Compounds useful for treating and preventing muscle wasting and other conditions

Abstract

The present invention relates to compounds of general formula I and pharmaceutically acceptable salts thereof; wherein the variables are as defined in the claims and the description. The present invention also relates to compounds of formula I for use as medicaments; pharmaceutical compositions comprising at least one compound of formula I, and methods for treating said conditions.

Description

Background of the Invention

[0002] The present invention relates to novel substituted 4-[2-oxo-chromen-7-yl)heteromethyl]benzoic acid compounds, in particular compounds of general formula I as described herein. Said compounds have important therapeutic properties and are suitable for the treatment and prevention of muscle wasting conditions, and for the treatment or prevention of myopathies, in particular myopathies associated with increased expression of MuscleRING Finger 1 (MuRF1).

[0003] Muscle wasting and weakness are debilitating complications observed in a variety of clinical conditions, including psychogenic cachexia, myocardial infarction, hypertension, cancer, cancer treatment with certain drugs, diabetes, kidney disease and renal failure, hereditary muscle wasting, and severe infections, but can also be observed in some medical conditions not related to a specific disease, such as immobilization, long-term mechanical ventilation, long-term weightlessness, or old age. Muscle wasting is characterized by a loss of muscle mass, which occurs in the limbs and respiratory muscles. Muscle wasting often leads to worse symptoms and prognosis, and prolongs recovery time.

[0004] The mechanisms underlying muscle atrophy are not fully understood. It is known that muscle atrophy involves the activation of so-called Atrogins, which promote the degradation of muscle proteins via the autophagosome and the ubiquitin-proteasome system (UPS).

[0005] Unfortunately, there are only a few reliable known strategies such as exercise training to prevent muscle protein loss caused by the above clinical conditions. The known strategies are generally aimed at downregulating or inactivating the expression of Atrogins and / or inhibiting their ubiquitination activity.

[0006] For example, WO2015 / 010107 A1 describes the use of small molecules to reduce muscle weakness and / or muscle atrophy and / or cachexia by inhibiting inflammatory cytokine-induced (IL-6-induced) tyrosine phosphorylation of signal transducer and activator of transcription 3 (Stat 3). Inhibition of Stat 3 phosphorylation prevents the increase in myostatin expression, which is known to prevent muscle growth by upregulating the expression of specific Atrogins.

[0007] Another promising strategy to prevent muscle protein loss is to inhibit the function of muscle RING finger 1 (MuRF1). MuRF1 is a muscle-specific ubiquitin E3 ligase that is considered to be a key step in transferring polyubiquitinated muscle proteins to the ubiquitin proteasome system for degradation. In many clinical situations, its expression is closely associated with muscle atrophy, while its genetic inactivation confers partial resistance to muscle wasting conditions. Numerous studies in the past 15 years have shown that many muscle-related diseases, i.e. myopathies such as critical illness myopathy, nemaline myopathy, chronic inflammatory myopathy, diabetes or pulmonary artery hypertension-induced myopathy (which leads to muscle weakness), may be associated with upregulated MuRF1 expression.

[0008] Hooijman et al., Am. J. Respir. Crit. Care. Med., 2015, 191(10), 1126-1138 describe a link between MuRF1 expression and diaphragm muscle fiber force loss and muscle protein degradation, which is often observed in critically ill patients receiving mechanical ventilation (critical illness myopathy). They observed that the muscle fiber force loss was attenuated in MuRF1-KO mice receiving mechanical ventilation, thus revealing a link between critical illness myopathy and MuRF1 expression.

[0009] Li and Granzier, Hum. Mol. Genet., Sep 15, 2015, 24(18), 5219-5233 describe a mouse model of nematode myopathy in which muscles rich in glycolytic fibers upregulate proteolytic pathways including MuRF-1 and undergo malnutrition, resulting in a reduced cross-sectional area (CSA), which exacerbates their strength deficits. Therefore, MuRF1 inhibition is predicted to protect myofiber CSA and myofiber force in nematode myopathy, particularly in muscle types rich in glycolytic fibers, such as the quadriceps.

[0010] Adams et al., J. Mol. Biol., 2008, 384, 48–59 describe that intraperitoneal injection of tumor necrosis factor-α (TNF-α) in C57B16 mice reduced soleus muscle force development at 150 Hz by 25%. In MuRF-1 knockout animals, TNF-α-induced muscle force loss was attenuated. Therefore, MuRF1 inhibition is expected to be useful in chronic inflammatory states where TNF-α levels are elevated.

[0011] De Man et al., Am. J. Respir. Crit. Care. Med., May 15, 2011, 183(10), 1411-1418 describe that the CSA of diaphragm fibers in rats with pulmonary hypertension (PH) was significantly less than that in controls. Consistent with the rat data, studies in PH patients showed a significant decrease in CSA and impaired contractile capacity of diaphragm fibers compared with control individuals. They further found that the decrease in diaphragm fiber CSA was associated with increased expression of the E3-ligases MAFbx and MuRF-1.

[0012] To date, there are a limited number of therapeutic approaches aimed at inhibiting MuRF1 function.

[0013] For example, Castillero et al., Metabolism, 2013, 62, 1495-1502 describe adenoviral knockout of Atrogin-1 and MuRF1, which can prevent dexamethasone-induced atrophy of cultured L6 myotubes.

[0014] Eddins et al., Cell Biochem. Biophys., 2011, 60, 113-118 describe targeted inhibition of MuRF1 ubiquitination by muscle-specific small molecules, which can significantly reduce muscle atrophy in a cellular atrophy model.

[0015] Although such therapeutic studies have shown that small molecule inhibition of MuRF1 function can prevent myotube atrophy, this approach has not yet translated to an in vivo setting, such as in a clinically relevant animal model. Thus, there remains a substantial unmet need in the art to provide compounds and methods for treating and / or preventing muscle wasting conditions.

[0016] Brief description of the invention

[0017] It is an object of the present invention to provide further small molecules which are suitable for the treatment and / or prevention of muscle wasting conditions or conditions which can be alleviated by improving muscle condition.

[0018] The above-mentioned and other objects are achieved by the following compounds of general formula I and their pharmaceutically acceptable salts:

[0019]

[0020] in

[0021] R 1 is hydrogen or the group -CH2R 1a , where R 1aselected from hydrogen, C1-C3-alkyl, phenyl, wherein phenyl is unsubstituted or may carry 1, 2 or 3 radicals independently selected from halogen, cyano, C1-C3-alkyl, C1-C3-haloalkyl and C1-C3-alkoxy; and containing as ring members independently selected from N, NR c , O and S 1 to 4 heteroatoms or hetero groups, wherein the 5- to 10-membered heteroaromatic ring is unsubstituted or may carry 1, 2 or 3 R 7 Group;

[0022] R 2 is hydrogen, methyl or fluorinated methyl;

[0023] R 3 is hydrogen, methyl or fluorinated methyl;

[0024] R 4 is hydrogen or C1-C4-alkyl;

[0025] R 5 each independently selected from halogen, cyano, C1-C3-alkyl, C1-C3-chloroalkyl, C1-C3-alkoxy and C1-C3-haloalkoxy;

[0026] R 6 each independently selected from halogen, cyano, C1-C3-alkyl, C1-C3-chloroalkyl, C1-C3-alkoxy and C1-C3-haloalkoxy;

[0027] R 7 each independently selected from halogen, cyano, C1-C3-alkyl, C1-C3-chloroalkyl, C1-C3-alkoxy and C1-C3-haloalkoxy;

[0028] X 1 YesNR a or O;

[0029] X 2 YesNR b , O or S;

[0030] Y represents an oxygen atom (so that C=Y is C=O) or two hydrogen atoms (so that C=Y is CH2);

[0031] R a , R b , R c are each independently selected from hydrogen and C1-C4-alkyl;

[0032] a is 0, 1, 2, 3 or 4; and

[0033] b is 0, 1, 2, or 3.

[0034] Therefore, the present invention relates to compounds of general formula I and pharmaceutically acceptable salts of compounds of general formula I.

[0035] In view of the pharmacological properties of compound I, the present invention also relates to a compound of formula I or a pharmaceutically acceptable salt thereof for use as a medicament.

[0036] In cell-based assays and clinically relevant animal models, the new compounds were found to reduce muscle atrophy and contractile dysfunction.

[0037] Therefore, the present invention relates to a compound of formula I or a pharmaceutically acceptable salt of a compound of formula I for use in treating or preventing muscle wasting conditions.

[0038] The present invention particularly relates to a compound of formula I or a pharmaceutically acceptable salt of a compound of formula I for use in treating or preventing skeletal muscle or cardiac muscle atrophy caused by one of the following diseases or conditions: congestive heart failure, chronic heart failure, cancer, cancer treatment with muscle toxic substances, congenital myopathy, AIDS, chronic obstructive pulmonary disease (COPD), chronic kidney disease, renal failure, diabetes, severe burns, muscle loss during aging, reduced blood supply, temporary or long-term immobilization, long-term mechanical ventilation, denervation, long-term weightlessness and malnutrition.

[0039] In addition, the compounds of the present invention are effective inhibitors of MuRF1 function by inhibiting the ubiquitin E3 ligase activity of MuRF1 and the binding of MuRF1 to target muscle proteins such as titin. Therefore, the present invention further relates to compounds of formula I or pharmaceutically acceptable salts of compounds of formula I for use in treating or preventing conditions associated with increased expression of muscle RING finger 1 (MuRF1), in particular myopathies associated with increased expression of MuRF1.

[0040] It has also been found that the compounds of general formula I have beneficial effects on cardiac conditions associated with systolic or diastolic dysfunction, which are not directly related to their effects on muscle conditions. Therefore, the present invention also relates to compounds of general formula I or pharmaceutically acceptable salts thereof for use in the treatment or prevention of cardiac conditions associated with systolic or diastolic dysfunction.

[0041] The compounds of general formula I have also been found to have beneficial effects on diabetes. Therefore, the present invention also relates to compounds of general formula I or pharmaceutically acceptable salts thereof for use in treating or preventing diabetes, especially type II diabetes.

[0042] The present invention also relates to a medicament comprising at least one compound of the formula I or at least one pharmaceutically acceptable salt of a compound of the formula I as described herein.

[0043] The present invention also relates to a pharmaceutical composition comprising a compound selected from the compounds of formula I as described herein, or at least one pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.

[0044] The present invention also relates to the use of a compound selected from the compounds of general formula I defined herein or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating or preventing muscle wasting conditions; or for the preparation of a medicament for treating or preventing the above conditions or patients.

[0045] The present invention also relates to a method for the treatment or prevention of one of the above mentioned conditions or disorders, said method comprising the step of administering to a subject in need thereof a compound selected from the compounds of general formula I as defined herein, or a pharmaceutically acceptable salt thereof. DETAILED DESCRIPTION OF THE INVENTION

[0047] The terms "compound of formula I" and "compound I" are used synonymously.

[0048] The term "pharmaceutically acceptable salt" refers to a cationic or anionic salt compound, wherein the counter ion is derived from a pharmaceutically acceptable non-toxic base or acid, including inorganic or organic bases and inorganic or organic acids. It should be understood that as used herein, references to compounds of Formula I are meant to also include pharmaceutically acceptable salts.

[0049] If the compound of formula I is alkaline, the salt can be prepared from pharmaceutically acceptable non-toxic acids (including inorganic and organic acids). The pharmaceutically acceptable acid addition salts include, but are not limited to, acetic acid, trifluoroacetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, etc. Particularly preferred are citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, fumaric acid and tartaric acid.

[0050] If the compound of formula I is acidic, salts can be prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Pharmaceutically acceptable alkali salts include non-toxic salts derived from alkalis, including metal cations such as alkali or alkaline earth metal cations, and amines. Examples of suitable metal cations include lithium, sodium, potassium, magnesium, calcium, zinc and aluminum. Examples of suitable amines include arginine, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethylamine, diethanolamine, dicyclohexylamine, ethylenediamine, glycine, lysine, N-methylglucamine, ethanolamine, 2-amino-2-hydroxymethyl-propane-1,3-diol and procaine. Preferred pharmaceutically acceptable alkali salts are lithium, sodium, potassium, magnesium, calcium, zinc, aluminum and diethanolamine.

[0051] For a discussion of useful acid addition and base salts, see, for example, SM Berge et al., "Pharmaceutical Salts," 66 J. Pharm. ScL 1-19 (1977).

[0052] The term "C1-C4-alkyl" refers to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl. Preferably, C1-C4-alkyl is selected from methyl, ethyl, n-propyl, isopropyl, in particular from methyl and ethyl.

[0053] The term "C1-C3-alkyl" refers to methyl, ethyl, n-propyl or isopropyl. Preferably, C1-C3-alkyl is selected from methyl and ethyl, in particular from methyl.

[0054] The term "halogen" refers to fluorine, chlorine, bromine and iodine. Preferably, halogen is fluorine and chlorine, especially fluorine.

[0055] The term "fluorinated methyl" refers to fluoromethyl, difluoromethyl, trifluoromethyl, preferably trifluoromethyl.

[0056] The term "fluorinated methoxy" refers to fluoromethoxy, difluoromethoxy, trifluoromethoxy, preferably trifluoromethoxy.

[0057] The term "C1-C3-alkoxy" refers to methoxy, ethoxy, n-propoxy and isopropoxy. Preferably, C1-C3-alkoxy is selected from methoxy and ethoxy, in particular from methoxy.

[0058] The term "C1-C3-haloalkyl" refers to a C1-C3-alkyl group as defined above, wherein at least one hydrogen atom, e.g. 1, 2, 3, 4, 5 or 6 hydrogen atoms of the C1-C3-alkyl group has been replaced by a halogen atom, preferably by a chlorine atom or a fluorine atom, in particular a fluorine atom. Examples of C1-C3-haloalkyl include chloromethyl, dichloromethyl, trichloromethyl, 2-chloroethyl, 2,2-dichloroethyl, 2,2,2-trichloroethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,1,2,2-tetrafluoroethyl, pentafluoroethyl, etc. In particular, "C1-C3-haloalkyl" is selected from fluorinated methyl as defined above, and is also selected from trichloromethyl, 2,2,2-trichloroethyl and 2,2,2-trifluoroethyl, in particular from trifluoromethyl.

[0059] The term "C1-C3-haloalkoxy" refers to a C1-C3-alkoxy group as defined above, wherein at least one hydrogen atom, for example 1, 2, 3, 4, 5 or 6 hydrogen atoms of the C1-C3-alkoxy group has been replaced by a halogen atom, preferably a chlorine atom or a fluorine atom, in particular a fluorine atom. Examples of C1-C3-haloalkoxy include chloromethoxy, dichloromethoxy, trichloromethoxy, 2-chloroethoxy, 2,2-dichloroethoxy, 2,2,2-trichloroethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, 2-fluoroethoxy, 2,2-difluoroethoxy, 2,2,2-trifluoroethoxy, 1-fluoroethoxy, 1,1-difluoroethoxy, 1,1,2,2-tetrafluoroethoxy, pentafluoroethoxy and the like. In particular, "C1-C3-haloalkoxy" is selected from the fluorinated methoxy groups defined above and also from the trichloromethoxy, 2,2,2-trichloroethoxy and 2,2,2-trifluoroethoxy groups, especially from the trifluoromethoxy groups.

[0060] The term "5- to 10-membered heteroaromatic ring" refers to a 5- to 6-membered monocyclic aromatic ring or a fused 8- to 10-membered bicyclic aromatic ring, wherein the monocyclic or bicyclic aromatic ring contains 1 to 4 independently selected from N, NR c , O and S heteroatoms or hetero groups, where R c is selected from hydrogen and C1-C4-alkyl. Examples of the 5- to 6-membered monocyclic aromatic ring include thienyl, furanyl, pyrrolyl, furazanyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,4-triazinyl, 1,3,6-triazinyl, etc. Examples of the 8- to 10-membered bicyclic aromatic ring include quinolyl, isoquinolyl, imidazopyridinyl, quinazolinyl, quinoxalinyl, pteridinyl, cinnolinyl, phthalazinyl, naphthyridinyl, indolyl, isoindolyl, azaindolyl, indolizinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, benzofuranyl, isobenzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzoxadiazolyl, benzothiadiazolyl, and the like.

[0061] For its intended use, the variable R in Formula I is 1 , R 2 , R 3 , R 4 , R 5 , R 6 , X 1 , X 2 , Y, R a , R b , R c, a and b have in particular the following meanings, wherein considered individually and in combination with at least one other or all specific embodiments of the compounds of formula I, the variables represent:

[0062] R 1 Preferably it is hydrogen or a group -CH2R 1a , where R 1a selected from hydrogen, methyl, phenyl, and containing as ring members independently selected from N, NR c , O and S 1 to 4 heteroatoms or hetero groups, wherein the 5- to 10-membered heteroaromatic ring is unsubstituted or may carry 1, 2 or 3 R 7 Group, where R c and R 7 has one of the general meanings given above, or in particular has one of the preferred meanings given below.

[0063] More preferably, R 1 Selected from hydrogen and the group -CH2R 1a , where R 1a selected from hydrogen, methyl and a 5- to 6-membered monocyclic heteroaromatic ring containing 1 to 3 heteroatoms, in particular 1 heteroatom, independently selected from N, O and S as ring members, wherein the 5- to 6-membered monocyclic heteroaromatic ring is unsubstituted or carries 1 R 7 Group, where R 7 is selected from halogen, C1-C3-alkyl and C1-C2-alkoxy.

[0064] In particular, R 1 Selected from hydrogen and the group -CH2R 1a , where R 1a An unsubstituted 5- to 6-membered monocyclic heteroaromatic ring selected from hydrogen and containing 1 to 3 heteroatoms, especially 1 heteroatom, independently selected from N, O and S as ring members.

[0065] More specifically, R 1 It is the group -CH2R 1a , where R 1a is an unsubstituted 5- to 6-membered monocyclic heteroaromatic ring containing 1 to 3 heteroatoms independently selected from N, O and S as ring members. In particular, R 1 It is the group -CH2R 1a , where R 1a is an unsubstituted 5- to 6-membered monocyclic heteroaromatic ring containing as ring member 1 heteroatom selected from N, O and S. Very especially, R 1 It is the group -CH2R 1a , where R 1a is furyl, thienyl or pyridyl.

[0066] In another more preferred embodiment, R1 Selected from hydrogen, methyl and groups CH2-I' or CH2-II'

[0067]

[0068] wherein * indicates the point of attachment to the urea nitrogen atom, and wherein

[0069] X 3 YesNR c , O or S;

[0070] c is 0, 1, 2, or 3; and

[0071] R 7 As defined in the text.

[0072] Specifically, R 1 Selected from CH2-I' and CH2-II'

[0073]

[0074] wherein * indicates the point of attachment to the urea nitrogen atom, and wherein R 7 , X 3 and c are as defined herein.

[0075] R 2 Preferably it is hydrogen or methyl. In particular, R 2 It's hydrogen.

[0076] R 3 Preferably it is hydrogen or methyl. In particular, R 3 It's hydrogen.

[0077] R 4 Preferably, it is methyl or ethyl. In particular, R 4 It's methyl.

[0078] Preferably, R 5 Each is independently selected from halogen, C1-C3-alkyl, C1-C2-haloalkyl and C1-C2-alkoxyfluoride. More preferably, R 5 Each is independently selected from halogen, C1-C3-alkyl and C1-C2-alkoxy. Even more preferably, R 5 are each independently selected from fluorine, methyl and trifluoromethyl. In particular, R 5 are each independently selected from fluoro and methyl.

[0079] Preferably, R 6 Each is independently selected from halogen, C1-C3-alkyl, C1-C2-haloalkyl and C1-C2-alkoxyfluoride. More preferably, R 6 Each is independently selected from halogen, C1-C3-alkyl and C1-C2-alkoxy. Even more preferably, R 6are each independently selected from fluorine, methyl and trifluoromethyl. In particular, R 6 are each independently selected from fluoro and methyl.

[0080] X 1 YesNR a Or O. Preferably, X 1 is NH or O.

[0081] Preferably, X 2 is O or S. In particular, X 2 It's O.

[0082] In a preferred embodiment, Y is an oxygen atom. In another preferred embodiment, Y represents two hydrogen atoms.

[0083] Preferably, R a , R b , R c Each is independently selected from hydrogen and C1-C3-alkyl. More preferably, R a , R b , R c are each independently selected from hydrogen and methyl. In particular, R a , R b , R c It's hydrogen.

[0084] Preferably, a is 0, 1, 2 or 3. More preferably, a is 0, 1 or 2. In particular, a is 0.

[0085] Preferably, b is 0, 1 or 2. More preferably, b is 0 or 1. In particular, b is 0.

[0086] In the group of formula CH2-I', the variable X 3 , c and R 7 (if present), considered alone and in combination with at least one other or all variables, has in particular the following meanings:

[0087] Preferably, R 7 Each is independently selected from halogen, C1-C3-alkyl, C1-C2-haloalkyl and C1-C2-alkoxy. More preferably, R 7 Each is independently selected from halogen, C1-C3-alkyl and C1-C2-haloalkyl. Even more preferably, R 7 are each independently selected from fluorine, methyl and trifluoromethyl. In particular, R 7 are each independently selected from fluoro and methyl.

[0088] Preferably, X 3 is O or S. In particular, X 3 is O. In another specific embodiment, X 3 It’s S.

[0089] Preferably, c is 0, 1 or 2. More preferably, c is 0 or 1. In particular, c is 0.

[0090] In the group of formula CH2-II', the variables c and R 7 (if present), considered alone and in combination with at least one other or all variables, has in particular the following meanings:

[0091] Preferably, R 7 Each is independently selected from halogen, C1-C3-alkyl, C1-C2-haloalkyl and C1-C2-alkoxy. More preferably, R 7 Each is independently selected from halogen, C1-C3-alkyl and C1-C2-haloalkyl. Even more preferably, R 7 are each independently selected from fluorine, methyl and trifluoromethyl. In particular, R 7 are each independently selected from fluoro and methyl.

[0092] Preferably, c is 0, 1 or 2. More preferably, c is 0 or 1. In particular, c is 0.

[0093] A preferred embodiment of the present invention relates to compounds of the general formula I, wherein:

[0094] R 1 is hydrogen or the group -CH2R 1a , where R 1a is hydrogen or an unsubstituted 5- to 6-membered monocyclic heteroaromatic ring containing 1 to 3 heteroatoms, in particular 1 heteroatom, independently selected from N, O and S as ring members;

[0095] R 2 is hydrogen or methyl;

[0096] R 3 is hydrogen or methyl;

[0097] R 4 is C1-C2-alkyl;

[0098] R 5 each independently selected from halogen, C1-C3-alkyl, C1-C2-haloalkyl and C1-C2-alkoxy;

[0099] R 6 each independently selected from halogen, C1-C3-alkyl, C1-C2-haloalkyl and C1-C2-alkoxy;

[0100] R 7 each independently selected from halogen, C1-C3-alkyl, C1-C2-haloalkyl and C1-C2-alkoxy;

[0101] X 1 YesNRa or O;

[0102] X 2 is O or S;

[0103] Y represents an oxygen atom or two hydrogen atoms;

[0104] R a is selected from hydrogen and C1-C3-alkyl;

[0105] a is 0, 1, 2, or 3; and

[0106] b is 0, 1, or 2;

[0107] and pharmaceutically acceptable salts thereof.

[0108] A more preferred embodiment of the present invention relates to compounds of formula I, wherein:

[0109] R 1 is hydrogen or the group -CH2R 1a , where R 1a is hydrogen or an unsubstituted 5- to 6-membered monocyclic heteroaromatic ring containing 1 heteroatom selected from N, O and S as a ring member;

[0110] R 2 is hydrogen or methyl;

[0111] R 3 It is hydrogen;

[0112] R 4 It is methyl;

[0113] X 1 is NH or O;

[0114] X 2 It is O;

[0115] Y represents an oxygen atom or two hydrogen atoms;

[0116] a is 0; and

[0117] b is 0;

[0118] and pharmaceutically acceptable salts thereof.

[0119] A specific embodiment of the present invention relates to compounds of formula I, wherein:

[0120] R 1 It is the group -CH2R 1a , where R 1a is an unsubstituted 5- to 6-membered monocyclic heteroaromatic ring containing as a ring member 1 heteroatom selected from N, O and S;

[0121] R 2 It is hydrogen;

[0122] R 3 It is hydrogen;

[0123] R 4 It is methyl;

[0124] X 1 is NH or O;

[0125] X 2 It is O;

[0126] Y represents an oxygen atom or two hydrogen atoms;

[0127] a is 0; and

[0128] b is 0;

[0129] and pharmaceutically acceptable salts thereof.

[0130] Another preferred embodiment of the present invention relates to compounds of general formula I, wherein:

[0131] R 1 is hydrogen, methyl or a group CH2-I' or CH2-II',

[0132] R 2 is selected from hydrogen and methyl;

[0133] R 3 is selected from hydrogen and methyl;

[0134] R 4 is C1-C2-alkyl;

[0135] R 5 each independently selected from halogen, C1-C3-alkyl, C1-C2-haloalkyl and C1-C2-alkoxy;

[0136] R 6 each independently selected from halogen, C1-C3-alkyl, C1-C2-haloalkyl and C1-C2-alkoxy;

[0137] R 7 each independently selected from halogen, C1-C3-alkyl, C1-C2-haloalkyl and C1-C2-alkoxy;

[0138] X 1 YesNR a or O;

[0139] X 2 is O or S;

[0140] X 3 is O or S;

[0141] Y represents an oxygen atom or two hydrogen atoms;

[0142] R a is selected from hydrogen and C1-C3-alkyl;

[0143] a is 0, 1, 2, or 3;

[0144] b is 0, 1, or 2; and

[0145] c is 0, 1, or 2;

[0146] and pharmaceutically acceptable salts thereof.

[0147] Another more preferred embodiment of the present invention relates to compounds of formula I, wherein

[0148] R 1 is hydrogen, methyl or a group CH2-I' or CH2-II',

[0149] R 2 is hydrogen or methyl;

[0150] R 3 It is hydrogen;

[0151] R 4 It is methyl;

[0152] R 5 are each independently selected from halogen, C1-C3-alkyl and C1-C2-alkoxy;

[0153] R 6 are each independently selected from halogen, C1-C3-alkyl and C1-C2-alkoxy;

[0154] R 7 are each independently selected from halogen, C1-C3-alkyl and C1-C2-alkoxy;

[0155] X 1 is NH or O;

[0156] X 2 It is O;

[0157] X 3 is O or S;

[0158] Y represents an oxygen atom or two hydrogen atoms;

[0159] a is 0, 1, or 2;

[0160] b is 0 or 1; and

[0161] c is 0 or 1;

[0162] and pharmaceutically acceptable salts thereof.

[0163] An even more preferred embodiment of the present invention relates to compounds of the general formula I, wherein

[0164] R 1 is hydrogen, methyl or a group CH2-I' or CH2-II',

[0165] R 2 is hydrogen or methyl;

[0166] R 3 It is hydrogen;

[0167] R 4 It is methyl;

[0168] R 5 are each independently selected from halogen, C1-C3-alkyl and C1-C2-alkoxy;

[0169] R 6 are each independently selected from halogen, C1-C3-alkyl and C1-C2-alkoxy;

[0170] R 7 are each independently selected from halogen, C1-C3-alkyl and C1-C2-alkoxy;

[0171] X 1 is NH or O;

[0172] X 2 It is O;

[0173] X 3 is O or S;

[0174] Y represents an oxygen atom or two hydrogen atoms;

[0175] a is 0;

[0176] b is 0; and

[0177] c is 0;

[0178] and pharmaceutically acceptable salts thereof.

[0179] Another specific embodiment of the present invention relates to compounds of formula I, wherein

[0180] R 1 is a group CH2-I' or CH2-II',

[0181] R 2 It is hydrogen;

[0182] R 3 It is hydrogen;

[0183] R 4 It is methyl;

[0184] X1 is NH or O;

[0185] X 2 It is O;

[0186] X 3 is O or S;

[0187] Y represents an oxygen atom or two hydrogen atoms;

[0188] a is 0;

[0189] b is 0; and

[0190] c is 0;

[0191] and pharmaceutically acceptable salts thereof.

[0192] Another group of preferred embodiments relates to compounds of the general formula I, in which the group CH2-I',

[0193] X 3 is O or S;

[0194] R 7 are each independently selected from halogen, C1-C3-alkyl, C1-C2-haloalkyl and C1-C2-alkoxy; and

[0195] c is 0, 1, or 2.

[0196] Another preferred embodiment relates to compounds of the general formula I, in which the group CH2-II',

[0197] R 7 are each independently selected from halogen, C1-C3-alkyl, C1-C2-haloalkyl and C1-C2-alkoxy; and

[0198] c is 0, 1, or 2.

[0199] An even more preferred group of embodiments relates to compounds of the general formula I, in which the group CH2-I',

[0200] X 3 is O or S;

[0201] R 7 are each independently selected from fluoro, methyl or trifluoromethyl; and

[0202] c is 0 or 1.

[0203] An even more preferred group of embodiments relates to compounds of the general formula I, in which the group CH2-II',

[0204] R 7 are each independently selected from fluoro, methyl or trifluoromethyl; and

[0205] c is 0 or 1.

[0206] A particularly preferred embodiment of the present invention relates to compounds of formula I, which correspond to formula IA,

[0207]

[0208] in

[0209] X 1 is NH or O; and

[0210] X 3 is O or S;

[0211] and pharmaceutically acceptable salts thereof.

[0212] Another particularly preferred embodiment of the present invention relates to compounds of formula I, which correspond to formula IB,

[0213]

[0214] in

[0215] X 1 is NH or O;

[0216] and pharmaceutically acceptable salts thereof,

[0217] Another particularly preferred embodiment of the present invention relates to compounds of formula I, which correspond to formula IC,

[0218]

[0219] in

[0220] X 1 is NH or O; and

[0221] X 3 is O or S;

[0222] and pharmaceutically acceptable salts thereof.

[0223] Another particularly preferred embodiment of the present invention relates to compounds of formula I, which correspond to formula ID,

[0224]

[0225] in

[0226] R 1 is selected from hydrogen or methyl; and

[0227] X 1 is NH or O;

[0228] and pharmaceutically acceptable salts thereof.

[0229] A particularly preferred compound of formula I is 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid [2-(2-furanylmethylcarbamoyl-amino)-2-oxo-ethyl] ester (i.e., compound I, wherein R 1 It is the group -CH2R 1a , where R 1a is furan-2-yl, R 2 Yes H, R 3 Yes H, R 4 is methyl, X 1 It's O, X 2 is O, Y is O, a is 0 and b is 0), and pharmaceutically acceptable salts thereof. The compound is also referred to hereinafter as MyoMed-946.

[0230] Another particularly preferred compound of formula I is N-[2-(2-furanylmethylcarbamoylamino)-2-oxo-ethyl]-4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzamide (ie, compound I, wherein R 1 It is the group -CH2R 1a , where R 1a is furan-2-yl, R 2 Yes H, R 3 Yes H, R 4 is methyl, X 1 Yes NH, X 2 is O, Y is O, a is 0 and b is 0), and pharmaceutically acceptable salts thereof.

[0231] Another particularly preferred compound of formula I is 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-benzoic acid [2-oxo-2-(2-thienylmethylcarbamoylamino)ethyl] ester (ie, compound I, wherein R 1 It is the group -CH2R 1a , where R 1a is thiophen-2-yl, R 2 Yes H, R 3 Yes H, R 4 is methyl, X 1 It's O, X 2 is O, Y is O, a is 0 and b is 0), and pharmaceutically acceptable salts thereof.

[0232] Another particularly preferred compound of formula I is 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-N-[2-oxo-2-(2-thienylmethylcarbamoylamino)ethyl]-benzamide (ie, compound I, wherein R 1 It is the group -CH2R 1a , where R 1a is thiophen-2-yl, R2 Yes H, R 3 Yes H, R 4 is methyl, X 1 Yes NH, X 2 is O, Y is O, a is 0 and b is 0), and pharmaceutically acceptable salts thereof.

[0233] Another particularly preferred compound of formula I is 4-[(4-methyl-2-oxo-chromen-7-yl)oxy-methyl]benzoic acid [2-oxo-2-(2-pyridylmethylcarbamoylamino)ethyl] ester (ie, compound I, wherein R 1 It is the group -CH2R 1a , where R 1a is pyridin-2-yl, R 2 Yes H, R 3 Yes H, R 4 is methyl, X 1 It's O, X 2 is O, Y is O, a is 0 and b is 0), and pharmaceutically acceptable salts thereof. The compound is also referred to as MyoMed-203 hereinafter.

[0234] Another particularly preferred compound of formula I is 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-N-[2-(2-thienylmethylcarbamoylamino)ethyl]benzamide (ie, compound I, wherein R 1 It is the group -CH2R 1a , where R 1a is thiophen-2-yl, R 2 Yes H, R 3 Yes H, R 4 is methyl, X 1 Yes NH, X 2 is O, Y represents two hydrogen atoms (ie, C=Y is CH2), a is 0 and b is 0), and pharmaceutically acceptable salts thereof. This compound is also referred to as MyoMed-205 hereinafter.

[0235] Another particularly preferred compound of formula I is 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid (1-methyl-2-oxo-2-ureido-ethyl) ester (ie, compound I, wherein R 1 Yes H, R 2 is methyl, R 3 Yes H, R 4 is methyl, X 1 It's O, X 2 is O, Y is O, a is 0 and b is 0), and pharmaceutically acceptable salts thereof. The compound is also referred to as MyoMed-946-5 hereinafter.

[0236] Another particularly preferred compound of formula I is 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-benzoic acid [1-methyl-2-(methylcarbamoyl-amino)-2-oxo-ethyl] ester (i.e., compound I, wherein R 1 It is the group -CH2R 1a , where R 1a Yes H, R 2 is methyl, R 3 Yes H, R 4 is methyl, X 1 It's O, X 2 is O, Y is O, a is 0 and b is 0), and pharmaceutically acceptable salts thereof. The compound is also referred to as MyoMed-946-8 hereinafter.

[0237] Compound I according to the present invention can be prepared by methods similar to those known in the literature. An important method for preparing the compounds of the present invention is to react a 4-bromoalkyl-substituted benzoic acid methyl ester compound II with a chromene-2-one compound III to form a 2-oxochromene-substituted benzoic acid methyl ester compound IV, which is hydrolyzed to an intermediate benzoic acid compound V to provide, as shown in the flow chart Figure 1 as shown in .

[0238] process Figure 1 :

[0239]

[0240] In process Figure 1 In the above example, the variable R 3 , R 4 , R 5 , R 6 , X 2 , a and b have the meanings as described above.

[0241] In process Figure 1 In step a), the bromide of formula (II) reacts with OH, SH or HNR of compound III under conditions suitable for nucleophilic substitution reaction. b Group (HX 2-group) reaction. The technician is familiar with the reaction conditions required for this type of nucleophilic substitution reaction. Generally, the reaction is carried out in the presence of a base. The appropriate base can be an inorganic base or an organic base. The example of a suitable inorganic base includes an alkali metal carbonate, such as Li2CO3, Na2CO3, K2CO3 or Cs2CO3, an alkali metal hydroxide such as LiOH, NaOH or KOH or a phosphate such as Li3PO4, Na3PO4, K3PO4 or Cs3PO4. The example of a suitable organic base is, for example, a tertiary amine such as trimethylamine, triethylamine, tripropylamine, ethyl diisopropylamine (DIPEA), etc., an alkaline N-heterocyclic compound such as morpholine, pyridine, lutidine, DABCO, DBU or DBN, or an alkoxide compound such as methoxide, ethoxide, propoxide, isopropoxide, butoxide or tert-butoxide such as its sodium or potassium salt.

[0242] In the presence of a strong base, for example an alkali metal hydroxide such as LiOH, NaOH or KOH, Figure 1 In step b), the methyl ester compound of formula (IV) thus obtained is saponified to obtain the benzoic acid compound of formula (V).

[0243] Then, the intermediate benzoic acid compound V is further reacted with the urea compound VIa or VIb to generate the compound I of the present invention, as shown in the flow chart. Figure 2 as shown in .

[0244] process Figure 2 :

[0245]

[0246] In process Figure 2 In the above example, the variable R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , X 2 , Y, R a , a and b have the meanings as described above.

[0247] In process Figure 2 In step c), the benzoic acid intermediate of formula (V) is reacted with a urea compound of formula (VIa) containing a chlorine group or a urea compound of formula (VIa) containing N(H)R a The urea compound of formula (VIb) of the group is reacted to produce the corresponding ester or amide compound of formula I. Figure 2In step c), under conditions suitable for nucleophilic substitution reaction, the reaction of the benzoic acid intermediate of formula (V) and the urea compound of formula (VIa) containing a chloro group is carried out. Those skilled in the art are familiar with the reaction conditions required for such reactions. Usually, the reaction is carried out in the presence of the above-mentioned base to neutralize the acid formed during the reaction. If necessary, the compound (VIa) can be further activated in situ by adding a suitable bromide salt or particularly an iodide salt to the nucleophilic substitution reaction. Suitable bromide salts or iodide salts are, for example, alkali metal bromides or iodides, and tetraalkylammonium bromides or tetraalkylammonium iodides. Examples include sodium bromide, sodium iodide, potassium bromide, potassium iodide, tetrabutylammonium bromide and tetrabutylammonium iodide.

[0248] The process was carried out under conditions suitable for amide bond formation. Figure 2 Step c) the benzoic acid intermediate of formula (V) is reacted with a N(H)R a The reaction of the urea compound of formula (VIb) with the amide bond is carried out by a person skilled in the art. The person skilled in the art is familiar with the reaction conditions required for such reactions. Usually, the formation of the amide bond is carried out in the presence of a coupling agent. Suitable coupling agents (activators) are well known and are, for example, selected from carbodiimides such as EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; also referred to as EDC), DCC (dicyclohexylcarbodiimide) and DIC (diisopropylcarbodiimide); benzotriazole derivatives such as HOAt (1-hydroxy-7-azabenzotriazole), HOBt (1-hydroxybenzotriazole), HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), HBTU ((O-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) and HCTU (1H-benzotriazolium-1-[bis(dimethylamino)methylene]-5-chlorotetrafluoroborate); Derivatized activators such as BOP ((benzotriazol-1-yloxy)-tris(dimethylamino) Hexafluorophosphate), Py-BOP ((benzotriazol-1-yloxy)-tripyrrolidinoyl Hexafluorophosphate) and Py-BrOP (bromotripyrrolidinoyl Hexafluorophosphate) and the like, such as COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylaminomorpholino-carbocation hexafluorophosphate). The above activators can also be used in combination with each other. Usually, the activator is used in at least an equimolar amount relative to the reactant not used in excess. Benzotriazole and The coupling agent is usually used in an alkaline medium. Alternatively, the carboxylic acid intermediate of formula (V) can first be converted into a so-called active ester, which is obtained in a formal sense by reacting a carboxylic acid with an alcohol (e.g., p-nitrophenol, N-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide or OPfp (pentafluorophenol)) that forms an active ester. Then, the active ester reacts with the amine 3 in the presence or absence of a coupling agent.

[0249] The compounds of formula (II) and formula (III) can be purchased or synthesized using methods well known to those skilled in the art.

[0250] Compounds of formula (VIa) and formula (VIb) can be purchased or prepared, for example, according to the process Figure 3 The method described was used for synthesis.

[0251] process Figure 3 :

[0252]

[0253] In process Figure 3 In the above example, the variable R 1 , R 2 and R a With the above meaning, in the process Figure 3 In step d), when Y represents an oxygen atom, the urea compound (VII) reacts with an acyl chloride (VIIIa) or (VIIIb) under nucleophilic acylation conditions. Those skilled in the art are familiar with the reaction conditions suitable for acylation reactions. If Y represents two hydrogen atoms, it is carried out under conditions suitable for nucleophilic substitution reactions. Those skilled in the art are familiar with the reaction conditions required for such reactions. If necessary, the compound (VIIIa) can be further activated in situ as described above. Typically, step d) is carried out in the presence of a base such as a tertiary amine as defined above to quench the hydrochloride or other acid formed during the reaction.

[0254] In certain specific cases, it may be necessary to use appropriate protecting groups to avoid side reactions with other reactive groups that may be present in compounds (VII), (VIIIa) or (VIIIb) and that may participate in or interfere with the reaction. In these cases, an additional deprotection step may be required to remove these protecting groups after the amide bond is formed. Those skilled in the art are familiar with appropriate protecting groups and methods for protecting and deprotecting different substituents using such protecting groups; see, for example, T. Greene and P. Wuts, Protective Groups in Organic Synthesis (3rd ed.) rd ed.)), John Wiley & Sons, NY (1999).

[0255] In addition, compound (VIc) can be prepared from compound (VIa), for example, according to the process Figure 4 Prepared according to the steps described in .

[0256] process Figure 4 :

[0257]

[0258] In process Figure 4 In the above example, the variable R 1 , R 2 and Y have the above meanings. Figure 4 In step e), the urea compound (VIa) is transferred to the azide compound (IX) in the presence of an azide source such as phosphoryl azide, hydrazoic acid or sodium azide, azide or by Staudinger reaction with PPh3 or other phosphorus reagents (as described in Zwierzak A., Phosphorus, Sulfur, and Silicon and the Related Elements (1993), 75: 1-4, 51-54). Typically, step e) is carried out in the presence of a base as defined above, such as a tertiary amine, to quench the hydrochloride formed during the reaction.

[0259] In process Figure 4 In step f), the azide group of compound (IX) is reduced with hydrogen in the presence of a hydrogenation catalyst, or reacted with a hydride to obtain amine compound (VIc). Those skilled in the art are familiar with reaction conditions suitable for such reactions.

[0260] If not otherwise stated, the above reactions are usually carried out in an organic solvent, including aprotic organic solvents such as substituted amides, lactams and ureas, for example dimethylformamide, dimethylacetamide, N-methylpyrrolidone, tetramethylurea; cyclic ethers, for example dioxane, tetrahydrofuran; halogenated hydrocarbons, for example dichloromethane and mixtures thereof, and mixtures thereof with C1-C6-alkanols and / or water.

[0261] The above reaction is usually carried out at a temperature between room temperature and the boiling temperature of the solvent used, depending on the reactivity of the compounds used.

[0262] The reaction mixture is worked up in a customary manner, for example mixed with water, the phases are separated and, if appropriate, the crude product is purified by chromatography. If intermediate products and end products are obtained as solids, they can also be purified by recrystallization or digestion.

[0263] Routine experimentation, including appropriate manipulation of reaction conditions, reagents and synthetic route sequences, protection of any chemical functionality that may be incompatible with the reaction conditions, and deprotection at appropriate points in the reaction sequence of the preparative methods are within the skill of the art.

[0264] As already mentioned above, the compounds of general formula I have been found to be able to reduce muscle atrophy and contractile dysfunction in cell-based assays and clinically relevant animal models.

[0265] The present invention therefore relates to compounds of general formula I as defined above for use in the treatment or prevention of muscle wasting conditions.

[0266] As used herein in connection with muscle wasting conditions and myopathies, the term "treating" refers to the treatment of the symptoms associated with the condition and / or disease, ie, muscle wasting or muscle weakness.

[0267] As used herein, the term "prevention" in relation to muscle wasting conditions and myopathies refers to prophylactic treatment with respect to a disease and / or condition, ie, treatment that prevents or reduces the risk of muscle wasting or muscle weakness.

[0268] As used herein, the term "muscle atrophy" must be understood to refer to a reduction in size (reduction in cross-sectional area) and number of muscle fibers, a visible loss of muscle mass, which is also referred to in the scientific and medical literature as muscle wasting.

[0269] The term "myopathies" as used herein refers to muscle diseases in which muscle fibers do not function properly. This results in muscle weakness. The term "myopathies" as used herein refers specifically to myopathies associated with increased expression of muscle RING finger 1 (MuRF1). However, it is believed that the effects of the compounds of the invention are not limited to this pathway.

[0270] Muscle atrophy is believed to be caused by a change in the balance between muscle protein synthesis and muscle protein degradation. In particular, it is believed that during atrophy, muscle protein degradation pathways such as ubiquitination of muscle protein are activated.

[0271] Muscle wasting is often seen in patients with cachexia, such as cachexia caused by heart disease (psychogenic cachexia). In addition, muscle wasting specifically affects skeletal muscle, causing it to deteriorate (skeletal muscle atrophy). However, muscle wasting can also affect the heart muscle.

[0272] Therefore, a preferred embodiment of the present invention relates to compounds of general formula I as defined above for use in the treatment or prevention of psychogenic cachexia, skeletal muscle atrophy or cardiac muscle atrophy.

[0273] For example, patients with myocardial infarction develop psychogenic cachexia, a wasting of myocardial and skeletal muscle, which often worsens their symptoms and prognosis and can significantly prolong their recovery time.

[0274] Therefore, a particular embodiment of the invention relates to compounds of general formula I as defined above for use in the treatment or prevention of cardiogenic cachexia, skeletal muscle atrophy or cardiac muscle atrophy, wherein the cardiogenic cachexia and cardiac or skeletal muscle atrophy are caused by myocardial infarction.

[0275] A large number of disease-related conditions are known, as well as a number of non-disease-related medical conditions, which can lead to cardiac or skeletal muscle atrophy and / or cachexia.

[0276] Disease-related conditions that can lead to myocardial or skeletal muscle atrophy and / or cachexia are, for example, congestive heart failure, chronic heart failure, myocardial infarction, cancer, congenital myopathies, AIDS, chronic obstructive pulmonary disease (COPD), multiple sclerosis, familial amyloid polyneuropathy, reduced blood supply, hormone deficiencies, chronic kidney disease, renal failure, diabetes, infectious diseases, chronic pancreatitis, and autoimmune diseases.

[0277] Physical conditions that can lead to skeletal muscle atrophy and / or cachexia and are not related to disease are, for example, severe burns, denervation, temporary or prolonged immobilization, prolonged mechanical ventilation, sarcopenia during aging, prolonged weightlessness, malnutrition and drug addiction.

[0278] Some chemotherapeutic drugs used in cancer treatment show cardiotoxicity and myotoxicity. For example, doxorubicin is an effective chemotherapeutic drug used in the treatment of various cancers, and its use is associated with early and chronic cardiotoxicity and myotoxicity (KM Cho et al., Oncotarget, 2017, 8(45), 79441–79452; DS Hydock et al., Characterization of the Effect of In Vivo Doxorubicin Treatment on Skeletal Muscle Function in the Rat, International Journal of Cancer Research and Treatment, 2011, 2028, 2023–2028; TA Nissinen et al., Sci. Rep., 2016, 6, 32695; MS Willis et al., Circulation: Heart Failure, 2019, 12(3), 1-12). In rodents, a single injection of doxorubicin can reduce the weight of the heart and skeletal muscle, followed by significant functional impairment.

[0279] A preferred embodiment of the invention relates to compounds of the general formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy caused by one of the following diseases or conditions: congestive heart failure, chronic heart failure, cancer, cancer treatment with myotoxic substances, congenital myopathies, AIDS, chronic obstructive pulmonary disease (COPD), chronic kidney disease, renal failure, diabetes, severe burns, muscle loss during aging, reduced blood supply, temporary or long-term immobilization, long-term mechanical ventilation, denervation, long-term weightlessness and malnutrition.

[0280] The present invention also relates to the treatment or prevention of diabetes, especially type II diabetes. Without wishing to be bound by theory, we believe that the positive effect on diabetes is based on MuRF1 inhibition, thereby enhancing the insulin sensitivity of muscle. S. Hirner et al., J. Mol. Biol., 2008, 379, 666-677 show that MuRF1 and insulin can be understood as functionally related in an antagonist manner, i.e. MuRF1 consumes glycogen stores and inhibits glycolysis, while insulin upregulates glycolysis and glycogen stores. Therefore, inhibition of MuRF1 can reduce insulin demand and constitute a strategy for treating diabetes.

[0281] The present invention particularly relates to:

[0282] - A compound of formula I as defined above for use in the treatment or prevention of skeletal muscle or cardiac muscle atrophy caused by congestive heart failure.

[0283] - A compound of the general formula I as defined above for use in the treatment or prevention of skeletal muscle or cardiac muscle atrophy caused by chronic heart failure.

[0284] - A compound of the general formula I as defined above for use in the treatment or prevention of cancer-induced skeletal muscle or cardiac muscle atrophy.

[0285] - A compound of formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy induced by the treatment of cancer with myotoxic and / or cardiotoxic substances such as doxorubicin.

[0286] - A compound of general formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy caused by congenital myopathy.

[0287] - A compound of formula I as defined above for use in the treatment or prevention of AIDS-induced skeletal or cardiac muscle atrophy.

[0288] - A compound of general formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy caused by chronic obstructive pulmonary disease (COPD).

[0289] - A compound of the general formula I as defined above for use in the treatment or prevention of skeletal muscle or cardiac muscle atrophy caused by chronic kidney disease.

[0290] - A compound of formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy caused by renal failure.

[0291] - A compound of formula I as defined above for use in the treatment or prevention of skeletal muscle or cardiac muscle atrophy caused by diabetes.

[0292] - A compound of formula I as defined above for use in the treatment or prevention of skeletal muscle or cardiac muscle atrophy caused by severe burns.

[0293] - A compound of formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy caused by sarcopenia during aging.

[0294] - A compound of formula I as defined above for use in the treatment or prevention of skeletal muscle or cardiac muscle atrophy caused by reduced blood supply.

[0295] - A compound of general formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy caused by temporary or prolonged immobilization.

[0296] - A compound of formula I as defined above for use in the treatment or prevention of skeletal muscle or cardiac muscle atrophy induced by long-term mechanical ventilation.

[0297] - A compound of general formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy caused by denervation.

[0298] - A compound of formula I as defined above for use in the treatment or prevention of skeletal muscle or cardiac muscle atrophy induced by long-term weightlessness.

[0299] - A compound of formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy caused by malnutrition.

[0300] - A compound of formula I as defined above for use in the treatment or prevention of diabetes.

[0301] The present invention also particularly relates to:

[0302] - A compound of general formula I as defined above for use in the treatment or prevention of psychogenic cachexia.

[0303] - A compound of general formula I as defined above for use in the treatment or prevention of tumour cachexia.

[0304] - A compound of formula I as defined above for use in the treatment of myocardial infarction.

[0305] - A compound of general formula I as defined above for use in the treatment of chronic heart failure.

[0306] - A compound of formula I as defined above for use in recovery from mechanical ventilation.

[0307] The present invention particularly relates to:

[0308] - A compound of general formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy caused by or associated with heart failure with reduced ejection fraction (HF-rEF).

[0309] - A compound of formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy caused by or associated with heart failure with preserved ejection fraction (HF-pEF).

[0310] - A compound of formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy caused by or associated with hypertension.

[0311] - A compound of formula I as defined above for use in the treatment or prevention of skeletal or cardiac muscle atrophy caused by or associated with tumour cachexia.

[0312] - A compound of formula I as defined above for use in the treatment or prevention of muscle wasting and / or cardiotoxicity induced by doxorubicin.

[0313] - A compound of formula I as defined above for use in the treatment or prevention of sarcopenia and / or cardiomyopathy caused by aging.

[0314] - A compound of formula I as defined above for use in the treatment or prevention of muscle atrophy caused by chronic kidney disease.

[0315] - A compound of formula I as defined above for use in the treatment or prevention of diaphragmatic weakness resulting from mechanical ventilation or congestive heart failure.

[0316] - A compound of general formula I as defined above for use in the treatment or prevention of congenital myopathies, in particular congenital muscular dystrophy.

[0317] - A compound of formula I as defined above for use in the treatment or prevention of diabetic muscle atrophy.

[0318] - A compound of formula I as defined above for use in the treatment or prevention of diabetes, in particular type II diabetes.

[0319] Because they are able to downregulate the function of MuRF1 by inhibiting the ubiquitin E3 ligase activity of MuRF1, and by inhibiting the binding of MuRF1 to target muscle proteins such as titin, the compounds of formula I and their pharmaceutically acceptable salts are also suitable for treating or preventing conditions associated with increased expression of muscle RING finger 1 (MuRF1); and in particular myopathies associated with dysregulated myosin synthesis and myosin degradation (which lead to muscle atrophy and myopathy), typically myopathies associated with increased expression of muscle RING finger 1 (MuRF1).

[0320] Therefore, the present invention further relates to compounds of formula I as defined above for use in the treatment or prevention of conditions associated with increased expression of Muscle RING Finger 1 (MuRF1).

[0321] In particular, the present invention further relates to compounds of formula I as defined above for use in the treatment or prevention of myopathies associated with increased expression of muscle RING finger 1 (MuRF1).

[0322] Myopathy associated with increased MuRF1 expression is, for example, selected from critical illness myopathy, nematode myopathy, inflammatory myopathy, myopathy caused by diabetes, myopathy caused by pulmonary hypertension, myopathy occurring during chronic heart failure, particularly heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF) subtypes, myopathy caused by renal failure, and myopathy caused by tumor cachexia.

[0323] For example, de Man et al., Am. J. Respir. Crit. Care Med., May 15, 2011, 183(10), 1411-1418 describe a link between increased expression of MuRF1 and myopathy caused by pulmonary hypertension.

[0324] For example, du Bois et al., Circ. Res., 2015 Aug 14, 117(5), 424-436 describe a link between increased MuRF1 expression and angiotensin II-promoted myopathy in chronic heart failure and / or renal failure.

[0325] Also, Bowen et al., Eur. J. Heart. Fail., Mar. 2015, 17(3), 263-272 describe a relationship between increased MuRF1 expression and myopathy that occurs during diastolic heart failure (HFpEF).

[0326] Upregulation of MuRF1 has been reported in patients with chronic renal failure, and this is closely related to muscle atrophy (J. Aniort et al., J. Cachexia Sarcopenia Muscle, 2019, 10 (2), 323-337; SH Lecker et al., J. Am. Soc. Nephrol., 2011, 22 (5), 821-824). Therefore, compounds that downregulate MuRF1 are expected to protect skeletal muscle during chronic renal failure. The concept of kidney damage and muscle atrophy has also been further verified in an animal model of acute gentamicin-induced kidney damage (J. Aniort et al., Int. J. Biochem. Cell Biol., 2016, 79, 505-516).

[0327] MuRF1 is upregulated during skeletal muscle aging (O. Rom et al., Free Radic. Biol. Med. 2016, 98, 218-230). This is related to mitochondrial dysfunction, which is believed to cause sarcopenia (myofibril loss) and cardiomyopathy (HW Liu et al., Biogerontology. 2020, 21 (3), 367-380). Rescue from mitochondrial dysfunction rescued the cardiomyopathy mouse model (YA Chiao et al., eLife, 2020, 9, 55513). Therefore, it is predicted that compounds that improve mitochondrial function and downregulate MuRF1 can prevent age-related sarcopenia and cardiomyopathy.

[0328] Mechanical ventilation (HW van Hees et al., Am. J. Physiol. Lung Cell Mol. Physiol., 2008, 294(6), L1260-8) or congestive heart failure (PE Hooijman et al., Am. J. Respir. Crit. Care Med., 2015, 191(10), 1126-1138) stress on the diaphragm activates the ubiquitin and proteasome systems, including MuRF1 (van Hees, loc. cit.; Hoojiman, loc. cit.). This is not just a correlational finding, but is supported by the finding that inhibition of the proteasome system by bortezomib (van Hees, loc. cit.) or elimination of MuRF1 using a knockout model (Hoojiman, loc. cit.) protects the diaphragm from weakening (Hoojiman, loc. cit.). Therefore, the compounds described herein are predicted to preserve diaphragmatic contractility in patients with congestive heart failure and critical illness (van Hees, loc. cit.; Hoojiman, loc. cit.).

[0329] Inherited muscular atrophy is associated with significant loss of muscle tissue and loss of muscle strength. Upregulation of MuRF1 is mechanistically related to this (J. Shin et al., Int. J. Biochem. Cell Biol., 2013, 45(10), 2266-2279). As a specific example of such nematode myopathy, it is worth mentioning that in glycolytically active muscle fibers, upregulation of MuRF1 at the fiber level is closely associated with those fibers and tissues that become the most atrophied (F. Li et al., Hum. Mol. Genet., 2015, 24(18), 5219-5233).

[0330] It has been shown that many types of metabolic stress, including diabetes, can activate MuRF1 expression (SH Lecker et al., FASEB J. 2004, 18 (1), 39-51). In animal models of diabetes (i.e., streptotozin-induced mouse diabetes), it has been shown to activate the Foxo-MuRF1 signaling axis, which in turn is closely related to muscle atrophy (BTO'Neill et al., Diabetes, 2019, 68 (3), 556-570).

[0331] Severe obesity is accompanied by changes in circulating factors (such as insulin and amino acids) that affect bone protein synthesis and degradation (CS Katsanos et al., Obesity, 2011, 19, 469-475). Insulin resistance accelerates muscle protein degradation by activating the ubiquitin-proteasome pathway (X. Wang, Endocrinology, 147 (9), 4160-4168). Previous studies have shown that up to 30% of weight loss may be due to the reduction of muscle mass in obese patients using different diets (DL Ballor et al., Int. J. Obes. Relat. Metab. Disord., 1994, 18, 35-40).

[0332] Therefore, a preferred embodiment of the present invention relates to compounds of formula I as defined above for use in the treatment or prevention of critical illness myopathy, nematode myopathy, inflammatory myopathy, myopathy caused by diabetes, myopathy caused by pulmonary hypertension, myopathy caused by chronic heart failure (especially HFrEF and HFpEF subtypes), myopathy caused by renal failure, myopathy caused by tumor cachexia, diaphragmatic weakness caused by pressure on the diaphragm caused by mechanical ventilation or congestive heart failure, congenital myopathy, especially congenital muscular atrophy; age-related sarcopenia and cardiomyopathy.

[0333] The present invention particularly relates to:

[0334] - A compound of formula I as defined above for use in the treatment or prevention of critical illness myopathy.

[0335] - A compound of formula I as defined above for use in the treatment or prevention of nematode myopathy.

[0336] - A compound of formula I as defined above for use in the treatment or prevention of inflammatory myopathies.

[0337] - A compound of formula I as defined above for use in the treatment or prevention of diabetic myopathy.

[0338] - A compound of formula I as defined above for use in the treatment or prevention of myopathy caused by pulmonary hypertension.

[0339] - A compound of formula I as defined above for use in the treatment or prevention of myopathy caused by chronic heart failure, in particular HFrEF and HFpEF subtypes.

[0340] - A compound of formula I as defined above for use in the treatment or prevention of myopathy caused by renal failure.

[0341] - A compound of formula I as defined above for use in the treatment or prevention of myopathy caused by tumour cachexia.

[0342] - A compound of formula I as defined above for use in the treatment or prevention of diaphragmatic weakness resulting from stress on the diaphragm caused by mechanical ventilation or congestive heart failure.

[0343] - A compound of formula I as defined above for use in the treatment or prevention of congenital myopathies, in particular congenital muscular dystrophy.

[0344] - A compound of formula I as defined above for use in the treatment or prevention of age-related sarcopenia.

[0345] - A compound of formula I as defined above for use in the treatment or prevention of age-related cardiomyopathy.

[0346] - A compound of formula I as defined above for use in the treatment or prevention of diabetes.

[0347] Furthermore, it has been found that, independently of their effects on muscle conditions, Compound I and its salts have beneficial effects on cardiac conditions associated with systolic or diastolic dysfunction. Therefore, the present invention also relates to Compound I and its salts for use in the treatment or prevention of cardiac conditions associated with systolic or diastolic dysfunction.

[0348] The present invention also relates to a pharmaceutical composition (ie a medicament) comprising at least one compound selected from the compounds of general formula I defined above, or at least one pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.

[0349] The pharmaceutically acceptable carrier is selected according to the pharmaceutical form and desired mode of administration.

[0350] The compounds of the present invention can be used to produce pharmaceutical compositions for parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracerebral injection or infusion, subcutaneous injection or implantation), oral, sublingual, intratracheal, intranasal, topical, transdermal, vaginal or rectal administration, and are administered to animals or humans in unit dosage forms mixed with conventional pharmaceutical carriers for the prevention or treatment of the above-mentioned conditions or diseases.

[0351] In pharmaceutical compositions, at least one compound of the invention may be formulated alone or with other active compounds into suitable dosage unit formulations containing conventional carriers, which are generally non-toxic and / or pharmaceutically acceptable. The carrier may be a solid, semi-solid or liquid material that acts as a solvent, carrier or medium for the active compound. Suitable carriers are listed in professional medical monographs. In addition, the preparation may contain pharmaceutically acceptable carriers or conventional auxiliary substances, such as glidants; wetting agents; emulsifiers and suspending agents; preservatives; antioxidants; anti-irritants; chelating agents; coating aids; emulsion stabilizers; film formers; gel formers; odor masking agents; flavoring agents; resins; hydrocolloids; solvents; solubilizers; neutralizers; diffusion accelerators; pigments; quaternary ammonium compounds; re-lipidating agents and super-lipidating agents; raw materials for ointments, creams or oils; silicone derivatives; development aids; stabilizers; bactericides; suppository bases; tablet aids, such as binders, fillers, glidants, disintegrants or coatings; propellants; desiccants; opacifiers; thickeners; waxes; plasticizers and white mineral oil. This aspect of the preparation is based on specialist knowledge, for example as described in Fiedler, HP, Lexikon der Hilfsstoffe für Pharmazie, Kosmetik und angrenzende Gebiete [Encyclopedia of auxiliary substances for pharmacy, cosmetics and related fields], 4th edition, Aulend of: ECV-Editio-Kantor-Verlag, 1996.

[0352] Suitable unit dosage forms include oral administration forms, such as tablets, gelatin capsules, powders, granules and solutions or suspensions for oral ingestion; sublingual, buccal, intratracheal or nasal administration forms, aerosols, implants; subcutaneous, intramuscular or intravenous injection forms; and rectal administration forms.

[0353] The compounds of the present invention may be used in creams, ointments or lotions for topical application.

[0354] If a solid composition is prepared in the form of tablets, the main ingredient is mixed with a pharmaceutically acceptable carrier such as gelatin, starch, lactose, magnesium stearate, talc, silicon dioxide and the like.

[0355] Tablets may be coated with sucrose, cellulose derivatives or other suitable substances, or treated in other ways so as to exhibit prolonged or delayed activity and to continuously release a predetermined amount of the essential active ingredient.

[0356] The formulation in the form of gelatin capsules is obtained by mixing the active ingredient with a filler and filling the resulting mixture into soft or hard gelatin capsules.

[0357] Preparations in the form of syrups or elixirs or for administration in the form of drops may contain the active ingredient and a sweetener (preferably caloric-free), methylparaben or propylparaben as preservatives, flavoring agents and appropriate coloring agents.

[0358] Water-dispersible powders or granules may contain the active ingredient mixed with a dispersant, a wetting agent or a suspending agent, such as polyvinyl pyrrolidone, and a sweetener or a flavoring agent.

[0359] Rectal administration is accomplished through the use of suppositories made with binders such as cocoa butter or polyethylene glycols that melt at rectal temperature.

[0360] Parenteral administration is achieved by using aqueous suspensions, isotonic saline solutions or sterile and injectable solutions containing pharmacologically suitable dispersants and / or wetting agents, such as propylene glycol or polyethylene glycol.

[0361] The active ingredient may also be formulated as microcapsules or liposomes / centrosomes, if appropriate, with one or more carriers or additives.

[0362] In addition to the compounds of general formula I and their pharmaceutically acceptable salts, the compositions of the present invention may contain other active ingredients that may be beneficial in treating the above-mentioned injuries or diseases.

[0363] Therefore, the present invention further relates to pharmaceutical compositions in which a plurality of active ingredients are present simultaneously, at least one of which is a compound according to the invention.

[0364] When producing pharmaceutical compositions, the compounds according to the invention are optionally mixed or diluted with one or more pharmaceutically acceptable carriers.

[0365] The present invention also relates to a compound selected from the compounds of general formula I as defined above, or a pharmaceutically acceptable salt thereof, for use in the preparation of a medicament for the treatment or prevention of a condition as defined above, such as a muscle wasting condition as defined above or a condition associated with increased expression of muscle RING finger 1 (MuRF1) as defined above, in particular myopathy or a heart disease associated with systolic or diastolic dysfunction, or diabetes.

[0366] For the preparation of said medicament, reference is made to the statements relating to the above-mentioned pharmaceutical composition.

[0367] The present invention also relates to a method for treating or preventing the above conditions, such as muscle wasting conditions or conditions as defined above, in particular myopathies, which are associated with increased expression of muscle RING finger 1 (MuRF1), or cardiac conditions associated with systolic or diastolic dysfunction, or diabetes, comprising the step of administering to an individual in need thereof a therapeutically effective amount of a compound selected from the group consisting of compounds of general formula I or a pharmaceutically acceptable salt thereof.

[0368] Likewise, the present invention relates to a method for protecting or enhancing diaphragmatic contractility under stress conditions such as long-term mechanical ventilation, surgery, chronic heart failure or primary myopathy, the method comprising the step of administering to an individual in need thereof a therapeutically effective amount of a compound selected from the compounds of general formula I or a pharmaceutically acceptable salt thereof.

[0369] The present invention particularly relates to:

[0370] - A method for treating or preventing skeletal or cardiac muscle atrophy and / or diaphragmatic weakness caused by congestive heart failure;

[0371] - A method for treating or preventing skeletal muscle or cardiac muscle atrophy caused by chronic heart failure;

[0372] - A method for treating or preventing skeletal muscle or cardiac muscle atrophy caused by cancer;

[0373] - A method for treating or preventing skeletal or cardiac muscle atrophy caused by the use of myotoxic and / or cardiotoxic substances such as doxorubicin for the treatment of cancer;

[0374] - A method for treating or preventing skeletal or cardiac muscle atrophy caused by congenital myopathy;

[0375] - A method for treating or preventing skeletal muscle or cardiac muscle atrophy caused by AIDS;

[0376] - A method of treating or preventing skeletal or cardiac muscle atrophy caused by chronic obstructive pulmonary disease (COPD);

[0377] - A method for treating or preventing skeletal muscle or cardiac muscle atrophy caused by chronic kidney disease;

[0378] - A method for treating or preventing skeletal muscle or cardiac muscle atrophy caused by renal failure;

[0379] - A method for treating or preventing skeletal muscle or cardiac muscle atrophy caused by diabetes;

[0380] - A method of treating or preventing skeletal or cardiac muscle atrophy caused by severe burns;

[0381] - A method for treating or preventing skeletal or cardiac muscle atrophy due to muscle loss during aging;

[0382] - A method of treating or preventing skeletal or cardiac muscle atrophy due to reduced blood supply;

[0383] - A method for treating or preventing skeletal or cardiac muscle atrophy caused by temporary or prolonged immobilization;

[0384] - A method for treating or preventing skeletal or cardiac muscle atrophy and / or diaphragmatic weakness caused by long-term mechanical ventilation;

[0385] - A method for treating or preventing skeletal or cardiac muscle atrophy caused by denervation;

[0386] - A method for treating or preventing skeletal muscle or cardiac muscle atrophy caused by long-term weightlessness;

[0387] - A method for treating or preventing skeletal muscle or cardiac muscle atrophy caused by malnutrition;

[0388] - A method for treating or preventing diabetes;

[0389] - A method of treating or preventing a cardiac condition associated with systolic or diastolic dysfunction;

[0390] The method comprises the step of administering to an individual in need thereof a therapeutically effective amount of a compound selected from the compounds of formula I or a pharmaceutically acceptable salt thereof.

[0391] The present invention also particularly relates to:

[0392] - A method of treating or preventing critical illness myopathy including preserving diaphragm contractility and function during intensive care;

[0393] - A method of treating or preventing nematode myopathy;

[0394] - A method of treating or preventing inflammatory myopathy;

[0395] - A method for treating or preventing myopathy caused by diabetes;

[0396] - A method of treating or preventing myopathy caused by pulmonary hypertension;

[0397] - A method for treating or preventing myopathy caused by chronic heart failure, especially HFrEF and HFpEF subtypes;

[0398] - A method for treating or preventing myopathy caused by renal failure;

[0399] - A method for treating or preventing myopathy caused by tumor cachexia;

[0400] The method comprises the step of administering to an individual in need thereof a therapeutically effective amount of a compound selected from the compounds of formula I or a pharmaceutically acceptable salt thereof.

[0401] The present invention particularly relates to:

[0402] - A method of treating or preventing skeletal or cardiac muscle atrophy caused by or associated with heart failure with reduced ejection fraction (HF-rEF).

[0403] - A method of treating or preventing skeletal or cardiac muscle atrophy caused by or associated with heart failure with preserved ejection fraction (HF-pEF).

[0404] - A method of treating or preventing skeletal or cardiac muscle atrophy caused by or associated with hypertension.

[0405] - A method of treating or preventing skeletal or cardiac muscle atrophy caused by or associated with tumor cachexia.

[0406] - A method for treating or preventing muscle atrophy and / or cardiotoxicity induced by doxorubicin.

[0407] - A method for treating or preventing sarcopenia and / or cardiomyopathy due to aging.

[0408] - A method for treating or preventing muscle atrophy caused by chronic kidney disease.

[0409] - A method of treating or preventing diaphragmatic weakness due to mechanical ventilation or congestive heart failure.

[0410] - A method for treating or preventing congenital myopathy, in particular homologous muscular dystrophy.

[0411] - A method for treating or preventing muscle atrophy caused by diabetes.

[0412] - A method of treating or preventing diabetes.

[0413] - A method of treating or preventing a cardiac condition associated with systolic or diastolic dysfunction.

[0414] As used herein, the term "individual in need thereof" refers to an individual suffering from one or more of the above conditions or diseases, or refers to an individual who may suffer from one or more of the above conditions or diseases. Preferably, the term "individual in need thereof" refers to a mammal, in particular a human, a production animal or a domestic animal. In particular, the term "individual in need thereof" refers to a human.

[0415] As used herein, the terms "effective amount" and "therapeutically effective amount" mean the amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. It will be appreciated that one skilled in the art can affect muscle wasting conditions and / or myopathy by treating a patient currently suffering from the condition or prophylactically treating a patient suffering from the condition with an effective amount of the compounds of the invention.

[0416] The term "treatment" as used herein in conjunction with the above methods refers to all processes in which the progression of the muscle wasting conditions and / or myopathy described herein may be delayed, interrupted, prevented, controlled or stopped, but this does not necessarily mean that all symptoms of the conditions or diseases are completely eliminated, the above conditions are treated prophylactically, especially in patients who are susceptible to the conditions or diseases.

[0417] The term "composition" as used herein is intended to include a product containing the specified ingredients in the specified amounts, as well as any product resulting directly or indirectly from the combination of the specified ingredients in the specified amounts. The term is related to pharmaceutical compositions and is intended to encompass products comprising active ingredients and inert ingredients constituting carriers, as well as any product resulting directly or indirectly from the combination, complexation or aggregation of any two or more of the ingredients, or from the dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Thus, the pharmaceutical compositions of the present invention include any composition prepared by mixing a compound of the present invention and a pharmaceutically acceptable carrier.

[0418] By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the subject.

[0419] The terms "administration of a compound" and / or "administering a compound" are to be understood as providing a compound of the invention or a prodrug of a compound of the invention to a subject in need of treatment.

[0420] In the treatment and / or prevention of the above-mentioned muscle wasting conditions and myopathies associated with increased MuRF1 expression, an appropriate dosage level is generally about 2 to 500 mg / kg body weight per day of the individual in need thereof, which can be administered in single or multiple doses. Preferably, the dosage level should be about 5 mg / kg to about 250 mg / kg per day.

[0421] For oral administration, the composition is preferably provided in the form of tablets containing 10 to 5000 mg of active ingredient, especially 50.0 mg, 100.0 mg, 200.0 mg, 500.0 mg, 1000.0 mg, 2000.0 mg, 3000.0 mg, 4000.0 mg and 5000.0 mg of active ingredient, for symptomatic adjustment of the dosage to an individual in need thereof.

[0422] The dosage regimen can be adjusted to provide the optimal therapeutic response. However, it should be understood that the specific dosage level and frequency of administration for any particular patient may vary and depend on a variety of factors, including the activity of the specific compound used, the metabolic stability and duration of action of the compound, age, body weight, general health, sex, diet, mode and time of administration, excretion rate, drug combination, severity of the particular condition, and the host being treated.

[0423] The compounds of the present invention can be administered by conventional routes of administration, including parenteral administration (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection or implantation), oral administration, inhalation spray administration, nasal administration, vaginal administration, rectal administration, sublingual administration or topical administration.

[0424] The compounds according to the invention are furthermore used in combination with other substances in a method for the treatment and / or prevention of the abovementioned diseases and conditions.

[0425] The compounds of the present invention can be used in combination with one or more other drugs for the treatment and / or prevention of the above-mentioned diseases and conditions, wherein the compounds of formula I or other drugs can have utility, wherein the combination is safer or more effective than the use of drugs alone. The other drugs can be administered simultaneously or sequentially by a route with the compounds of formula I in the amounts commonly used. When the compounds of formula I are administered simultaneously with one or more other drugs, a pharmaceutical composition in a unit dosage form containing the other drugs and the compounds of formula I is preferred. However, combination therapy can also include therapies in which the compounds of formula I and one or more other drugs are administered in different overlapping schemes. It can also be envisioned that when used in combination with one or more other active ingredients, the compounds of the present invention and other active ingredients can be used in an amount lower than the dosage when used alone. The above-mentioned combination includes not only the combination of the compounds of the present invention with one other active compound, but also the combination with two or more other active compounds.

[0426] The weight ratio of the compound of the present invention to the second active ingredient can be varied and depends on the effective dose of each ingredient. Generally, an effective dose is used for each drug. Thus, for example, when the compound of the present invention is combined with another active agent, the weight ratio of the compound of the present invention to the other active agent is generally about 1000:1 to about 1:1000, preferably about 200:1 to about 1:200. The combination of the compound of the present invention and other active ingredients is also generally within the above range, but in each case, an effective dose of each active ingredient should be used. In the combination, the compound of the present invention and other active substances can be administered alone or in combination. In addition, the administration of an active substance can be performed before, at the same time, or after the administration of other active agents.

[0427] The following examples and figures are intended to further illustrate the present invention.

[0428] abbreviation:

[0429] ALPHA Amplified Luminescence Near Uniform Analysis;

[0430] DSF differential scanning fluorimetry;

[0431] GST glutathione-S-transferase;

[0432] UBE1 recombinant human His6 ubiquitin activating enzyme;

[0433] DMSO dimethyl sulfoxide;

[0434] CDI 1,1'-Carbonyldiimidazole;

[0435] DMEM Dulbecco's modified Eagle's medium;

[0436] Sham saline-treated;

[0437] DEX dexamethasone;

[0438] PBS phosphate-buffered saline;

[0439] MCT monocrotaline;

[0440] TA tibialis anterior;

[0441] TL tibia length;

[0442] EDL extensor digitorum longus;

[0443] CSA cross-sectional area;

[0444] LAD left anterior dexterior coronary artery ligation;

[0445] MI myocardial infarction;

[0446] WB Western blot;

[0447] BW body weight;

[0448] CHF Chronic heart failure;

[0449] LV left ventricle;

[0450] LVEDD left ventricular end-diastolic diameter;

[0451] LVEF left ventricular ejection fraction;

[0452] LVESD left ventricular end-systolic diameter;

[0453] LVFS Left ventricular (ejection) fraction shortened;

[0454] MRPS-5 mitochondrial ribosomal protein 5;

[0455] Nox 2 NADPH oxidase 2;

[0456] CS citrate synthase;

[0457] SDH succinate dehydrogenase;

[0458] TOM-20, mitochondrial outer membrane translocase 20;

[0459] GAPDH glyceraldehyde 3-phosphate dehydrogenase;

[0460] AU arbitrary unit;

[0461] HPRT hypoxanthine phosphoribosyltransferase;

[0462] FRET fluorescence resonance energy transfer;

[0463] MAFbx muscle atrophy F-box;

[0464] CARP cardiac adriamycin-responsive protein;

[0465] GAPDH glyceraldehyde-3-phosphate dehydrogenase;

[0466] eIF2B-delta Translation initiation factor eIF-2B subunit delta;

[0467] AS3MT arsenite methyltransferase;

[0468] ATPAF1 ATP synthase mitochondrial F1 complex assembly factor 2;

[0469] GHDC GH3 domain-containing protein;

[0470] BAX apoptosis regulator BAX.

[0471] Attached photos:

[0472] Figure 1 : Results of cytotoxicity assay based on the release of lactate dehydrogenase (LDH) activity in cultured myoblasts in the presence of increasing concentrations of compounds MyoMed-946, MyoMed-946-5, MyoMed-946-8.

[0473] Figure 2 : Results of cytotoxicity assay based on the release of lactate dehydrogenase (LDH) activity in cultured myocytes in the presence of increasing concentrations of compounds MyoMed-946, MyoMed-946-5, MyoMed-946-8.

[0474] Figures 3 to 7 : Physical characteristics of mice fed normal chow (MCT; n=27) or fed MyoMed-946 compound (MCT+MyoMed-946; n=27) treated with monocrotaline. The data demonstrate that MCT treatment induces cardiac cachexia independent of the administered diet, as evidenced by impaired weight gain ( Figure 3 ), increased pulmonary congestion ( Figure 4 ) and heart weight relative to body weight (BW) increase ( Figure 5 ), and right ventricular (RV) hypertrophy ( Figure 6 ) as visualized in representative H&E-stained medial cardiac cross sections ( Figure 7 ). *P<0.01vs. sham-treated group.

[0475] Figures 8 to 12 : In the absence or presence of the MuRF1 inhibitor MyoMed-946, the extensor digitorum longus (EDL) ( Figure 8 ), soleus muscle( Fig. 9 ), tibialis anterior (TA)( Fig.10 ) skeletal muscle wet weight (normalized to tibia length TL). In addition, the fiber cross-sectional area (CSA) of TA muscle is also provided ( Fig.11 ), the latter visualized by representative H&E staining ( Fig.12 ). *P<0.05vs. sham-treated group; § P<0.01 vs. sham-treated group and MCT+MyoMed-946.

[0476] Figures 13 to 18 : Physical characteristics of sham-treated (n=10) and monocrotaline-treated mice fed with normal chow (MCT; n=10) or MuRF1 inhibitors MyoMed-946 (MCT+MyoMed-946; n=10), MyoMed-203 (MCT+MyoMed-203; n=10), and MyoMed-205 (MCT+MyoMed-205; n=10). Data are presented as mean ± standard error of the mean. Fig.13 : ***p<0.001vs. start; Fig.14 : ***p<0.001 vs. sham treatment **p<0.01 vs. sham treatment; Fig.15 : ***p<0.001, **p<001 vs. sham treatment, §§p<0.01, §p<0.05 vs. MCT; Figure 16-18: *P<0.01 vs. sham treatment. The data demonstrate that MCT treatment induces psychogenic cachexia independent of the administered food, as evidenced by impaired weight gain ( Fig.13 ), increased pulmonary congestion ( Fig.14 ) and heart weight relative to body weight (BW) increase ( Fig.15 ), and MuRF1 inhibitors MyoMed-946, MyoMed-203, and MyoMed-205 reduced right ventricular (RV) hypertrophy ( Figure 16-18 ).

[0477] Fig.19 and 20 : Extensor digitorum longus (EDL) ( Fig.19 ) and tibialis anterior (TA) ( Fig. 20 ) of skeletal muscle wet weight (normalized to tibia length TL).

[0478] Fig.21 : Maximum diaphragm force of MCT-treated mice in the absence (sham) or presence of the MuRF1 inhibitors MyoMed-946, MyoMed-203, and MyoMed-205.

[0479] Fig. 22 and 23 : MuRF1 ( Fig. 22 ) and telethonin ( Fig.23 ) in the tibialis anterior muscle tissue.

[0480] Figure 24-27 :Isometric contraction of diaphragm muscle fiber bundles in mice with chronic heart failure (CHF) and reduced ejection fraction (HFrEF) after myocardial infarction (MI) Fig.24 ) and isotonic contraction ( Fig.25 ) during the evaluation of diaphragm function, thereby determining the maximum diaphragm force ( Fig.26 ) and diaphragm peak power ( Fig. 27 ). Data are expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001 vs. CHF, §P < 0.05 and §§P < 0.01 vs. CHF + MyoMed-946.

[0481] Figures 28-30 :Isometric contraction( Fig.28) and isotonic contraction to assess in vitro skeletal muscle contractile function, thereby determining shortening velocity ( Fig.29 ) and power ( Fig.30 ). MCT treated mice showed approximately 20% impairment in shortening velocity and power compared to the sham treated group. The impairment was substantially prevented in MCT mice fed the MyoMed-946 compound. *P<0.05 vs sham treated group; §P<0.01 vs sham treated group and MCT+MyoMed-946.

[0482] Fig.31 : Tibialis anterior (TA) muscle wet weight of B16F10 cell-inoculated mice receiving regular chow (tumor), and B16F10 cell-inoculated mice fed MyoMed-946 (tumor+MyoMed-946) and MyoMed-205 (tumor+MyoMed-205).

[0483] Fig.32 : Wire suspension test of B16F10 cell inoculated mice (melanoma cell model) on days 9, 16 and 23 after inoculation. Tumor growth resulted in a significant decrease in muscle function (tumor group) compared to control (sham) mice. The decrease in muscle function was attenuated in mice fed the compounds MyoMed-946 (tumor + MyoMed-946) and MyoMed-205 (tumor + MyoMed-205).

[0484] Figures 33-35 : Western blot analysis of eIF2B subunit-δ ( Fig.33 ) and BAX( Fig.34 ) expression levels, representative blots ( Fig.35 ). *P<0.05vs sham-treated group; § P<0.01vs.MCT.

[0485] Figures 36-38 : MuRF1 ( Fig.36 )、MAFBx( Fig.37 ) and CARP( Fig.38 ) protein expression levels and representative western blots. MCT treatment resulted in increased expression of MuRF1 and CARP, but the expression was attenuated in mice fed the MyoMed-946 compound. No changes in MAFBx levels were detected. *P<0.05 vs sham and MCT MyoMed-946.

[0486] Fig.39: MRPS-5 protein expression levels and representative western blots in diaphragm tissues of sham-treated mice, CHF mice, and CHF+MyoMed-946 mice. Data are presented as mean ± standard error of the mean.

[0487] Figure 40-42 : MuRF1 ( Fig.40 )、MuRF2( Fig.41 ) and continuous proteins ( Fig.42 ) protein expression levels and representative western blots. CHF resulted in increased expression of MuRF1 and MuRF2 and decreased protein expression in mice fed MyoMed-946 compound, but the effects were attenuated in mice fed MyoMed-946 compound. Data are presented as mean ± standard error of the mean.

[0488] Figures 43-46 : Western blots (WB) analysis of muscle tissue of B16F10 cell-inoculated mice fed with regular chow (tumor) or compound MyoMed-946 (tumor+MyoMed-946) or MyoMed-205 (tumor+MyoMed-205) ( Fig.43 ) measured MuRF1, Nox 2 ( Fig.44 ) and LC3 I / II( Fig.46 ) protein expression levels and the level of reactive oxygen marker nitrotyrosine ( Fig.45 ). Tumor growth resulted in a significant increase in the protein expression levels of MuRF1 and Nox 2 and nitrotyrosine levels in the tumor group, and a significant decrease in the expression level of LC3 I / II compared to the control (sham) mice. The changes were attenuated in mice fed the compound MyoMed-946 (tumor + MyoMed-946) or MyoMed-205 (tumor + MyoMed-205). Data are presented as mean ± standard error of the mean.

[0489] Figures 47-49 : Expression of citrate synthase ( Fig.47 ), succinate dehydrogenase ( Fig.48 ) and mitochondrial complex I ( Fig.49 ) enzyme activity. The data showed that the activities of citrate synthase, succinate dehydrogenase and mitochondrial complex I were significantly downregulated in CHF compared with the sham-treated group, but the downregulation was attenuated in mice fed the compound MyoMed-946. The data are expressed as mean ± standard error of the mean.

[0490] Fig.50 and 51 :The protein expression levels of mitochondrial porins in the mitochondrial outer membrane in diaphragm tissue samples of sham-treated mice, chronic heart failure (CHF) mice, and CHF+MyoMed-946 mice ( Fig.50 ) and TOM-20( Fig.51 ). The data showed that the expression of porin and TOM-20 was significantly downregulated in CHF compared with the sham-treated group, but the downregulation was attenuated in mice fed the compound MyoMed-946. The data are expressed as mean ± standard error of the mean.

[0491] Fig.52 and 53 : Citrate synthase ( Fig.52 ) and mitochondrial complex I ( Fig.53 ) enzyme activity. The data showed that the activities of citrate synthase and mitochondrial complex I in the tumor group were significantly downregulated compared with the sham-treated group, but the downregulation was attenuated in mice fed with compounds MyoMed-946 or MyoMed-205. The data are expressed as mean ± standard error of the mean.

[0492] Fig.54 : MuRF1 expression at the mRNA level in myotubes after 24 h of incubation with dexamethasone (DEX; 10 μmol / L). Shown are the fold changes of untreated cells (DEX) and cells pretreated for 2 h with 0.1 μmol / L and 10 μmol / L of the compound MyoMed-946. Pretreated cells showed a decrease in MuRF1 mRNA levels. *P<0.05 vs. CON. § P<0.05vs.DEX.

[0493] Fig.55 : Differential scanning fluorimetry (DSF) melting curves, plotting the ratio of the fluorescence signal at 350 nm to the fluorescence signal at 330 nm versus the thermal gradient, for the MuRF1 central fragment in PBS buffer (solid line), for the MuRF1 central fragment in PBS buffer + 1% DMSO (dashed line), and for the MuRF1 central fragment in a 10 mM stock solution of the compound MyoMed-946 in PBS buffer + 1% DMSO (final concentration of MyoMed-946: 100 μM) (dotted line).

[0494] Fig.56: Schematic diagram of the experimental study design of the HFpEF rat model.

[0495] Fig.57 : Results of echocardiographic and invasive hemodynamic measurements of myocardial function in a HFpEF rat model.

[0496] Fig.58 : Results of skeletal muscle mass and function measurements in the HFpEF rat model.

[0497] Fig.59 : Cachexia and physical stress outcomes in a mouse model of doxorubicin-induced muscle atrophy and cardiotoxicity.

[0498] Fig.60 : Echocardiographic findings in a mouse model of doxorubicin-induced muscle atrophy and cardiotoxicity.

[0499] Fig.61 : Wire suspension test in type 2 diabetic obese mice during diet-induced weight loss. MyoMed-205 treatment attenuates the loss of muscle function.

[0500] Fig.62 : Blood glucose levels after 6 h of fasting in MyoMed-203-treated DIO mice compared with those in DIO control mice.

[0501] Fig.63 and 64 : Results of oral glucose tolerance test on days 14 and 28 of the experiment in MyoMed-203-treated DIO mice compared with DIO control mice.

[0502] Fig.65 : Results of insulin tolerance test in MyoMed-203-treated DIO mice compared with DIO control mice.

[0503] I. Synthesis of Compound I

[0504] 1. analyze

[0505] If not stated otherwise, compounds were measured by NMR in d6-dimethylsulfoxide (DMSO-d6) on a 400 MHz NMR instrument (Bruker AVANCE III). 1 H NMR and finally 13 C NMR characterization.

[0506] Nuclear magnetic resonance spectroscopy (NMR) refers to chemical shifts (δ) expressed in parts per million (ppm). 1The relative area of ​​the shifts in the H NMR spectrum corresponds to the number of hydrogen atoms of one particular functional type in the molecule. In terms of multiplicity, the nature of the shifts is indicated as singlet (s), broad singlet (s.br.), doublet (d), broad doublet (d br.), triplet (t), broad triplet (tbr.), quartet (q), quintet (quint.), and multiplet (m).

[0507] The compounds were further characterized by HPLC-MS and / or UPLC-MS with fast gradient on C18-material (electrospray ionization (ESI) mode). If not stated otherwise, ESI MS-data were recorded in positive mode. MS data refer to protonated compounds (M+H) + , expressed as mass-to-charge ratio (m / z), where z is 1.

[0508] HPLC-MS Description:

[0509] HPLC-MS instrument: Agilent 1100 Series LC / MSD system equipped with DAD\ELSD and Agilent LC\MSDVL(G1956A),SL(G1956B) mass spectrometer or Agilent 1200 Series LC / MSD system equipped with DAD\ELSD and Agilent LC\MSD SL(G6130A),SL(G6140A) mass spectrometer. All LC / MS data were acquired by positive / negative mode switching.

[0510] Acquisition method: Column: Zorbax SB-C18 1.8μm 4.6x15mm Rapid Resolution cartridge (PN 821975-932). Mobile phase A: acetonitrile + 0.1% formic acid; B: water containing 0.1% formic acid. Flow rate: 3ml / min; Injection volume: 1μl.

[0511] Solvent gradient:

[0512] 0-0.01 min 100% B;

[0513] 0.01-1.5 min 100% to 0% B, linear gradient;

[0514] 1.5-1.8 minutes 0% B;

[0515] 1.8-1.81 min 0% to 100% B.

[0516] Ionization mode: atmospheric pressure chemical ionization (APCI);

[0517] Scan range: m / z 80-1000.

[0518] UPLC-MS Description:

[0519] UPLC-MS instrument: Agilent Infinity 1290 equipped with a single quadrupole, electrospray ionization mass spectrometer;

[0520] Acquisition method: Column: Acquity UPLC BEH C18; 1.7 μm; 2.1 x 50 mm; T = 40 ° C. Mobile phase: А: water + 0.1% trifluoroacetic acid; В: MeCN + 0.1% trifluoroacetic acid. Flow rate: 1 ml / min; Injection volume: 3 μl; Run time 3 min.

[0521] Solvent gradient (3 min gradient):

[0522] 0-2.3 min 5%-100% B, linear gradient;

[0523] 2.3-2.5 minutes 100% B;

[0524] 2.5-2.6 min 100-5% B, linear gradient;

[0525] 2.6-3.0 min 100% B.

[0526] If not stated otherwise, ESI MS-data were recorded in positive mode. MS-data refer to protonated compounds (M+H) + , expressed as mass-to-charge ratio (m / z), where z is 1.

[0527] 2. synthesis

[0528] 2.1 Preparative HPLC-Purification

[0529] The results were analyzed by HPLC (H2O-MeOH or H2O-CH3CN; Agilent 1260 Infinity systems) equipped with DAD and mass spectrometry-detector using a Waters SunFire C18 OBD preparative column ( 5μm, 19mm X 100mm, equipped with SunFire C18 Prep Guard Cartridge, 10μm, 19mm x 10mm), preparative HPLC-purification was completed. The starting compound was dissolved in 0.7mL DMSO. Flow rate: 30mL / min. The purity of the obtained fractions was checked by analytical LCMS. The spectrum of each fraction was recorded as it was obtained directly in solution after chromatography. The solvent was evaporated in a N2 flow at 80°C. The individual fractions were combined based on the post-chromatographic LCMS analysis. The solid fraction was dissolved in 0.5mL MeOH / CH3CN and transferred to a pre-weighed labeled vial. The obtained solution was evaporated again in a N2 flow at 80°C. After drying, the product was finally analyzed by LC-MS and 1 H-NMR characterization.

[0530] 2.2 Intermediates

[0531] 2.2.1 4-[(4-Methyl-2-oxo-chromen-7-yl)oxymethyl]acetic acid

[0532] A mixture of 80.6 g (0.458 mol) of 7-hydroxy-4-methylcoumarin, 110.1 g of methyl 4-(bromomethyl)benzoate (0.480 mol) and anhydrous K2CO3 (95 g, 0.686 mol) in anhydrous acetone (800 mL) was heated to reflux for 3 hours. The mixture was then cooled, filtered, and the filtrate was concentrated in vacuo. The residue was dissolved in DMSO (450 mL), and aqueous potassium hydroxide solution (200 mL, 20% KOH) was added. The resulting mixture was stirred at ambient temperature for 72 h. After the hydrolysis was complete, 1 L of water was added and the solution was acidified to pH = 1-2 with 10% hydrochloric acid. The precipitate formed was filtered and dried in vacuo to obtain the pure product. The yield of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]acetic acid was 75% (106 g).

[0533] HPLC-MS (positive mode): m / z 311 (M+H) + ; Retention time: 1.11min.

[0534] 1 H NMR (400MHz, DMSO-d6, ppm): δ = 13.01 (br s,1H),8.0-7.97(m,2H),7.71(d,J=7.0Hz,1H),7.60-7.58(m,2H),7.09-7.05(m,2H),6.23(s,1H),5.34(s,2H),2.40(s,3H).

[0535] 2.2.2 2-Amino-N-(2-furylmethylcarbamoyl)acetamide

[0536] A mixture of 1.46 g (6.74 mmol) of 2-chloro-N-(2-furanylmethyl-carbamoyl)acetamide and 90 mg of sodium azide (NaN3, 2 eq.) in 15 ml of ethanol was stirred at 70°C overnight. After cooling to room temperature, the solids were removed and the filtrate obtained was concentrated in vacuo (1 / 3 volume). The residue was partitioned between EtOAc and water. The organic layer was washed with brine, dried over Na2SO4 and evaporated in vacuo. The residue was dissolved in MeOH and Pd / C was added thereto. The mixture was degassed and stirred at RT overnight under a hydrogen atmosphere. The catalyst was removed and the filtrate was evaporated and dried in vacuo to obtain the crude amine, which was used in the next step without further purification. (X=O 0.8 g 60%; X=O 0.7 g 62% (over 2 steps)).

[0537] 2.2.3 2-Amino-N-(2-thienylmethylcarbamoyl)acetamide

[0538] 2-Amino-N-(2-thienylmethylcarbamoyl)acetamide was prepared analogously to Example 2.2.2, except that 1.26 g (5.41 mmol) of 2-chloro-N-(2-thienylmethylcarbamoyl)-acetamide was used as starting material instead of 2-chloro-N-(2-furanylmethyl-carbamoyl)acetamide. The crude 2-amino-N-(2-thienylmethyl-carbamoyl)acetamide thus obtained was used in the next step without further purification. Yield: 0.7 g 62% (over 2 steps).

[0539] 2.3 Compound I

[0540] Example 1

[0541] Synthesis of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid [2-(2-furanylmethylcarbamoylamino)-2-oxo-ethyl] ester (MyoMed-946)

[0542] A mixture of 310 mg (1.0 mmol) of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-acetic acid, 228 mg (1.05 mmol) of 2-chloro-N-(2-furanylmethylcarbamoyl)acetamide, 75 mg (0.5 mmol) of NaI and 155 mg (1.2 mmol) of DIPEA was dissolved in 6 mL of DMSO. The resulting slurry was stirred at room temperature for 72 h to complete the reaction; the conversion was controlled by LC-MS. The reaction mixture was then poured into 50 mL of water and the resulting precipitate was filtered and subsequently washed with additional portions of water, isopropanol and hexane. The solid product was dried in vacuo. The yield of 4-[(4-methyl-2-oxo-chromen-7-yl)oxy-methyl]benzoic acid [2-(2-furanylmethylcarbamoylamino)-2-oxo-ethyl] ester (MyoMed-946) was 61% (289 mg).

[0543] After several months of storage at room temperature, 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid [2-(2-furanylmethylcarbamoylamino)-2-oxo-ethyl] ester (MyoMed-946) showed no signs of decay (confirmed by NMR and LC-MS).

[0544] HPLC-MS (positive mode): m / z 491 / 492 (M+H) + ; Retention time: 1.436min.

[0545] 1 H NMR (400MHz, DMSO-d6, ppm): δ = 10.50 (br.s, 1H, NH), 7.85 (br.s, 1H, NH), 7.55 (d, J = 8 .0Hz,2H,CH+CH),7.22(d,J=8.4Hz,1H,CH),7.15(d,J=8.0Hz,2H,CH+CH),7.09(s,1H ,CH),6.60(m,2H,CH+CH),5.90(m,1H,CH),5.78(d,J=1.4Hz,1H,CH),5.74(s,1H,CH) ,4.87(s,2H,OCH2),4.44(s,2H,OCH2),3.88(d,J=5.2Hz,2H,NCH2),1.91(s,3H,CH3).

[0546] Further analysis and characterization of MyoMed-946

[0547] The purity and identity of the compounds were further evaluated using 1D and 2D NMR and UPLC-MS as follows:

[0548] For NMR analysis, 2 mg of MyoMed-946 was dissolved in 1 ml of d6-DMSO containing trace amounts of CCl4. 1 H- and 13 C-spectrum, and for 1 COSY and HSQC 2D-NMR spectra of H-NMR peak assignments.

[0549] 1 H NMR (500MHz, DMSO-d6+CCl4): δ = 10.68 (s, 1H), 8.34 (s, 1H), 8.05 (d, J = 7.37Hz, 2H), 7.53-7.45 (m, 4H), 7.1 4-6.91(m,2H),6.40(s,1H),6.30-6.16(m,2H),5.37(s,2H),4.94(s,2H),4.44-4.23(m,2H),2.41(s,3H).

[0550] 13 C NMR (126MHz, DMSO-d6+CCl4): δ=169.0,165.1,161.0,160.0,154.6,153.3,152.5,151.8,142.3(2C),1 29.6(2C),128.5,127.6(2C),126.6,113.5,112.6,111.4,110.4,107.0,101.8,69.1,62.7,36.0,18.1.

[0551] For UPLC-MS analysis, a small amount of MyoMed-946 was dissolved in acetonitrile (MeCN), and 3 μl of the solution was injected into a C18 UPLC column (Acquity UPLC BEH C18; 1.7 μm; 2.1 x 50 mm). The UPLC-MS system and analysis method were as described above.

[0552] UPLC-MS (positive mode): m / z = 491.1 (M+H) + ; Retention time: 1.7min.

[0553] Embodiment 2:

[0554] Synthesis of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid (1-methyl-2-oxo-2-ureido-ethyl) ester (MyoMed-946-5)

[0555] The synthesis was carried out similarly to Example 1 except that N-carbamoyl-2-chloro-propionamide was used instead of 2-chloro-N-(2-furanylmethyl-carbamoyl)acetamide.

[0556] HPLC-MS (positive mode): m / z 439 (M+H) + ; Retention time 1.388min.

[0557] 1 H NMR (400MHz, DMSO-d6, ppm): δ = 10.63 (br.s, 1H), 8.05-7.98 (m, 3H), 7.72-7.68 (m, 1H), 7.68-7.59 (m, 2H) ,7.05-7.01(m,2H),6.23(s,1H),5.35(s,2H),5.22-5.14(m,1H),2.70(d,3H),2.39(s,3H),1.50(d,3H).

[0558] Embodiment 3:

[0559] Synthesis of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid [1-methyl-2-(methylcarbamoylamino)-2-oxo-ethyl] ester (MyoMed-946-8)

[0560] The synthesis was carried out similarly to Example 1 except that 2-chloro-N-(methylcarbamoyl)propionamide was used instead of 2-chloro-N-(2-furanylmethyl-carbamoyl)acetamide.

[0561] HPLC-MS (positive mode): m / z 425 (M+H) + ; Retention time 1.327min.

[0562] 1 H NMR (400MHz, DMSO-d6, ppm): δ = 10.51 (br.s, 1H), 8.06-7.97 (m, 2H), 7.73-7.67 (m, 1H), 7.67-7.59 (m, 2H), 7.54 (br ,s,1H),7.33(br,s,1H),7.09-7.02(m,2H),6.22(s,1H),5.35(s,2H),5.22-5.14(m,1H),2.39(s,3H),1.51(d,3H).

[0563] Embodiment 4:

[0564] Synthesis of N-[2-(2-furanylmethylcarbamoylamino)-2-oxo-ethyl]-4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzamide

[0565] To a cooled solution of 2-amino-N-(2-furanylmethylcarbamoyl)acetamide (0.6 mmol) obtained in Example 2.2.2, 558 mg (1.8 mmol) of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-acetic acid, 109 mg of 1-hydroxy-7-azabenzotriazole (HOAt, 0.8 mmol) in 2 mL of DMF, 124 mg (0.8 mmol) of EDC was added dropwise, and the mixture was stirred at room temperature overnight. The precipitate formed was collected, washed with methanol, then with water, washed with methanol again, and dried to give 110 mg of the title compound. Yield: 37%.

[0566] 1 H NMR (400MHz, DMSO-D6, ppm): δ = 10.53 (s, 1H), 8.82 (t, J = 5.7Hz, 1H), 8.51 (br.s, 1H), 7.89 (d, J = 8.0Hz, 2H), 7.70 (d, J = 8.6Hz, 1H), 7.56 (m, 3H),7.06(m,2H),6.37(s,1H),6.24(d,J=2.5Hz,1H),6.21(s,1H),3.50(s,2H),4.35(d,J=5.7Hz,2H),3.99(d,J=5.0Hz,2H),2.38(s,3H).

[0567] Embodiment 5:

[0568] Synthesis of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid [2-oxo-2-(2-thienylmethylcarbamoylamino)ethyl] ester

[0569] A mixture of 310 mg (1.0 mmol) of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-acetic acid, 250 mg (1.05 mmol) of 2-chloro-N-(2-thienylmethylcarbamoyl)acetamide, 75 mg (0.5 mmol) of NaI and 155 mg (1.2 mmol) of DIPEA was dissolved in 6 mL of DMSO. The resulting slurry was stirred at room temperature for 72 h to complete the reaction; the conversion was monitored by LC-MS spectroscopy. The reaction mixture was then poured into 50 mL of water, the precipitate formed was filtered, and then washed with another portion of water, isopropanol and hexane in that order. The product was dried under vacuum. The yield of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid [2-oxo-2-(2-thienylmethylcarbamoyl-amino)ethyl] ester was 61% (310 mg).

[0570] HPLC-MS (positive mode): m / z 507 / 508 (M+H) + ; Retention time 1.399min.

[0571] 1 H NMR (400MHz, DMSO-d6, ppm): δ = 10.67 (br s, 1H), 8.47 (br s,1H),8.03(d,J=8.0,2H),7.71(d,J=8.0,1H),7.64(d,J=8.0,2H),7.39(d,J=4.0,1H),7 .08-6.95(m,4H),6.22(s,1H),5.35(s,2H),4.92(s,2H),4.52(d,J=4.6,2H),2.39(s,3H).

[0572] Embodiment 6:

[0573] 4-[(4-Methyl-2-oxo-chromen-7-yl)oxymethyl]-N-[2-oxo-2-(2-thienylmethyl-carbamoylamino)ethyl]benzamide

[0574] The title compound was prepared analogously to Example 3. 90 mg of the title compound were obtained. Yield: 30%.

[0575] 1 H NMR (400MHz, DMSO-D6, ppm): δ = 10.52 (s, 1H), 8.82 (t, J = 5.7Hz, 1H), 8.63 (br.s, 1H), 7.89 (d, J = 8.0Hz, 2H), 7.68 (d, J = 8.7Hz, 1H), 7.55 (d, J = 8.0Hz, 2 H),7.39(d,J=4.2Hz,1H),7.07(m,2H),6.98(s,1H),6.95(m,1H),6.21(s,1 H),5.31(s,2H),4.23(d,J=5.2Hz,2H),4.00(d,J=5.7Hz,2H),2.39(s,3H).

[0576] Example 7

[0577] Synthesis of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid [2-oxo-2-(2-pyridylmethylcarbamoylamino)ethyl] ester (MyoMed-203)

[0578] Preparation of 2-chloro-N-(2-pyridylmethylcarbamoyl)acetamide:

[0579] To a stirred solution of 2-pyridylmethylamine (7.84 g, 72.5 mmol) in anhydrous dichloromethane (100 mL) cooled to -10°C was added chloroacetyl isocyanate (8.66 g, 72.5 mmol) and the reaction mixture was stirred at rt for 2 h. The precipitated solid was collected by filtration, washed with dichloromethane (2 x 30 ml) and dried to give 12.0 g (52.7 mmol, yield: 73%) of 2-chloro-N-(2-pyridylmethylcarbamoyl)acetamide.

[0580] 1 H NMR (500 MHz, chloroform-d): δ = 9.45 (s, 1H), 9.05 (s, 1H), 8.59 (s, 1H), 7.67 (t, J = 7.1 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.24-7.18 (m, 1H), 4.65 (d, J = 5.2 Hz, 2H), 4.13 (s, 2H).

[0581] HPLC-MS (negative mode) m / z 226 (MH) + ; Retention time 0.565min.

[0582] Preparation of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid:

[0583] To a solution of 7-hydroxy-4-methyl-chromene-2-one (80.6g, 458mmol) in acetone (1000mL), K2CO3 (94.9g, 687mmol) and 4-(bromomethyl) methyl benzoate (110g, 480mmol) were added, and the reaction mixture was refluxed for 3h. Then, it was cooled to rt and filtered. The filtrate was evaporated under reduced pressure, and the residue was mixed with water (1000mL). The insoluble solid was collected by filtration, washed with water, 2-propanol and hexane, dried and dissolved in DMSO (500mL). A 20% KOH aqueous solution (150mL) was added to the obtained solution of 4-[(4-methyl-2-oxo-chromene-7-yl)oxymethyl] methyl benzoate, and the mixture was stirred overnight at rt. After the reaction was completed, it was poured into water (3000mL) and acidified to pH 1-2 with 10% hydrochloric acid. After stirring for 30 min, the precipitated solid was collected by filtration, washed with water, 2-propanol and hexane, and dried to give 106 g (342 mmol, yield 75%) of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid.

[0584] Methyl 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate:

[0585] 1 H NMR (500MHz, DMSO-d6): δ = 7.98 (d, J = 8.1Hz, 2H), 7.68 (d, J = 8.5Hz, 1H), 7.60 (d, J = 8.1Hz,2H),7.12–6.96(m,2H),6.20(s,1H),5.32(s,2H),3.85(s,3H),2.38(s,3H).

[0586] 4-[(4-Methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid:

[0587] 1 H NMR (400MHz, DMSO-d6): δ = 12.99 (br s,1H),7.96(d,J=7.2Hz,2H),7.67(d,J=8.4Hz,1H),7.57(d,J=7.2Hz,2H),7.11–6.99(m,2H),6.20(s,1H),5.31(s,2H),2.37(s,3H).

[0588] HPLC-MS (positive mode) m / z 311 (M+H) + ; Retention time 1.242min.

[0589] Preparation of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid [2-oxo-2-(2-pyridylmethylcarbamoylamino)ethyl] ester

[0590] A mixture of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid (9.28 g, 29.9 mmol), 2-chloro-N-(2-pyridinylmethylcarbamoyl)acetamide (7.49 g, 32.9 mmol), DIPEA (4.64 g, 35.9 mmol) and NaI (0.900 g, 6.00 mmol) in DMSO (100 mL) was stirred overnight at rt and then poured into cold water (500 mL). The precipitated solid was collected by filtration, washed with water, 2-propanol and hexane, and dried to give 13.8 g (27.5 mmol, yield: 92%) of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid [2-oxo-2-(2-pyridinylmethylcarbamoyl-amino)ethyl] ester (MyoMed-203).

[0591] 1H NMR (400MHz, DMSO-d6): δ = 10.65 (br s, 1H), 8.69 (br s,1H),8.47(d,J=4.6Hz,1H),8.01(d,J=8.1Hz,2H),7.73(t,J=8.4Hz,1H),7.67(d,J=8.7Hz,1H),7.61(d,J=8.1Hz,2H),7.28(d ,J=7.7Hz,1H),7.26–7.20(m,1H),7.08–6.97(m,2H),6.19(s,1H),5.33(s,2H),4.91(s,2H),4.46(d,J=5.3Hz,2H),2.36(s,3H).

[0592] HPLC-MS (negative mode) m / z 502 (MH) + ; Retention time 1.176min.

[0593] Example 8

[0594] Synthesis of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-N-[2-(2-thienylmethyl-carbamoylamino)ethyl]benzamide (MyoMed-205)

[0595] Preparation of tert-butyl N-[2-(2-thienylmethylcarbamoylamino)ethyl]carbamate:

[0596] To a suspension of CDI (42.8 g, 264 mmol) in anhydrous acetonitrile (400 mL) was added 2-thienylmethylamine (14.9 g, 132 mmol) and the reaction mixture was kept in an ultrasonic bath at rt for 1 h. Then, water (2.5 mL) was added and the mixture was kept in an ultrasonic bath for another 30 min. After the solution was degassed, N-boc-ethylenediamine (21.1 g, 132 mmol) was added and the reaction was stirred at 50 ° C for 2 h. Then, the mixture was cooled to rt and evaporated under reduced pressure. The residue was triturated with water (100 mL), filtered, and dried to give 35.2 g (118 mmol, 89%) of tert-butyl N-[2-(2-thienylmethylcarbamoylamino)-ethyl]carbamate.

[0597] 1H NMR (400MHz, DMSO-d6): δ = 7.35 (d, J = 5.8Hz, 1H), 6.97–6.88 (m, 2H), 6.83–6.72 (m, 1H), 6.55–6.38 ( m,1H),6.07–5.93(m,1H),4.34(d,J=5.9Hz,2H),3.11–3.00(m,2H),2.99–2.88(m,2H),1.37(s,9H).

[0598] HPLC-MS (positive mode) m / z 300 (M+H) + ; Retention time 1.156min.

[0599] Preparation of 2-(2-thienylmethylcarbamoylamino)ethylammonium chloride:

[0600] To a solution of tert-butyl N-[2-(2-thienylmethylcarbamoylamino)-ethyl]carbamate (19.5 g, 65.1 mmol) in anhydrous dichloromethane (200 mL) was added 10% dioxane / HCl (50 mL) and the reaction was stirred at rt for 2 h. The precipitated solid was collected by filtration and dried in vacuo to give 14.3 g (60.6 mmol, 95%) of 2-(2-thienylmethylcarbamoylamino)ethylammonium chloride.

[0601] 1 H NMR (500MHz, DMSO-d6): δ = 8.08 (br s, 3H), 7.35 (s, 1H), 6.93 (s, 2H), 6.78 (brs, 1H), 6.51 (br s,1H),4.38–4.29(m,2H),3.31–3.19(m,2H),2.88–2.74(m,2H).

[0602] HPLC-MS (positive mode) m / z 200 (M+H) + ; Retention time 0.428min.

[0603] Preparation of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid:

[0604] To a solution of 7-hydroxy-4-methyl-chromene-2-one (80.6g, 458mmol) in acetone (1000mL), K2CO3 (94.9g, 687mmol) and 4-(bromomethyl)benzoic acid methyl ester (110g, 480mmol) were added, and the reaction mixture was refluxed for 3h. Then, it was cooled to rt and filtered. The filtrate was evaporated under reduced pressure, and the residue was mixed with water (1000mL). The insoluble solid was collected by filtration, washed with water, 2-propanol and hexane, dried and dissolved in DMSO (500mL). A 20% KOH aqueous solution (150mL) was added to the obtained solution of 4-[(4-methyl-2-oxo-chromene-7-yl)oxymethyl]benzoic acid methyl ester, and the mixture was stirred at rt overnight. After the reaction was completed, it was poured into water (3000mL) and acidified to pH 1-2 with 10% hydrochloric acid. After stirring for 30 min, the precipitated solid was collected by filtration, washed with water, 2-propanol and hexane, and dried to give 106 g (342 mmol, yield 75%) of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid.

[0605] Methyl 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate:

[0606] 1 H NMR (500MHz, DMSO-d6): δ = 7.98 (d, J = 8.1Hz, 2H), 7.68 (d, J = 8.5Hz, 1H), 7.60 (d, J = 8.1Hz,2H),7.12–6.96(m,2H),6.20(s,1H),5.32(s,2H),3.85(s,3H),2.38(s,3H).

[0607] 4-[(4-Methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid:

[0608] 1 H NMR (400MHz, DMSO-d6): δ = 12.99 (br s,1H),7.96(d,J=7.2Hz,2H),7.67(d,J=8.4Hz,1H),7.57(d,J=7.2Hz,2H),7.11–6.99(m,2H),6.20(s,1H),5.31(s,2H),2.37(s,3H).

[0609] HPLC-MS (positive mode) m / z 311 (M+H) + ; Retention time 1.242min.

[0610] Preparation of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-N-[2-(2-thienyl-methylcarbamoylamino)ethyl]benzamide:

[0611] To a solution of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid (12.6 g, 40.6 mmol) in DMA (150 mL) was added CDI (7.26 g, 44.8 mmol) and the mixture was stirred at rt for 30 min. Then, 2-(2-thienylmethylcarbamoylamino)-ethylammonium chloride (10.1 g, 42.7 mmol) and triethylamine (4.90 g, 48.4 mmol) were added and the reaction was stirred at 50° C. for 16 h. The mixture was cooled to rt and water (600 mL) was added. The precipitated solid was collected by filtration, washed with water, 2-propanol and hexane, and dried to give 13.3 g (27.1 mmol, 67%) of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-N-[2-(2-thienyl-methylcarbamoylamino)ethyl]benzamide (MyoMed-205).

[0612] 1 H NMR (400MHz, DMSO-d6): δ=8.61–8.47(m,1H),7.86(d,J=7.9Hz,2H),7.70(d,J=8.7Hz,1H),7.55(d,J=7.9Hz,2H),7.38–7.27(m,1H),7.12 –7.02(m,2H),6.51(t,J=6.0Hz,1H),6.22(s,1H),6.17–6.06(m,1H),5.30(s,2H),4.36(d,J=5.9Hz,2H),3.32–3.17(m,4H),2.39(s,3H).

[0613] HPLC-MS (positive mode) m / z 492 (M+H) + ; Retention time 1.249min.

[0614] II. Biochemical Analysis

[0615] 1. MuRF1–titin interaction assay:

[0616] A small molecule screen was performed using the ALPHA screen to identify compounds that inhibit the interaction between MuRF1 and titin. The screen is based on studies of the interaction between MuRF1 and titin that identified the interaction between the MuRF1 B-box-coiled domain and titin A169 (see, e.g., Mresk et al., Biochemistry 2008, 47, 10722-10730). An example of the ALPHA screening assay is described in WO2009 / 077618.

[0617] method:

[0618] These interacting fragments were expressed as GST and biotin fusion proteins, allowing the formation of the complex to be monitored using glutathione acceptor and avidin donor beads, respectively, as described in WO 2009 / 077618. A study of 280,000 compounds (in-house library, EMBL Chemistry Core Facility) identified a total of 40 molecules with Ki values ​​of 5-25 μmol / L for the MuRF1-titin interaction.

[0619] 2. Inhibition of MuRF1 Determination of E3 ligase activity:

[0620] The effects of the compounds on MuRF1 E3 ligase activity (either against titin or against MuRF1 itself (autoubiquitination)) were then evaluated by mixing 75 nmol / L UBE1 (Boston Biochem), 1 μmol / L UbcH5c (Boston Biochem), 100 μmol / L ubiquitin, 4 mmol / L ATP, 100 nmol / L titin A168-170 with 20-100 μmol / L of the corresponding compound. Reactions were initiated by the addition of 220 nmol / L MuRF1 and then reacted for 1 h at 37°C and analyzed by SDS PAGE and Western blot using MuRF1- and titin-specific antibodies. All reactions also included 5% DMSO. Based on the ubiquitination pattern, MyoMed-946, MyoMed-946-5, and MyoMed-946-8 were tested and determined to significantly inhibit MuRF1 E3 ligase activity.

[0621] 3. Differential scanning fluorescence of the central fragment of MuRF1 in the absence and presence of the compound MyoMed-946 Determination method (DSF):

[0622] The effect of compound MyoMed-946 on the stability of MuRF1 protein was determined in vitro using differential scanning fluorimetry (DSF).

[0623] method:

[0624] The "MuRF1 central fragment" was expressed as described previously (Mrosek M et al., FASEB J, 2007, 21, 1383-1392) and used in DSF experiments at a final concentration of 75 μM. A 10 mM DMSO stock solution of compound MyoMed-946 was diluted to 100 μM with PBS as DSF assay buffer to form a final concentration of 1% DMSO. After preincubation at room temperature for 1 hour, the protein aqueous solution was aspirated into capillaries and placed in a Prometheus NT.48 nanoDSF device (NanoTemper Technologies, Munich, Germany). The changes in intrinsic tryptophan or tyrosine fluorescence of the protein after LED laser excitation after thermal gradient unfolding were detected at 330 nm and 350 nm, respectively. The changes in intrinsic protein fluorescence of the thermal gradient were monitored at 350 nm and 330 nm, respectively. The transition midpoint (Tm) of single and multiple transition states was calculated using the first derivative of the fluorescence wavelength ratio of 350 / 330 nm during thermal protein unfolding.

[0625] result:

[0626] from Fig.55 It can be seen that in PBS, the Tm of the central fragment of MuRF1 is 65.2°C (solid line) and is only negligibly shifted to 65.8°C (dashed line) by the addition of 1% DMSO. In contrast, a strong effect on the thermal unfolding of MuRF1 can be observed by the addition of the compound MyoMed-946 (dotted line). The compound MyoMed-946 destabilizes MuRF1, as shown by the significantly reduced main Tm of 52.5°C.

[0627] III. Biological Research

[0628] 1. Cell culture experiments

[0629] Mouse C2C12 myoblasts (CRL-1772, ATCC) were cultured in medium supplemented with 10% fetal calf serum (FCS; Invitrogen, Carlsbad, CA) in DMEM (Lonza; Basel, Switzerland). To induce differentiation into myotubes, subconfluent cultures were switched to DMEM containing 2% horse serum (Sigma-Aldrich; Seelze, Germany). Subsequently, myotubes were pretreated with increasing compound concentrations (0.1-10 μmol / L, dissolved in DMSO) or with an equal volume of DMSO for 2 h and then treated with 10 μmol / L dexamethasone (DEX; Sigma-Aldrich; Seelze, Germany) for 24 h. Myotube diameter was then assessed by image analysis software (Analysis 3.0, Olympus Soft Imaging Solutions GmbH, Münster, Germany). To determine the cytotoxicity of the selected compounds, myoblasts or myotubes were incubated with increasing concentrations for 24 h. Subsequently, the concentration of lactate dehydrogenase (LDH) activity in the cell culture supernatant was quantified as a measure of cell damage as described in Bellocci et al., Anal Biochem, 2008, 374, 48-55. Figure 1 and Figure 2 It can be seen that the compound MyoMed-946 shows low toxicity in myoblasts and myotubes (muscle cells).

[0630] 2. Animal experiments

[0631] 2.1 Pulmonary hypertension mouse model:

[0632] 2.1.1 Test Series 1 with the MuRF1 Inhibitor MyoMed-946:

[0633] Animal experiments Karlsruhe (35-9185.81 / G-141 / 13) and Approval from the Leipzig (TVV 40 / 16). Three groups of mice were included in this study, including: 1) saline-treated (sham, n=20); 2) monocrotaline (MCT)-treated (MCT; n=27) fed a normal chow; and 3) MCT-treated (MCT+compound; n=27) fed a MuRF1 inhibitor chow. Briefly, C57BL / 6 mice (8 weeks old) were injected subcutaneously with MCT (600 mg / kg) or a matching volume of saline weekly for 6 weeks, a period of time known to induce psychogenic cachexia due to pulmonary hypertension and subsequent RV dysfunction rather than anorexia as described in Ahn et al., PLoS One, 2013, 8:e62702. Mice were exposed to identical conditions, 12:12 h light / dark cycle, with free access to food and water. One week before MCT injection, the MCT+compound group began to receive inhibitor food, while the sham and MCT groups were given the same food, but the selected compound was not added. The body weight of each mouse was recorded every week. Mice were killed after deep anesthesia by intraperitoneal injection of fentanyl (0.05mg / kg), medetomidine (0.5mg / kg), midazolam (5mg / kg) and ketamine (100mg / kg). At the time of killing, the heart and lung were dissected, cleaned, blotted and weighed, and the heart was fixed with 4% PBS-buffered formalin. The left tibialis anterior (TA), soleus, extensor digitorum longus (EDL) and costal diaphragm slices were also dissected, weighed, and fixed with 4% PBS-buffered formalin, and the remaining muscle parts were immediately frozen in liquid N2 for molecular analysis.

[0634] For histological evaluation, paraffin-embedded TA muscle sections (3 μm) were stained with H&E, and the fiber cross-sectional area (CSA) was evaluated by imaging software (Analysis 3.0, Olympus Soft Imaging Solutions GmbH, Münster, Germany). In addition, medial cross-sections (3 μm) of the heart were mounted on slides and subsequently stained with H&E to assess RV wall thickness.

[0635] from Figures 3 to 5 As can be seen, body weight gain, lung weight, and heart weight were almost identical in MCT and MCT+MyoMed-946 fed mice, suggesting that disease progression in both groups was independent of compound feeding. Figure 6 and Figure 7 It can be seen that the MuRF1 inhibitor MyoMed-946 slows the progression of right ventricular hypertrophy. Figures 8 to 12 As can be seen, MCT treated mice showed a progressive loss of skeletal muscle mass, however MCT + compound fed mice did not follow this trend and were protected - the most pronounced effect was seen in the TA muscle.

[0636] 2.1.2 Test Series 2 with MuRF1 Inhibitors MyoMed-946, MyoMed-203 and MyoMed-205:

[0637] Test series 2 was performed similar to test series 1. Briefly, three groups of mice were included in this study: 1) saline-treated mice (sham group, n=10); 2) monocrotaline (MCT)-treated mice fed normal chow (MCT; n=10); and 3) MCT-treated mice fed MuRF1 inhibitor chow (MCT+compound; n=10 for each compound). Eight-week-old C57BL / 6 mice were injected subcutaneously with MCT (600 mg / kg) or a matching volume of saline weekly for 8 weeks. One week before the MCT injection, the MCT+compound group began to receive the inhibitor chow, while the sham and MCT groups were given the same chow without the addition of the selected compound. The compound concentration in the chow was 0.1% by weight, resulting in a daily intake of approximately 3 mg of compound per mouse. The body weight of each mouse was recorded weekly. Mice were sacrificed after 8 weeks of treatment. At the time of sacrifice, examination and tissue analysis were completed as described in test series 1.

[0638] from Figures 13 to 15 As can be seen, weight gain, lung weight, and heart weight were similar in MCT and MCT+compound-fed mice, suggesting that disease progression in both groups was independent of compound feeding. Figures 16 to 18 It can be seen that the MuRF1 inhibitors MyoMed-946, MyoMed-203, and MyoMed-205 slowed the progression of right ventricular hypertrophy. Figures 19 to 21 It can be seen that while the MCT-treated mice showed a progressive loss of skeletal muscle mass, the MCT+compound-fed mice did not follow the described trend and were protected – the most pronounced effect was seen with compound MyoMed-203. In addition, the MuRF1-expression levels in TA muscle tissue were significantly reduced in the MCT+compound-fed mice, with the most pronounced decrease being seen with compound MyoMed-205, as shown in Figure 2. Fig. 22 In MCT+compound-fed mice, the expression levels of telethonin (a MuRF1 target protein) were essentially normalized, as shown in Figure 2. Fig.23 shown.

[0639] 2.2 Myocardial infarction LAD mouse model:

[0640] As described below, a myocardial infarction mouse model of heart failure with reduced ejection fraction (HFrEF) was established by ligating the left anterior dexterior coronary artery (LAD) to induce acute myocardial infarction (MI), followed by the development of chronic (systolic) heart failure (CHF).

[0641] LAD ligation procedure:

[0642] Small animal surgery was performed for 12-week-old C57 / BL6 mice at the Leipzig Heart Center following established and well-known methods in the art (see, e.g., Bowen et al., J Appl Physiol, 2015, 118, 11–19; Mangner et al., J Cachexia Sarcopenia Muscle, 2015, 6, 381–390). Briefly, mice in the LAD group were anesthetized by intraperitoneal injection of MMF, medetomidine (0.5 mg / kg body weight), midazolam (5.0 mg / kg body weight), and fentanyl (0.05 mg / kg body weight). Comatose mice were fixed on the operating table. The ventral part of the chest was shaved, cleaned, and disinfected. Then, the comatose mice were intubated and ventilated with normal room air using an animal respirator (TSE GmbH, Siemensstr. 21, 61352 Bad Homburg; product: http: / / tinateb.com / wp-content / uploads / 2016 / 06 / TSE_Respirator-Compact_20080724_HR.pdf). For LAD surgery, the chest cavity was opened by incising the skin about 1 cm above the xiphoid process and about 1 cm beside the left sternum. Without further injury, the pectoralis major muscle below was moved to the side wall to contact the chest wall. The intercostal muscles between the two ribs were moved to one side without breaking the ribs. The intrathoracic passage created thereby was enlarged by inserting a surgical retractor above the pericardium. The pericardium was cut open with two dissecting forceps. If the thymus is present in the surgical field of view, the thymus is pushed to one side with a surgical swab to access the aortic root. The heart was carefully removed from the pericardium with a hook, and the left atrium was put aside with a surgical swab. The LAD was then ligated with 5.0 Prolene suture (Ethicon, see http: / / www.ethicon.com / healthcare-professionals / products / wound-closure / non- absorbable-sutures / prolene-polypropylene ). Ligation was performed close to the aortic root and the sutures were tightened until pallor of the anterior coronary artery wall was observed. After ligation, the chest wall and skin were sutured in sequence with 4.0 Prolene sutures (Company: Ethicon).

[0643] As a control, a sham operation (or "fake treatment") was performed. For the sham control group, the subsequent operation was exactly the same as the above operation except that the 5.0 prolene suture was loosened around the LAD instead of tightening.

[0644] The surgery ended with extubation and intravenous injection of Atipamezol (2.5 mg / kg body weight) and Flumazenil (0.5 mg / kg body weight) to counter anesthesia. The mouse was placed on a warm partner until it woke up and then transferred back to the animal cage. The entire process took about 30 minutes for the expert to complete.

[0645] One week after LAD ligation, M-mode echocardiography was performed to determine MI, i.e., to assess left ventricular end-diastolic (LVEDD) and systolic (LVESD) diameters to allow calculation of left ventricular (LV) fractional shortening (LVFS = [LVEDD - LVESDLVEDD] x 100). Subsequently, only mice with large infarction (left ventricular ejection fraction (LVEF) < 20%) were randomly divided into two groups, i.e., one group received normal chow (CHF, n = 11), while the second group received chow supplemented with compound (0.1% compound MyoMed-946, CHF + MyoMed-946, n = 12). Sham animals received normal chow only (n = 15). After 9 weeks, echocardiography was performed again.

[0646] Table 1: Animal characteristics after 10 weeks of intervention

[0647]

[0648]

[0649] *P<0.05 vs. sham operation; ***P<0.001 vs. sham operation.

[0650] Animals were sacrificed to collect tissues, especially diaphragm tissue, for functional and molecular characterization. All experiments and manipulations were approved by the local animal research committee, the University of Leipzig and Approval of Sachsen (TVV 36 / 15).

[0651] 2.3 Contraction function:

[0652] In order to provide a direct functional assessment, the contractility of skeletal muscle fiber bundles, i.e., fiber bundles from the diaphragm of MCT mice and CHF mice, was measured as follows. As described in Bowen et al., FASEB J, 2017, 31, fiber bundles were isolated from the diaphragm, thereby evaluating in vitro contractile function using a length-controlled lever system (301B, Aurora Scientific Inc., Aurora, Canada). In brief, the muscle bundle was vertically loaded in an organ bath (~22 ° C) filled with buffer, set to the optimal length, and after 15min, stimulated by a force-frequency scheme of 1-300Hz (600mA; 500ms training duration; 0.25ms pulse width). Then, the muscle undergoes a force-speed scheme, whereby after stimulating 300ms at 150Hz, the muscle is allowed to shorten under external load (80-10% of the maximum tetanic force; 1min interval each time). 10ms after the first change in length, and the shortening speed is determined on a transient linear cross section (DMA software, Aurora Scientific). Force (N) was calculated relative to muscle cross-sectional area (CSA; cm) by dividing muscle mass (g) by the product of Lo (cm) and estimated muscle density (1.06). 2 ) normalized, which allows calculation of N / cm 2 The shortening rate is relative to the optimal muscle length (L o / s) and the power of each load is calculated as the product of shortening speed and specific force (W / cm 2 ).

[0653] from Figures 24 to 27 It can be seen that diaphragm muscle fiber bundles from chronic heart failure mice (CHF mice) generated less force during electrical stimulation ( Fig.24 ), and also reduces the maximum force ( Fig.25 and 26 ) and peak power ( Fig. 27 ). Thus, CHF mice in the control chow group developed diaphragmatic myopathy at week 10 compared to sham-operated animals. Feeding with the compound MyoMed-946 protected mice from the post-infarction diaphragmatic weakness described above. Mice fed with the compound MyoMed-946 had significantly reduced chronic heart failure-induced loss of diaphragmatic function and diaphragmatic maximum strength compared to untreated CHF mice. This suggests that selective inhibition of MuRF1 with the compound MyoMed-946 has a beneficial effect on diaphragmatic function following myocardial infarction-induced heart failure with reduced ejection fraction (HFrEF).

[0654] Likewise, from Figures 28-30As can be seen, when the MCT mice were fed the compound MyoMed-946 (MCT+MyoMed-946), the contractile dysfunction of the diaphragm (in terms of shortening velocity and power) was also substantially prevented in mice with MCT-induced pulmonary hypertension.

[0655] Thus, taken together, the above results suggest that selective inhibition of MuRF1 by the compound MyoMed-946 produces beneficial effects on both the quantity (i.e., amount) and quality (i.e., contractile function) of skeletal muscle in chronic heart failure and psychogenic cachexia.

[0656] 2.4 Tumor mouse model:

[0657] Eight-week-old female C57BL / 6N mice were inoculated with B16F10 melanoma cells (9×10 5 cells) or saline (sham-treated group, n=10). Within the first 3 days after tumor inoculation, both B16F10 mice and sham-treated mice received regular food. Then, the B16F10 mice were randomly divided into three groups, wherein the first group received MyoMed-946 food (tumor+MyoMed-946, n=10), the second group received MyoMed-205 food (tumor+MyoMed-205, n=10), and the third group received the same food but without the addition of the selected compound (tumor, n=10). The concentration of the compound in the food was 0.1% by weight, resulting in an intake of approximately 3 mg of compound per mouse per day. In addition, the sham-treated group was fed the same food but without the addition of the selected compound. On days 9, 16, and 23 after tumor inoculation, each mouse was subjected to a wire suspension test (muscle function). To this end, a standard wire suspension structure was used, with the wire (length: 40 cm, diameter: 2.5 mm) 70 cm above the ground. Below the center of the wire, a large box filled with sawdust was placed. For the test, mice were suspended from the wire by two limbs and the hanging time was recorded. Each animal was tried three times, each for a maximum of 180 seconds. After three attempts, the maximum holding impulse was calculated (hanging time x body weight).

[0658] Twenty-five days after tumor inoculation, mice were sacrificed and, at the time of sacrifice, examination and tissue analysis were completed as described above.

[0659] from Fig.31 It can be seen that tumor-induced skeletal muscle atrophy (based on TA muscle weight) was at least somewhat attenuated in tumor+MyoMed-946 and tumor+MyoMed-205 mice compared to tumor mice. Fig.32), where administration of compounds MyoMed-946 and MyoMed-205 (tumor+MyoMed-946 mice and tumor+MyoMed-205 mice) significantly attenuated the loss of muscle function compared to tumor-bearing mice fed a normal diet.

[0660] 2.5 Myocardial and skeletal muscle changes in heart failure with preserved ejection fraction (HFpEF) – rat model

[0661] A schematic diagram of the study design is shown in Fig.56 For this experiment, ZSF1 rats were used. After 20 weeks of age, ZSF1 rats lose diastolic compliance, resulting in an increase in end-diastolic volume, thus mimicking human HFpEF (also known as diastolic heart failure).

[0662] Female ZSF1 lean mice (control group, n = 25) and ZSF1 obese mice (n = 40) were included in the study. At 20 weeks of age, the development of HFpEF was confirmed by echocardiography / invasive hemodynamic measurements, and tissue material was collected from one group of animals (control group n = 10; ZSF1 obese n = 10). The remaining control group rats (control group, n = 15) were left alone for another 12 weeks, while the remaining ZSF1 obese animals (n = 30) were randomly divided into the following groups: (1) rats receiving normal chow (HFpEF group, n = 15) or rats receiving chow supplemented with the compound MyoMed-205 (0.1% MyoMed-205, HfpEF+MyoMed-205, n = 15). The rats were exposed to the same conditions, with a 12-h light / dark cycle, and food and water were provided freely. Twelve weeks after randomization, echocardiography and invasive hemodynamic measurements were completed to clarify the extent of diastolic dysfunction. The rats were subsequently sacrificed (thoracotomy under deep anesthesia), and skeletal and cardiac muscle tissues were collected for functional and molecular analyses (formalin fixation and quick freezing in liquid nitrogen). All experiments and manipulations were approved by the local animal research committee, the Technical University of Dresden, and Approval of Sachsen (TVV42 / 2018).

[0663] Echocardiography

[0664] As previously described, rats were anesthetized with isoflurane (1.5-2%) and transthoracic echocardiography was performed using a Vevo 3100 system and a 21-MHz transducer (Visual Sonic, Fujifilm) to assess cardiac function (TS Bowen et al., Eur. J. Heart Fail., 2015, 17, 263-272). For systolic function, B and M modes were measured in the parasternal long and short axes at the level of the papillary muscles. Diastolic function was assessed in the apical 4-chamber view at the level of the basal septal segment of the left ventricular septal wall using pulsed wave Doppler (for measuring the early (E) wave and atrial (A) wave of the mitral valve velocity) and tissue Doppler (for measuring myocardial velocity (E′ and A′)). Functional parameters (ie, LV ejection fraction (LVEF) and stroke volume (SV), as well as the ratios of [E / E'] and [E / A]) were obtained using Vevo LAB 2.1.0 software.

[0665] Invasive hemodynamic measurements:

[0666] As a final step, invasive hemodynamic pressure measurements were completed. In rats that were anesthetized (ketamine, xylazine) but breathing spontaneously, the right carotid artery was cannulated with a rat PV catheter (SPR-838, ADInstruments Limited), and the cannula was gently placed in the middle of the left ventricle. For left ventricular diastole, LV end-diastolic and end-systolic pressures, maximum rate of pressure rise (dP / dtmax), maximum rate of pressure decrease (dP / dtmin), and time constant (τ) were measured, and then the catheter was withdrawn to the aorta, and the phase pressure and mean arterial pressure were measured. The mean arterial pressure of the ascending aorta was measured. The data were recorded in LabChart8 software (ADInstruments).

[0667] Skeletal muscle function

[0668] The right EDL and left soleus muscles were segmented and mounted vertically in Krebs-Henseleit buffer between a hook and a force transducer, and the output was continuously recorded and digitized (1205A: Isolated Muscle System-Rat, Aurora Scientific Inc., Ontario, Canada). In vitro muscle function was assessed by stimulating the muscles with supramaximal current (700 mA, 500 ms training duration, 0.25 ms pulse width) using platinum electrodes with a high-power bipolar stimulator (701C; Aurora Scientific Inc., Ontario, Canada) (700 mA, 500 ms training duration, 0.25 ms pulse width). The muscle fascicle was set to an optimal length (Lo) equal to the maximum twitch force produced, and the bath temperature was then raised to 25°C, followed by 15 minutes of thermal equilibration. Then, force-frequency protocols were completed at frequencies of 1 Hz, 15 Hz, 30 Hz, 50 Hz, 80 Hz, and 120 Hz, with 1 minute rest intervals. After a 5 minute rest period, the muscles were then subjected to a fatigue protocol for 5 minutes (every 2 seconds, 40 Hz). The forces generated during the fatigue protocol were normalized to the initial forces generated to provide a relative assessment of fatigue performance.

[0669] result:

[0670] Animal characteristics at 20 weeks (random time point)

[0671] To verify the progression of HFpEF, echocardiography, invasive hemodynamic measurements, and skeletal muscle function measurements were performed on 10 ZSF1-lean and 10 ZSF1-obese animals before randomization into different treatment groups. ZSF1-obese animals showed weight gain (a sign of obesity), and signs of cardiac hypertrophy (increased heart weight when normalized to tibia length) were evident. Regarding markers of diastolic function, the E / é ratio and left ventricular end-diastolic pressure (LVEDP) were significantly increased in ZSF1-obese animals. Despite the diastolic dysfunction, the left ventricular ejection fraction (LVEF) was normal (>60%) and even slightly higher than that of the lean control group. For mean arterial blood pressure (MABP), a significant increase was found in ZSF1-obese animals. The increase in MABP is a sign of the hyperosmotic state of the animals, a feature that is well known in patients with HFpEF. Regarding peripheral skeletal muscle, ZSF1-obese animals developed muscle atrophy and skeletal muscle dysfunction. In summary, 20-week-old animals presented features consistent with the diagnosis of HFpEF.

[0672] Effects of MyoMed-205 Treatment on Cardiac Parameters in HFpEF

[0673] To evaluate the effects of MyoMed-205 on myocardial function, echocardiography and invasive hemodynamic measurements were performed. Fig.57 As shown, when compared with control ZSF1-lean animals, the left ventricular ejection fraction (LVEF) of untreated HFpEF animals ( Fig.57 A) was significantly reduced. After 12 weeks of treatment with MyoMed-205, the reduction was significantly attenuated. Regarding the diastolic function parameter E / é ratio ( Fig.57 B) and LVEDP( Fig.57 C), Treatment with MyoMed-205 attenuated the increases seen in untreated ZSF1-obese animals. For MABP, MyoMed-205 was found to have no therapeutic effect ( Fig.57 D). Taken together, the results indicate that MyoMed-205 treatment significantly improves both systolic and diastolic function and that the effects are not mediated by regulation of blood pressure.

[0674] Effects of MyoMed-205 treatment on skeletal muscle mass and function

[0675] At the time point when randomized to the different treatment groups, ZSF1-obese animals had impaired bone mass and skeletal muscle function. This study tested whether MyoMed-205 could modulate skeletal muscle mass and function. Results can be found in Fig.58 . Tibialis anterior (TA) muscle weight analysis ( Fig.58 A) shows a significant decrease in muscle wet weight in untreated HFpEF animals. MyoMed-205 attenuated this muscle atrophy. Fig.58 C) and soleus ( Fig.58 In EDL muscles, however, treatment with MyoMed-205 resulted in a small but significant increase in muscle weight ( Fig.58 C).

[0676] In addition to measuring muscle weight, muscle function evaluation is also very important for the progression of muscle atrophy. Fig.58 As shown in D, untreated ZSF1-obese animals developed skeletal muscle dysfunction when compared to ZSF1-lean controls. When absolute specific muscle force was measured, the decrease in muscle force was evident in the soleus muscle ( Fig.58 D) Treatment of HFpEF animals with MyoMed-205 for 12 weeks attenuates functional loss.

[0677] in conclusion:

[0678] Conclusions: MyoMed-205 attenuates the development of myocardial diastolic dysfunction and reduces skeletal muscle atrophy and dysfunction in HFpEF.

[0679] 2.6 Doxorubicin-induced muscle atrophy and cardiotoxicity

[0680] Doxorubicin (DOX) is an effective chemotherapeutic agent used in the treatment of various cancers. However, its use is associated with early and chronic cardiotoxicity and myotoxicity. In rodents, a single injection of doxorubicin reduces cardiac and skeletal muscle mass, followed by significant functional impairment.

[0681] The effect of MyoMed-205-enriched diet was evaluated in a mouse model treated with doxorubicin. C57bl / 6 mice were acclimated in an animal house for 3 days and then randomly divided into 4 groups as follows: 1. Control group (placebo diet + intraperitoneal injection of 0.9% saline); 2. MyoMed-205 (MyoMed-205-diet, diet supplemented with 1g / kg MyoMed-205; + 0.9% saline injection); 3. DOX (placebo diet + DOX injection); 4. DOX+MyoMed-205 (MyoMed-205 diet + DOX injection). Animals were pre-fed with MyoMed-205 diet or placebo for 7 days before the first DOX injection. The total dose of DOX treatment was 25 mg / kg, injected 5 times on days 10, 12, 16, 25 and 28, respectively. The body weight and food intake of the animals were measured daily at the same time of the day to assess food consumption and weight gain. Cardiac function was evaluated by echocardiography on days 19 and 42. Fig.59 As shown, MyoMed-205 feeding significantly reduced the characteristics of cachexia and physical stress at day 43: MyoMed-205-fed mice had higher lean body mass ( Fig.59 A), retain more body fat ( Fig.59 B), and interstitial edema and free fluid decreased by approximately 2-fold ( Fig.59 C). * means p < 0.05 vs. control; $ means p < 0.05 vs. MyoMed-205; § means p < 0.05 vs. DOX+MyoMed-205 (Tukey post-test).

[0682] After the cumulative DOX dose of 25 mg / Kg and on day 15, echocardiography showed cardiotoxicity and failure, which could be seen from Fig.60 As can be seen, the heart weight was reduced (corrected for tibia length) ( Fig.60 C), decreased ejection fraction (calculated from the long axis distribution diagram) ( Fig.60 A), and the reduced fraction (calculated from the short axis distribution plot) ( Fig.60B). MyoMed-205 treatment did not prevent this until day 43. In conclusion, the administration of MyoMed-205 helped protect the heart during DOX chemotherapy. * refers to p<0.05v. control; $ refers to p<0.05v.MyoMed-205; § refers to p<0.05v.DOX+MyoMed-205 (Tukey post-test).

[0683] 2.7 Muscle function in type 2 diabetic obese mice during diet-induced weight loss (DIO mouse model: diabetes with insulin resistance and obesity)

[0684] The effect of compound feeding was tested using a diabetic mouse model that also develops myopathy during the progression of diabetes. In DIO mice, diabetes was induced for 4 months using a high fructose, high fat diet (HFD). Body weight increased by 22.2%. The mice developed increased fasting blood glucose and insulin resistance. Control animals received a normal rodent diet (regular diet). Then, during the 30-day test period, weight loss was induced. In the first phase, the animals were fed a normal rodent diet for 16 days. Then, in the second phase, the animals received a low-calorie diet for 14 days. At the end point, the average weight loss was 12.4%. Two days after the start of the first phase, MyoMed-205 (1g compound / 1kg food) was added to the diet. The muscle strength of compound-fed mice and control mice was compared using a wire suspension test at the beginning of the first phase and then on days 3, 7, 14, 21, and 28 of the test period.

[0685] Experimental design: ICR-DIO male mice were randomly divided into groups of 8-10 animals each, including three control groups: a control group of mice without obesity (control group; regular diet (RD); n = 10); obese mice, fed a high-fructose high-fat diet (DIO HFD; n = 8) throughout the study; and obese mice that began to lose weight (DIO control; n = 8).

[0686] Wire hanging test: For the wire hanging test (WHT), a standard wire hanging structure (length: 40 cm, diameter: 2.5 mm) was used, at a height of 70 cm above the ground. Below the center of the wire, a large box filled with sawdust was placed. For the test, mice were hung on the wire with two limbs, and the time the mouse was able to hold the wire before falling off was recorded. Three attempts were made for each animal, with a maximum of 180 seconds. After three attempts, the maximum holding impulse (hanging time x body weight) was calculated.

[0687] result:

[0688]

[0689] Fig.61Graph showing the results of WHT in MyoMed-205-treated DIO mice compared to DIO control mice. As can be seen, MyoMed-205 significantly improved muscle strength compared to DIO control mice by week 4 (28 days). Muscle strength was also significantly improved compared to DIO HFD mice and was not significantly different from control (RD) mice at week 4 ( Fig.61 not shown).

[0690] 2.8 Glucose and insulin regulation during diet-induced weight loss in type 2 diabetic obese mice (DIO mouse model: diabetes with insulin resistance and obesity

[0691] Similar to Example 2.7, diabetes was induced in DIO mice with a high fructose, high fat diet (HFD) for 4 months. The mice developed increased fasting blood glucose and insulin resistance. The control group animals received a normal rodent diet (regular diet). Then, weight loss was induced during the 30-day test period. In the first phase, the animals were fed a normal rodent diet for 16 days. Then, in the second phase, the animals received a low calorie diet for 14 days. Two days after the start of the first phase, MyoMed-203 (1 g compound / 1 kg food) was added to the diet.

[0692] Experimental design: ICR-DIO male mice were randomly divided into groups of 8-10 animals each, including three control groups: a control group of mice without obesity (control group; regular diet (RD); n = 10); obese mice, fed a high-fructose high-fat diet (DIO HFD; n = 8) throughout the study; and obese mice that began to lose weight (DIO control; n = 8).

[0693] Blood glucose level measurement:

[0694] After fasting for 6 hours, blood glucose levels were measured. An On Call Plus blood glucose meter (Acon Laboratories, Inc., USA) and special test strips (REF G133-111) were used to measure blood glucose levels. Blood was collected from the tail vein by cutting the tip of the tail. 5 to 6 μl of blood was used for each test.

[0695] Blood glucose levels in MyoMed-203-treated animals were significantly lower than those in the DIO control group throughout the treatment period, with significant changes in fasting blood glucose values ​​on days 2 and 21 of the study. Fig.62 Shown are comparisons of MyoMed-203-treated DIO mice to DIO control mice.

[0696] Glucose tolerance test

[0697] For the oral glucose tolerance test (OGTT), mice were orally treated with glucose at a dose of 2 g / kg in a dose volume of 10 ml / kg after 6 hours of fasting. Glucose measurements were performed before treatment, 15 minutes, 30 minutes, 60 minutes, and 120 minutes after glucose administration. The AUC describing the blood glucose output rate of each mouse during the test was calculated using the following formula:

[0698]

[0699] MyoMed-203 showed a mild hypoglycemic effect in obese mice after weight loss and 14 days of treatment, according to OGTT data. Blood glucose levels at 30 minutes of the OGTT test tended to be lower in this group of animals compared to the DIO control group. The same was true for the calculated AUCs describing glucose elimination. On day 28 of the study, a similar trend continued: a significant reduction in blood glucose levels at 120 minutes was observed in the MyoMed-203-treated group compared to all control groups; the calculated area under the glucose elimination curve also tended to be lower. Fig.63 and Fig.64 OGTT data of MyoMed-203-treated DIO mice and DIO control mice on days 14 and 28, respectively, are shown.

[0700] Insulin tolerance test

[0701] For the insulin tolerance test (ITT), mice were injected intraperitoneally with a recombinant human insulin solution (Eli Lilly, France; REF C620001K) at a dose of 0.75 IU / g body weight or 0.60 IU / g body weight in a volume of 5 ml / kg after 6 hours of fasting. Glucose measurements were performed before treatment, 15 min, 30 min, 60 min and 120 min after insulin administration. The results of blood glucose output rate were expressed as the area under the curve (AUC).

[0702] A significant decrease in blood glucose levels was observed in MyoMed-203-treated mice 120 minutes after insulin injection compared to all control groups. Fig.65 ITT results of MyoMed-203-treated DIO mice and DIO control mice are shown.

[0703] 3. Organizational Analysis

[0704] 3.1 Proteomics and Western Blot Analysis:

[0705] 3.1.1 MCT mice:

[0706] Proteomic analysis:

[0707] Proteins from frozen diaphragm samples of sham, MCT and MCT+compound mice (n=3 per group) were crushed under liquid N2. Then, mass spectrometry analysis was completed at the DZHK Core Facility in Bad Nauheim, as described in Konzer et al., Methods in molecular biology, 2013, 1005, 39-52. The relative ratios of MCT / sham and MCT+compound / MCT were determined, and only hits were considered to be differentially expressed, which were further studied by Western blot and were highly significant (P<0.01).

[0708] Western blot analysis:

[0709] Western blot analysis involved homogenization of frozen TA muscle samples in RIPA buffer (50 mmol / L Tris, 150 mmol / L NaCl, 1 mmol / L EDTA, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 1% Triton X-100; pH 7.4) or relaxation buffer (90 mmol / L HEPES, 126 mmol / L KCl, 1 mmol / L MgCl, 50 mmol / L EGTA, 8 mmol / L ATP, 10 mmol / l creatine phosphate; pH 7.4) containing a protease inhibitor mix (inhibitor mix M, Serva, Heidelberg, Germany), sonication, and centrifugation at 16,000 x g for 5 min. The protein concentration of the supernatant was determined (BCA assay, Pierce, Bonn, Germany), and the samples (5-20 μg) were separated by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene fluoride membranes (PVDF) and incubated overnight at 4°C with the following primary antibodies: MAFbx (1 / 2000, Eurogentec, Seraing, Belgium), MuRF1 (1 / 1000, Myomedix Ltd., Neckargemünd, Germany), CARP (1 / 500, Myomedix Ltd., Neckargemünd, Germany), BAX (1 / 1000; Abcam, Cambridge, UK) and eIF2B-delta (1 / 200; Santa Cruz Biotechnolgy, Santa Cruz, USA). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and specific bands were visualized by enzymatic chemiluminescence (Super Signal West Pico, Thermo Fisher Scientific Inc., Bonn, Germany) and the density was quantified using the ID scanning software package (Scanalytics Inc., Rockville, USA). The blots were then normalized to the loading control GAPDH (1 / 30000; HyTest Ltd, Turku, Finland). All data are presented as fold change relative to the sham-treated group.

[0710] The expression levels of five proteins were found to be specifically responsive to the compounds, as shown by comparing the MCT and MCT+MyoMed-946 proteomes. Table 2 summarizes the expression levels of the proteins.

[0711] Table 2

[0712]

[0713] This included upregulation of eIF2B (δ subunit) and downregulation of BAX, which was subsequently confirmed by Western blotting and was seen in Figures 33-35 . The eIF2B pathway is a known translation regulator of protein synthesis. Previously, eIF2B was identified as a MuRF1 interactor, indicating that MuRF1-mediated depletion of the translation initiation factor eIF2B under MCT stress, but the compound alleviated this situation. In addition, the compound also regulates the pro-apoptotic regulator BAX, which is upregulated in MCT mice compared to the sham treatment group, but normal in the MCT+MyoMed-946 group. In human patients with chronic heart failure, apoptosis increases in skeletal muscle and is closely related to the degree of atrophy (see, for example, Adams et al., J Am Coll Cardiol, 1999, 33, 959-965 and Vescovo et al., Heart, 2000, 84, 431-437). Indeed, it has been previously noted that BAX is elevated in psychogenic cachexia and is associated with increased MuRF1 expression (see e.g. Dalla Libera et al., Am J Physiol Cell Physiol, 2004, 286, C138-144 and Rezk et al., PLoS One, 2012, 7, e30276).

[0714] from Figures 36-38 It can be further seen that proteomic analysis confirmed that the increase in MuRF1 protein expression in MCT mice was prevented by the compound MyoMed-946, while no effect was observed on MAFBx (another key atrogin E3 ligase). This suggests that the underlying mechanism of the compound appears to be MuRF1 specific, which is expected based on preliminary in vitro studies. MuRF1 is also known to interact with many substrates, one of which is CARP (a member of the muscle anchor protein repeat protein (MARP) family), which is a nuclear- and sarcomere (titin)-based protein with a purported transcriptional function (see, e.g., Miller et al., J Mol Biol, 2003, 333, 951-964). CARP is known to be upregulated under stress-related conditions and is associated with contractile dysfunction and muscle atrophy (see, e.g., Laure et al., The FEBS journal, 2009, 276, 669-684 and Moulik et al., J Am Coll Cardiol, 2009, 54, 325-333). Consistent with this evidence, increased expression of CARP was observed in MCT-stressed mice, whereas this effect was abolished in MyoMed-946-fed mice, suggesting that this compound may attenuate CARP expression through inhibition of MuRF1, which in turn contributes to the maintenance of muscle mass and function.

[0715] 3.1.2 CHF mice:

[0716] To analyze the molecular mechanisms underlying the physiological changes and benefits observed in MyoMed-946-treated mice with CHF, comparative quantitative proteomic analysis and Western blot analysis were completed on diaphragm tissues of sham, CHF, and CHF+MyoMed-946-treated mice.

[0717] Proteomic analysis:

[0718] Mass spectrometry-based proteomic analysis was performed at the DZHK Core Facility, Bad Nauheim, Germany. The obtained MS raw data were processed by MaxQuant (1.6.0.1) using the Andromeda search engine and the Uniprot mouse database (as of April 20, 2017). At a false discovery rate of 1% (peptide and protein level), >2600 protein groups were identified. The reductive dimethylation protocol used (see Boersema et al., Nat Protoc, 2009, 4, 484-494) generated paired relative comparative quantifications (ratios) between proteins under CHF+MyoMed-946, CHF, CHF and sham conditions, and statistical queries were performed on their significant differences using standard statistical tests. Several proteins were determined to be statistically different (e.g., TNNT3, Timm9, Ccdc5, Adi1, Ptges3 and Ndufa3). Using multiple hypothesis testing (Benjamini–Hochberg; corrected P < 0.05) to compare CHF mice with and without compound feeding, only Mrps5 (mitochondrial ribosomal protein 5) remained a significantly upregulated protein (P = 0.02; Figure 4 A), Mrps5 is a mitochondrial-cytoplasmic shuttling protein responsible for protein initiation and elongation in mitochondrial ribosomes and was further investigated by Western blot analysis as described below.

[0719] from Fig.39 As can be seen, Western blot analysis of the diaphragm of all animals included in the CHF study showed a significant decrease in Mrps5 expression in the CHF group compared to the sham-treated control group. This could be reversed by feeding the compound MyoMed-946. Data are presented as mean ± standard error of the mean.

[0720] Western blot analysis:

[0721] For western blot analysis, frozen diaphragms were homogenized in relaxation buffer (90 mmol / L HEPES, 126 mmol / L KCl, 36 mmol / L NaCl, 1 mmol / L MgCl, 50 mmol / L EGTA, 8 mmol / L ATP and 10 mmol / L creatine phosphate, pH 7.4) containing a protease inhibitor mix (Inhibitor Mix M, Serva, Heidelberg, Germany) and sonicated. The protein concentration of the supernatant was determined (bicinchoninic acid assay, Pierce, Bonn, Germany), and samples (5–20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene fluoride membranes and incubated overnight at 4°C with the following primary antibodies: porin and contiguous (both 1 / 1000, Abcam, Cambridge, UK), MRPS-5 (1 / 500, Thermo Fisher, Rockford, IL, USA), MuRF1 and MuRF2 (both 1 / 1000; commercially available from Myomedix, Neckargemünd, Germany), and Tom20 (1:200, Santa Cruz Biotechnologies, Heidelberg, Germany). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, and specific bands were visualized by enzyme chemiluminescence (Super Signal West Pico, Thermo Fisher Scientific Inc., Bonn, Germany) and quantified by densitometry using a one-dimensional scanning software package (Scanalytics Inc., Rockville, MD, USA). The measurements were normalized to the loading control GAPDH (1 / 30 000; HyTest Ltd, Turku, Finland) or α-tubulin (1:1000, Santa Cruz Biotechnologies). All data are expressed as fold change relative to the sham treated group.

[0722] from Figures 40-42 It can be seen that in the CHF group, the expression of MuRF1 and MuRF2 was significantly upregulated, while treatment with the compound MyoMed-946 prevented the upregulation ( Fig.40 and 41 ). Quantifying the expression of continuum (a MuRF1 target protein), a trend toward decreased expression was observed in the CHF group (P = 0.08), which was not significant in the MyoMed-946-treated group ( Fig.42 ).

[0723] 3.1.3 B16F10 mice (tumor-bearing mice):

[0724] As described above for MCT and CHF mice, the protein expression of MuRF1, Nox 2, and LC3 I / II was determined in muscle tissue of B16F10 mice. In addition, the level of the reactive oxygen species marker nitrotyrosine was determined.

[0725] from Figures 43-46 It can be seen that in the tumor group, MuRF1 expression was significantly upregulated, and treatment with compounds MyoMed-946 and MyoMed-205 prevented the upregulation ( Fig.43 In addition, the expression of the known endogenous reactive oxygen species (ROS) Nox 2 and the tissue level of nitrotyrosine were also significantly upregulated in the tumor mice group, while the amount of these species in muscle tissue was significantly reduced in the tumor+MyoMed-946 and tumor+MyoMed-205 mouse groups ( Fig.44 and 45 On the other hand, the expression level of LC3I / II, a key protein involved in autophagy, was downregulated in the tumor group, which was not observed in the tumor+MyoMed-946 and tumor+MyoMed-205 groups ( Fig.46 ).

[0726] 3.2 Enzyme activity assay:

[0727] 3.2.1 CHF mice:

[0728] Since the proteomic analysis data indicated that mitochondrial homeostasis was impaired, the enzymatic activities of key mitochondrial enzymes were determined in diaphragm muscle tissue of CHF mice.

[0729] Diaphragm tissue was homogenized in relaxation buffer and samples were used to determine enzyme activity. The enzyme activities of lactate dehydrogenase (EC 1.1.1.27), pyruvate kinase (EC 2.7.1.40), succinate dehydrogenase (SDH, EC 1.3.5.1), citrate synthase (CS, EC 2.3.3.1), β-hydroxyacyl-COA dehydrogenase (EC 1.1.1.35), and mitochondrial complex I were determined spectrophotometrically as described in detail previously (Mukherjee et al., J Biol Chem, 1976, 251, 2155–2160; Vanderlinde et al., Ann Clin Lab Sci, 1985, 15, 13–31; Dzeja et al., Mol Cell Biochem, 1999, 201, 33–40; Takashi et al., Biochim Biohphys Acta, 1979, 574, 258–267; Schwarzer et al., J Biol Chem, 1986, 257, 258–267). Physiol, 2014, 592, 3767–3782). The enzyme activity data were expressed as fold change relative to the sham treatment group.

[0730] from Figures 47 to 51 It can be seen that when compared with the sham-treated group, mitochondrial enzymes including citrate synthase ( Fig.47 ), succinate dehydrogenase ( Fig.48 ) and mitochondrial complex I ( Fig.49 ) were significantly reduced by 21%, 28% and 27% respectively. No difference in creatine kinase was found. Fig.50 ) and TOM-20( Fig.51 ) protein expression assessment, the amount of mitochondria in the diaphragm tissue of CHF mice was also significantly reduced. Consistent with the effect on mitochondrial function, treatment with the compound MyoMed-946 partially improved the activities of citrate synthase, succinate dehydrogenase, and mitochondrial complex I enzymes ( Figures 47-49 ), and produced near-normal porins, as well as a modest but statistically significant improvement in TOM-20 expression ( Fig.50 and 51 ).

[0731] In contrast, when the cytoplasmic enzymes of glycolysis and fatty acid metabolism were evaluated (glycolysis: pyruvate kinase and lactate dehydrogenase; fatty acid metabolism: β-hydroxyacyl-COA dehydrogenase), no differences among the three groups were detected.

[0732] 3.2.2 B16F10 mice (tumor-bearing mice):

[0733] The enzyme activities of citrate synthase and mitochondrial complex I were determined in muscle tissue of B16F10-inoculated mice as described previously for CHF mice.

[0734] from Fig.52 and 53 It can be seen that when compared with the sham treated group, the mitochondrial enzyme citrate synthase ( Fig.52 ) and mitochondrial complex I ( Fig.53 Treatment with compounds MyoMed-946 and MyoMed-205 partially or completely restored the enzyme activities of citrate synthase and mitochondrial complex I in the muscle tissue of B16F10-inoculated mice ( Fig.52 and 53 ).

[0735] 4. C2C12 myotube cell culture and reverse transcription PCR

[0736] C2C12 myotubes were incubated for 20 minutes at a final concentration of 10 μmol / L with or without the compound MyoMed-946. After the incubation period, total RNA was isolated from C2C12 cells and reverse transcribed to cDNA using random hexamers and Sensiscript reverse transcriptase (Qiagen, Hilden, Germany). A portion of cDNA was used for quantitative RT-PCR using the Light Cyclersystem (Roche Diagnostics, Mannheim, Germany). The expression of specific genes was standardized relative to the expression of hypoxanthin-phosphoribosyl-transferase (HPRT)-mRNA. For quantification of MuRF-1 expression, fluorescence resonance energy transfer (FRET) technology was used with the following primers (TIB MolBiol, Berlin, Germany) and conditions: HPRT: 5′-CTCATggACTgATTATggACAggAC-3′ (SEQ ID NO: 1) and 5′-gCAggTCAgCAAAgAACTTATAgCC-3′ (SEQ ID NO: 2), annealing at 60° C.; MuRF-1: 5′-gATgTgCAAggAACACgAA-3′ (SEQ ID NO: 3), 5′-CCTTCACCTggTggCTATTC-3′ (SEQ ID NO: 4), LC640-gCACAAggAgCAAgTAggCACCTCAC-PH (SEQ ID NO: 5), 5′-gCCTggTgAgCCCCAAACACCT-FL (SEQ ID NO: 6), annealing at 58° C. LC640 stands for LC Red 640, a fluorescent dye. FL stands for fluorescein. PH stands for phosphate group (blocking free 3'-hydroxyl group, preventing unwanted extension by polymerase).

[0737] from Fig.54 It can be seen that the compound MyoMed-946 prevented the increase in MuRF1 protein expression in MCT mice.

[0738] 5. Statistical analysis:

[0739] Data are expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by Bonferroni post hoc was used to compare groups, while two-way repeated measures ANOVA followed by Bonferroni post hoc was used to evaluate contractile function (GraphPad Prism). P < 0.05 was considered significant.

Claims

1. Compounds of Formula I in R 1 is hydrogen or the group -CH2R 1a , where R 1a is selected from hydrogen and an unsubstituted 5- to 6-membered monocyclic heteroaromatic ring containing as ring member 1 heteroatom selected from N, O and S; R 2 is selected from hydrogen and methyl; R 3 It is hydrogen; R 4 It is methyl; X 1 is NH or O; X 2 It is O; Y represents an oxygen atom or two hydrogen atoms; a is 0; and b is 0; and pharmaceutically acceptable salts thereof.

2. The compound according to claim 1, wherein R 1 It is the group -CH2R 1a , where R 1a is an unsubstituted 5- to 6-membered monocyclic heteroaromatic ring containing 1 heteroatom selected from N, O and S as a ring member; and pharmaceutically acceptable salts thereof.

3. The compound according to claim 1, wherein R 1 Selected from the group CH2-I' or CH2-II' Where * indicates the point of attachment to the urea nitrogen atom; X 3 is O or S; and c is 0; and pharmaceutically acceptable salts thereof.

4. The compound according to claim 3, wherein R 2 It is hydrogen; and pharmaceutically acceptable salts thereof.

5. A compound of formula I as defined in any one of claims 1 to 4, corresponding to formula IA, in X 3 is O or S; and pharmaceutically acceptable salts thereof, Or corresponding to Formula IB, and pharmaceutically acceptable salts thereof, Or corresponding to the formula IC, in X 3 is O or S; and pharmaceutically acceptable salts thereof, Or corresponding to the formula ID, in R 1 is hydrogen or methyl; and pharmaceutically acceptable salts thereof.

6. The compound 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid [2-(2-furanylmethylcarbamoylamino)-2-oxo-ethyl] ester, and pharmaceutically acceptable salts thereof.

7. The compound 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-N-[2-(2-thienylmethylcarbamoylamino)ethyl]benzamide, and pharmaceutically acceptable salts thereof.

8. The compound 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid [2-oxo-2-(2-pyridylmethylcarbamoylamino)ethyl] ester, and pharmaceutically acceptable salts thereof.

9. A compound of formula I as defined in claim 1 selected from N-[2-(2-furanylmethylcarbamoylamino)-2-oxo-ethyl]-4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzamide, 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-benzoic acid [2-oxo-2-(2-thienylmethylcarbamoylamino)ethyl]ester and 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-N-[2-oxo-2-(2-thienylmethylcarbamoylamino)ethyl]-benzamide, and pharmaceutically acceptable salts thereof.

10. A compound of formula I as defined in claim 1, selected from 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid (1-methyl-2-oxo-2-urea-ethyl) ester and 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid [1-methyl-2-(methylcarbamoylamino)-2-oxo-ethyl] ester, and pharmaceutically acceptable salts thereof.

11. A pharmaceutical composition comprising at least one compound of formula I as defined in any one of claims 1 to 10 or at least one pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

12. Use of a compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing muscle wasting conditions.

13. Use of a compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing skeletal or cardiac muscle atrophy caused by one of the following diseases or conditions: congestive heart failure, chronic heart failure, cancer, cancer treatment with myotoxic and / or cardiotoxic substances, congenital myopathy, AIDS, chronic obstructive pulmonary disease, chronic kidney disease, renal failure, diabetes, severe burns, sarcopenia during aging, reduced blood supply, temporary or long-term immobilization, long-term mechanical ventilation, denervation, long-term weightlessness and malnutrition.

14. Use of a compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing a condition associated with increased muscle RING finger 1 expression.

15. The use of claim 14, wherein the condition associated with increased muscle RING finger 1 expression is a myopathy associated with increased muscle RING finger 1 expression.

16. The use of claim 15, wherein the myopathy is selected from critical illness myopathy, nematode myopathy, inflammatory myopathy, myopathy caused by diabetes, myopathy caused by pulmonary hypertension, myopathy caused by chronic heart failure, myopathy caused by renal failure, and myopathy caused by tumor cachexia.

17. Use of the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing a heart disease associated with systolic or diastolic dysfunction.

18. Use of the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing diabetes.

19. Use of a compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof in the preparation of a medicament, wherein the medicament is used to treat or prevent - Skeletal or myocardial atrophy caused by or associated with heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, hypertension, or tumor cachexia; - Doxorubicin-induced muscle atrophy and / or cardiotoxicity; - Sarcopenia and / or cardiomyopathy caused by aging; - Muscle wasting caused by chronic kidney disease; - Diaphragmatic weakness due to mechanical ventilation or congestive heart failure; - Congenital myopathy; - Muscle wasting caused by diabetes; and / or -diabetes.

20. The use according to claim 19, wherein the congenital myopathy is homologous muscular dystrophy.

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