaction at 90 °C for 12 h. TLC analysis showed consumption of the starting material. Dissolve the reaction mixture in ethyl acetate (50 mL), wash with water (20 mL) and brine solution (20 mL). Dry the organic layer over anhydrous sodium sulfate and concentrate under reduced pressure to obtain a crude residue. Purify the residue thus obtained by silica gel column chromatography (230 - 400 mesh), eluting with 0 - 15% EtOAc in petroleum ether to give 4-(benzothiophen-2-yl)-N-methylaniline (3b) as a white solid (300 mg, 1.136 mmol, 40.5% yield). LCMS (ESI, +ve mode): 90.65%, observed: C 15 H 13 The [M+H]+ of C 15 H 13 NS is 240.1, RT: 3.00 min

[0329] Synthesis of 4-acetamido-N-(4-(benzothiophen-2-yl)phenyl)-N-methylbutyramide (AT037) (5b)

[0330]

[0331] Stir a solution of 4-(benzo[b]thiophen-2-yl)-N-methylaniline (3b) (100 mg, 0.418 mmol) in N,N-dimethylformamide (6 mL). Add 4-acetamidobutyric acid (4b) (60.7 mg, 0.418 mmol) and DIPEA (0.298 mL, 1.671 mmol) at 0 °C. Then add HATU (318 mg, 0.836 mmol) and stir at room temperature for 12 h. LCMS analysis showed consumption of the starting material. Dissolve the reaction mixture in DCM (15 mL) and wash with 10% sodium bicarbonate solution (10 mL) and water (10 mL). Dry the organic layer over anhydrous sodium sulfate and concentrate under reduced pressure to give a crude residue. Purify the residue thus obtained by preparative HPLC (0.1% NH4HCO3 in ACN) and lyophilize to give 4-acetamido-N-(4-(benzothiophen-2-yl)phenyl)-N-methylbutyramide (AT037) (5b) as a white solid (16 mg, 0.041 mmol, 10% yield). 1 1H-NMR (400 MHz, DMSO-d6): δ 8.00 (d, J = 7.20 Hz, 1H), 7.92. LCMS (ESI, +ve mode): 99.73%, observed: C 21 1 22 1N2O2S (M+H) is 367.1, RT: 2.51 min. HPLC: 99.92%, RT: 4.20 min.

[0332] Example 3:

[0333] Synthesis of N-(4-(benzo[d]thiazol-2-yl)phenyl)-4-propionamidobutyramide (AT045) (5c)

[0334]

[0335] Synthesis of methyl 4-propionamidobutyrate (2c)

[0336]

[0337] At 0 °C, triethylamine (1.372 mL, 9.77 mmol) and propionyl chloride (301 mg, 3.27 mmol) were added to a solution of methyl 4-aminobutyrate hydrochloride (1c) (500 mg, 3.26 mmol) in DCM (10 mL). The mixture was stirred at room temperature for 16 h. LCMS analysis showed consumption of the starting material. The reaction mixture was diluted with DCM (15 mL) and washed with water (10 mL) and sodium bicarbonate solution (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give methyl 4-propionamidobutyrate (2c) as a pale yellow liquid (190 mg, 1.096 mmol, 33.7% yield). It was carried on to the next step without further purification. LCMS (ELSD +ve mode): 99.94%, observed: C8H 15 NO3(M+H) was 174.1, RT: 1.23 min.

[0338] Synthesis of 4-propionamidobutyric acid (4c)

[0339]

[0340] A solution of methyl 4-propionamidobutyrate (3c) (180 mg, 1.039 mmol) in a mixture of water (4 mL) and THF (4 mL) was stirred and sodium hydroxide (125 mg, 3.12 mmol) was added thereto at 0 °C. The mixture was stirred at room temperature for 2 h. LCMS analysis showed consumption of the starting material. The reaction mixture was concentrated under reduced pressure to remove THF, acidified with 1.5 N HCl (pH 3 - 4), and extracted with DCM (2 x 15 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 4-propionamidobutyric acid (4c) as a pale yellow liquid (75 mg, 0.405 mmol, 38.9% yield). It was carried on to the next step without further purification. LCMS (ESI, +ve mode): 86%, observed: C7H 13 NO3(M+H) was 160.3, RT: 0.94 min.

[0341] Synthesis of N-(4-(benzothiazol-2-yl)phenyl)-4-propionamidobutanamide (AT045) (5c)

[0342]

[0343] Stir a solution of 4-(benzo[b]thiophen-2-yl)-N-methylaniline (3b) (100 mg, 0.418 mmol) in N,N-dimethylformamide (6 mL). At 0 °C, add 4-acetamidobutyric acid (4b) (60.7 mg, 0.418 mmol) and DIPEA (0.298 mL, 1.671 mmol). Then add HATU (318 mg, 0.836 mmol), and stir at room temperature for 12 h. LCMS analysis showed consumption of the starting material. Dissolve the reaction mixture in DCM (15 mL), and wash with 10% sodium bicarbonate solution (10 mL) and water (10 mL). Dry the organic layer over anhydrous sodium sulfate and concentrate under reduced pressure to obtain a crude residue. Purify the residue thus obtained by preparative HPLC (0.1% NH4HCO3 in ACN) and lyophilize to give 4-acetamido-N-(4-(benzo[b]thiophen-2-yl)phenyl)-N-methylbutyramide (AT037) (5b) as a white solid (16 mg, 0.041 mmol, 10% yield). 1 1H-NMR (400 MHz, DMSO-d6): δ 8.00 (d, J = 7.20 Hz, 1H), 7.92. LCMS (ESI, +ve mode): 99.73%, observed: C 21 1 22 17H16N2O2S (M+H) is 367.1, RT: 2.51 min. HPLC: 99.92%, RT: 4.20 min.

[0344] Stir a solution of 4-propionamidobutyric acid (4c) (70 mg, 0.440 mmol) in DCM (5 mL). At 0 °C, add 4-(benzo[d]thiazol-2-yl)aniline (3c) (100 mg, 0.44 mmol) and triethylamine (0.062 mL, 0.40 mmol) thereto. Then add a solution of T3P in EtOAc (0.259 mL, 0.440 mmol, 50%), and stir at room temperature for 12 h. TLC analysis showed consumption of the starting material. Dilute the reaction mixture with DCM (20 mL), and wash with 10% sodium bicarbonate solution (10 mL) and brine solution (10 mL). Dry the organic layer over anhydrous sodium sulfate and concentrate under reduced pressure to obtain a crude residue. Purify the residue obtained by preparative HPLC (0.1% NH4HCO3 in ACN) and lyophilize to give N-(4-(benzo[d]thiazol-2-yl)phenyl)-4-propionamidobutyramide (AT045) (5c) as a white solid (63 mg, 0.171 mmol, 39.0% yield). 1H-NMR (400 MHz, DMSO-d6): δ 10.25 (s, 1H), 8.13. LCMS (ESI, +ve mode): 97.3%, observed: C 20 H 21 N3O2S (M+H) is 368.1, RT: 2.40 minutes. HPLC: 99.94%, RT: 3.86 minutes.

[0345] Example 4

[0346] Cytotoxicity 50 assay protocol (CC50)

[0347] Preparation

[0348] MB200 cells were grown to 80% confluence in F10 medium supplemented with rhFGF basic (10 ng / ml), 15% fetal bovine serum, 1% penicillin-streptomycin, 1% amphotericin B, and 1 μM dexamethasone. Then, adherent MB200 cells grown on a 10 cm plate were trypsinized with 1 ml of trypsin. Once the cells were detached from the culture plate, F10 medium (10 mL) was added, and the cells were filtered through a 70 μm cell strainer.

[0349] Cells were counted using a mixture of 10 μL of cell suspension and 10 μL of trypan blue. After counting the total number of cells, a dilution of 200 k / mL was prepared, and cells were seeded at 10 k cells / well in a 96-well plate in triplicate, ensuring to avoid seeding cells at the plate edges to avoid edge effects. Using a multi-channel pipette, 200 k / mL cell dilution (50 μL / well) was added to wells B2 - B11, C2 - C11, and D2 - D11 in triplicate.

[0350] Cell treatment with inhibitor

[0351] In a deep well block (DWB), medium for the appropriate cell line (450 μL / well) was added to wells 1 - 10. A 10 mM drug solution (13.5 μL) was added to DWB well 2, and then an additional volume of medium (225 μL) was added. The well was pipetted up and down four times. A portion (225 μL) was transferred to well 3, and the process was repeated until well 10. Using a multi-channel pipette, a smaller portion (50 μL / well) was taken from all wells on the DWB and then dispensed on top of each row of cells. After adding the drug, the plate was incubated at 37 °C for 72 hours.

[0352] Reading of light intensity

[0353] Using a multi-channel pipette, add pre-warmed Cell Titer-Glo reagent (Promega) (100 μL) to each well. After 5 minutes, measure the total luminescence using a luminometer. Determine the CC50 using a GraphPad Prism template.

[0354] Example 5

[0355] EC 50 Protocol

[0356] Preparation of transfection complex

[0357] This example provides a representative protocol for one well on a 96-well plate. It can be scaled up as needed for handling multiple wells.

[0358] For the negative control, use wells containing reporter DNA, Renilla DNA, and Turbofect (without the expression vector). For the positive control, use wells containing cells that have not been treated with any inhibitor. Transfect them with the complete DNA-lipid complex as described below.

[0359] Prepare the DNA-lipid complex for one well on a 96-well plate by adding 100 ng of the transfection factor (TF) expression vector, 100 ng of TF reporter DNA, 10 ng of Renilla reporter DNA, 0.4 μL of Turbofect transfection reagent, and 25 μL of medium without any serum or penicillin / streptomycin to a test tube. Gently shake the test tube to mix the mixture, then transfer the mixture to the well on the 96-well plate. Then gently tap the plate to evenly distribute the mixture on the bottom of the well. Incubate the well for 30 minutes.

[0360] Cell preparation

[0361] Culture HEK293 cells to 80% confluence in DMEM supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% amphotericin B. Digest the adherent cells growing on a 10 cm plate with 1 mL of pre-warmed trypsin. Once the cells are detached from the plate, add 10 mL of medium and filter the cells through a 70 μm cell strainer.

[0362] Count the cells using a mixture of 10 mL of cell suspension and 10 mL of trypan blue. After counting the total number of cells, prepare a dilution of 15, k / mL.

[0363] Transfection

[0364] After the transfection complex incubation was completed, 155 mL of HEK293s (total cell number 23.25 k) was gently dispensed into the wells containing 25 mL of the transfection complex to give a total volume of approximately 180 mL. The mixture was then incubated for 24 hours.

[0365] Cell treatment with inhibitor

[0366] To treat the cells with each inhibitor, the inhibitors were prepared in the same growth medium. In deep well blocks, regular medium containing serum was added to well 1 (135 mL) and wells 2 to 10 (100 mL). 15 mL of 10 nM inhibitor was added to the first well. The final concentration of the drug in well 1 was 1 nM. Using a pipette, aspirate up and down in the well and transfer 50 mL from well 1 to well 2 (1 / 3 dilution). Mix the well and repeat the serial dilution with the remaining wells. After the serial dilution was completed, 20 mL of each inhibitor was transferred to the designated wells in a 96-well plate using a multi-channel pipette. After the inhibitor was added, the cells were incubated at 37 °C for 24 hours.

[0367] Measurement of relative luciferase activity using luciferase assay system

[0368] Before the assay, a sufficient amount of 1x passive lysis buffer was prepared. The luciferase substrate buffer (Promega) was completely thawed and then mixed with the lyophilized luciferase substrate. The stop buffer (Promega) was also completely thawed.

[0369] (1) Cell lysis: After the cell treatment was completed, the 96-well plate was inverted and tapped, dried on a dry paper towel to remove all cell culture medium. Immediately thereafter, 1x passive lysis buffer (25 μL) was added. The plate was then placed on a rocker at medium speed for 15 minutes.

[0370] (2) Measuring luciferase signal: The luciferase substrate solution for all wells was placed in a solution basin. Then, using a multi-channel pipette, the luciferase substrate solution was added to each well (100 μL / well). Then immediately read the total luminescence using a plate reader.

[0371] (3) Measuring Renilla signal: Buffer was mixed with 50X stop solution substrate (Promega) to make a final 1x solution to prepare "complete stop solution" in a solution basin. Using a multi-channel pipette, the complete stop solution was added to each well (50 μL / well). Then immediately read the total luminescence using a plate reader.

[0372] Measurement of relative luciferase units

[0373] Using a spreadsheet such as Excel, divide the luciferase signal by the renilla signal to obtain the relative luciferase units.

[0374] Example 6

[0375] Synthesis and Activity of Exemplary Compounds

[0376] Table 6-1 shows other exemplary compounds prepared according to the methods of the foregoing examples and the activities of the compounds.

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407] Legend: A => 10 mM; B = 1 to 10 mM; C = < 1 mM

[0408] Example 7

[0409] Protocol for RNA isolation and qPCR to measure MBD3L2 RNA levels

[0410] This protocol describes the process of isolating RNA from cultured cells, converting the RNA into complementary DNA (cDNA), and measuring the expression of target genes using quantitative polymerase chain reaction (qPCR).

[0411] Cell treatment with compound

[0412] Seed FSHD cells were dispensed into six-well culture dishes. One day after seeding, DUX4 inhibitor (11 μM or 3.6 μM / well) was added to the cells. After another 48 hours, the old medium was removed from the cells. The cells were washed with 2 mL of warm phosphate-buffered saline (PBS).

[0413] The PBS was aspirated, and the cells in each well were lysed with 350 μL of RLT buffer + 3.5 μL of β-mercaptoethanol (BME). The lysate was added to a fresh, RNase-free, labeled Eppendorf tube. Then, the cells were physically disrupted at level 4 of a bead beater for 60 minutes to ensure the release of RNA.

[0414] RNA collection

[0415] RNA was collected according to the standard procedure described below (i.e., Qiagen kit instructions).

[0416] Add 350 μl of 70% ethanol to each sample in a separate Eppendorf tube to precipitate the RNA. Mix each suspension by pipetting up and down.

[0417] Transfer the solution to a pink spin column (700 μL). Transfer up to 700 μL of the sample (including any precipitate formed) to an RNeasy spin column placed in a 2 mL collection tube. Centrifuge the tube at 13K RPM for 15 seconds and discard the flowthrough.

[0418] Add buffer RW1 (700 μL) to the RNeasy spin column. Centrifuge the tube at 13K RPM for 15 seconds and discard the flowthrough.

[0419] Add buffer RPE (500 μL) to the RNeasy spin column. Centrifuge the tube at 13K RPM for 15 seconds and discard the flowthrough.

[0420] Add an additional buffer RPE (500 μL) to the RNeasy spin column. Centrifuge the tube at 13K RPM for 15 seconds and discard the flowthrough.

[0421] Place the column in a fresh collection tube and centrifuge at 13K RPM for 2 minutes.

[0422] Then elute and collect the RNA from the column. Add water (30 μL) to the center of the column and incubate at room temperature for 5 minutes. Then centrifuge the column at 14K RPM for 1 minute, and then measure the RNA concentration.

[0423] RNA concentration measurement

[0424] Use 2 μL of nuclease-free water to calibrate the NanoDrop measurements as a blank. Add the RNA sample (2 μL) to the NanoDrop and measure the RNA concentration. Once the concentration is determined, prepare the sample for reverse transcription.

[0425] Reverse transcription

[0426] Normalize the RNA concentration to 200 μg / μL. Dilute the RNA (2 μg) in a new Eppendorf tube to 200 μg / μL RNA with RNase-free water in a PCR tube to a final volume of 9.5 μL.

[0427] Remove any DNA contamination with DNase. The DNase master mix was made as follows: 1.5 μL / reaction of DNase solution (1 unit / μL, Promega), 3 μL / reaction of 5X RT buffer (i.e., 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2 and 500 μL of 0.1 M DTT; Promega MMLV), and 1 μL / reaction of RNase inhibitor solution (RNasin, 40 units / μL, Promega). Add the DNase master mix to the normalized RNA samples (5.5 μL / sample). Mix the samples by vortexing and centrifuge briefly. Heat the samples to 37 °C for 60 minutes, 80 °C for 5 minutes, and then cool to 4 °C. Store the samples on ice until the next batch of reagents is added.

[0428] The RT reaction master mix was made as follows: 5 μL / reaction of 5X RT buffer, 2 μL / reaction of dNTP solution (2.5 mM each nucleotide), 1 μL / reaction of ribonuclease (RNase) inhibitor solution (RNasin, 40 units / μL, Promega), 1.6 μL / reaction of M-MLV reverse transcriptase (200 units / μL, Promega), and 13.4 μL / reaction of deionized, RNase-free water. Cool the RT reaction master mix on ice until use.

[0429] Add 50 μM of random primer 6 random hexanucleotide solution (2 μL) to each sample. Mix the samples by vortexing and centrifuge briefly. Heat the samples to 70 °C for 5 minutes and then cool to 4 °C.

[0430] Add the RT reaction master mix (23 μL) to each sample. Mix the samples by vortexing and centrifuge briefly. To convert the RNA to cDNA, heat the samples to 42 °C for 60 minutes, 95 °C for 5 minutes, and then cool to 4 °C. Dilute the cDNA product mixture 5:1 with deionized water (40 μL + 160 μL).

[0431] Running qPCR

[0432] Detect the samples in triplicate with methyl-CpG-binding domain protein 3-like 2 (MBD3L2) target primers and eukaryotic translation elongation factor 1-alpha (EEF1A) control primers.

[0433] Prepare qPCR master mix for each primer and store it on ice (4 °C) until use. The qPCR master mix includes: 10 μL / reaction SYBR Green Mix (2X) (ThermoFisher), 1 μL / primer mix (i.e., MBD3L2 PCR primer mix or EEF1A primer mix), and 4 μL / RNase-free deionized water.

[0434] To set up the qPCR plate, add 15 μL of the appropriate qPCR master mix to the appropriate wells for each target / primer. After adding all the targets / primers to the wells, add 5 μL of the sample to the corresponding wells to make the total volume in each well 20 μL. Cover the plate with a transparent paper.

[0435] Set up the qPCR run (QuantStudio 5) with the following cycles:

[0436] Hold stage (1 cycle): 50 °C – 2 min (1.6 °C / s); 95 °C – 10 min (1.6 °C / s)

[0437] PCR stage (for 50 cycles): 95 °C – 15 s (1.6 °C / s) denaturation; 60 °C – 1 min (1.6 °C / s) amplification

[0438] All publications and patent applications cited in this specification are hereby incorporated by reference into this application as if each individual publication or patent application were specifically and individually recited as being incorporated by reference. Although the claimed subject matter has been described in accordance with various embodiments, those skilled in the art will understand that various modifications, substitutions, omissions, and changes can be made without departing from its spirit. Accordingly, the scope of this application is limited only by the scope of the appended claims, including their equivalents.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof: Wherein: Each R 1 is independently selected from H and R 2 ; m is an integer from 0 to 4; The wavy bond represents a single bond connection from L to the free site of the thiazole or benzothiazole ring; L 1 Selected from a single bond, C 1-6 alkyl, and -(C=O)-; L 2 Selected from -(C=O)(NR 3 )-, -(C=O)alkyl-, and -(NR 3 )(C=O)-; C y Selected from C 3-9 cycloalkyl, C 3-9 heterocyclic group, C3-C9 heteroaryl and C 6-10 aryl; Each R 2 is independently selected from halogen, C 1-3 alkoxy, C 1-3 alkyl, cyano and R 5 ; n is an integer from 0 to 2; Each R 3 is independently selected from H and C 1-3 alkyl; R 4 is C 1-6 alkylene, and R 4 is substituted with 0 to 4 R 7 groups; R 5 selected from -O(CO)R 6 、-N(R 3 )(CO)R 3 、-N(R 3 )(CO)R 6 、-OR 6 、-(CO)R 6 、-(CO)N(R 3 )R 3 、-(CO)N(R 3 )R 6 、C 3-7 cycloalkyl, C 3-9 heterocyclic group, C 6-10 aryl and C3-C9 heteroaryl; Alternatively, R 4 and R 5 are linked together to form a 5- to 8-membered cycloalkyl or heterocyclic group, which may be substituted with 0 to 4 R 7 groups; Each R 6 is independently selected from C 1-6 alkyl, C 3-7 cycloalkyl, C 3-9 heterocycloalkyl, C 6-10 aryl, and C3-C9 heteroaryl; R 6 is substituted with 0 to 4 R 7 groups; Each R 7 is independently selected from halogen, C 1-3 alkoxy, and C 1-3 alkyl.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound has formula II: Wherein: Y is selected from CH, CR 2 , -N=CH-, -N=CR 2 -, O, S and N; wherein Y and Z 1 are not both N at the same time; Z 1 selected from CH, CR 2 , -N=CH-, -N=CR 2 -, and N; wherein Y and Z 1 are not simultaneously N.

3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound has formula III: Wherein: Z 1 and Z 2 each independently selected from CH, CR 2 or N; provided that Z 1 and Z 2 are not both N at the same time.

4. The compound or a pharmaceutically acceptable salt thereof according to claim 3, wherein the compound has formula IV: Wherein: R 1 selected from H, a halogen or C 1-3 alkyl; Each R 2 is independently selected from halogen, C 1-3 alkoxy, and C 1-3 alkyl; R 4 is C 1-6 an alkylene group; R 5 Selected from -O(CO)R 6 、-NH(CO)R 6 、-OR 6 、-(CO)R 6 、C 3-7 cycloalkyl and C 3-9 heterocyclic group; R 6 selected from C 1-6 alkyl, C 3-7 cycloalkyl, C 3-9 heterocyclic group, and C 3-9 heteroaryl.

5. The compound according to claim 3 or 4, wherein Z 1 and Z 2 are each independently selected from CH and N.

6. The compound according to any one of claims 1-5, wherein R 1 is H or methyl.

7. The compound according to any one of claims 1-6, wherein n is 0.

8. The compound according to any one of claims 1-7, wherein R 4 is substituted with zero R 2 groups.

9. The compound according to any one of claims 1-8, wherein R 5 is selected from -NH(CO)CH3, -O(CO)CH3, -(CO)CH3 and -OCH2CH3.

10. The compound according to claim 1, wherein the compound is selected from:

11. The compound according to claim 10, wherein the compound is selected from:

12. The compound according to claim 1, wherein the compound is selected from the compounds in Table 6-1.

13. The compound according to claim 12, wherein the compound inhibits the production of MBD3L2 RNA at 11 mM.

14. A compound according to claim 12 or 13, wherein the compound has a DUX4 EC 50 less than 10 mM.

15. The compound according to claim 14, wherein the compound DUX4 EC 50 is less than 1 mM.

16. A pharmaceutical composition comprising: the compound according to any one of claims 1-15, and a pharmaceutically acceptable excipient, carrier or diluent.

17. The pharmaceutical composition according to claim 16, wherein the composition is an oral preparation.

18. A method of treating a patient, comprising administering an effective therapeutically amount of the compound or composition according to any one of claims 1-17.

19. The method according to claim 18, wherein the patient is a human.

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