COMPOUNDS SUITABLE FOR THE TREATMENT AND PROPHYLAXIS OF MUSCLE WASTING AND OTHER CONDITIONS

Abstract

The present invention relates to a compound of the general formula I and the pharmaceutically acceptable salts thereof; where the variables are as defined in the claims and the description. The invention also relates to the compounds of formula I for use in the treatment or prophylaxis of muscle wasting conditions, of skeletal or cardial muscle atrophy, of conditions, in particular of myopathies, which are associated with an increased Muscle RING Finger 1 (MuRF1) expression and of other conditions; to the compounds of formula I for use as a medicament; to a pharmaceutical composition comprising at least one compound of formula I and to a method for treating said conditions.

##STR00001##

Claims

1. A compound of the general formula I ##STR00013## wherein R.sup.1 is hydrogen or a group —CH.sub.2R.sup.1a, where R.sup.1a is selected from the group consisting of hydrogen, C.sub.1-C.sub.3-alkyl, phenyl, where phenyl is unsubstituted or may carry 1, 2 or 3 radicals independently selected from halogen, cyano, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl and C.sub.1-C.sub.3-alkoxy; and a 5- to 10-membered heteroaromatic ring containing 1 to 4 heteroatoms or hetero-groups independently selected from the group consisting of N, NR.sup.c, O and S as ring member, where the 5- to 10-membered heteroaromatic ring is unsubstituted or may carry 1, 2 or 3 radicals R.sup.7; R.sup.2 is selected from the group consisting of hydrogen, methyl and fluorinated methyl; R.sup.3 is selected from the group consisting of hydrogen, methyl and fluorinated methyl; R.sup.4 is selected from the group consisting of hydrogen and C.sub.1-C.sub.4-alkyl; each R.sup.5 is independently selected from the group consisting of halogen, cyano, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy and C.sub.1-C.sub.3-haloalkoxy; each R.sup.6 is independently selected from the group consisting of halogen, cyano, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy and C.sub.1-C.sub.3-haloalkoxy; each R.sup.7 is independently selected from the group consisting of halogen, cyano, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy and C.sub.1-C.sub.3-haloalkoxy; X.sup.1 is NR.sup.a or O; X.sup.2 is NR.sup.b, O or S; Y represents an oxygen atom or two hydrogen atoms; R.sup.a, R.sup.b, R.sup.c are each independently selected from the group consisting of hydrogen and C.sub.1-C.sub.4-alkyl; a is 0, 1, 2, 3 or 4; and b is 0, 1, 2 or 3; and the pharmaceutically acceptable salts thereof.

2. The compound as claimed in claim 1, wherein R.sup.1 is selected from the group consisting of hydrogen and a group —CH.sub.2R.sup.1a, where R.sup.1a is selected from the group consisting of hydrogen, methyl and a 5- to 6-membered monocyclic heteroaromatic ring containing 1 to 3 heteroatoms, in particular 1 heteroatom, independently selected from the group consisting of N, O and S, as ring member, where the 5- to 6-membered monocyclic heteroaromatic ring is unsubstituted or carries 1 radical R.sup.7, where R.sup.7 is selected from the group consisting of halogen, C.sub.1-C.sub.3-alkyl and C.sub.1-C.sub.2-alkoxy.

3. The compound as claimed in claim 2, wherein R.sup.1 is selected from the group consisting of hydrogen and a group —CH.sub.2R.sup.1a, where R.sup.1a is selected from the group consisting of hydrogen and an unsubstituted 5- to 6-membered monocyclic heteroaromatic ring containing 1 to 3 heteroatoms, in particular 1 heteroatom, independently selected from the group consisting of N, O and S, as ring member; where R.sup.1 is in particular a group —CH.sub.2R.sup.1a, where R.sup.1a is an unsubstituted 5- to 6-membered monocyclic heteroaromatic ring containing 1 to 3 heteroatoms, in particular 1 heteroatom, independently selected from the group consisting of N, O and S, as ring member.

4. The compound as claimed in any of claims 1 to 3, wherein R.sup.2 is hydrogen or methyl; R.sup.3 is hydrogen; R.sup.4 is methyl; each R.sup.5 is independently selected from the group consisting of halogen, C.sub.1-C.sub.3-alkyl and C.sub.1-C.sub.2-alkoxy; each R.sup.6 is independently selected from the group consisting of halogen, C.sub.1-C.sub.3-alkyl and C.sub.1-C.sub.2-alkoxy; X.sup.1 is NH or O; X.sup.2 is O; a is 0, 1 or 2; and b is 0 or 1.

5. The compound as claimed in claim 1, wherein R.sup.1 is selected from the group CH.sub.2—I′ or CH.sub.2—II′ ##STR00014## wherein * indicates the point of attachment to the urea nitrogen atom; each R.sup.7 is independently selected from the group consisting of halogen, cyano, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-haloalkyl, C.sub.1-C.sub.3-alkoxy and C.sub.1-C.sub.3-haloalkoxy; X.sup.3 is NR.sup.c, O or S; R.sup.c is selected from the group consisting of hydrogen and C.sub.1-C.sub.4-alkyl; and c is 0, 1, 2 or 3.

6. The compound as claimed in claim 5, wherein R.sup.2 is hydrogen or methyl; R.sup.3 is hydrogen; R.sup.4 is methyl; each R.sup.5 is independently selected from the group consisting of halogen, C.sub.1-C.sub.3-alkyl and C.sub.1-C.sub.2-alkoxy; each R.sup.6 is independently selected from the group consisting of halogen, C.sub.1-C.sub.3-alkyl and C.sub.1-C.sub.2-alkoxy; each R.sup.7 is independently selected from the group consisting of halogen, C.sub.1-C.sub.3-alkyl and C.sub.1-C.sub.2-alkoxy; X.sup.1 is NH or O; X.sup.2 is O; X.sup.3 is O or S; a is 0, 1 or 2; b is 0 or 1; and c is 0 or 1.

7. The compound as claimed in claim 6, wherein R.sup.2 is hydrogen; R.sup.3 is hydrogen; R.sup.4 is methyl; X.sup.1 is NH or O; X.sup.2 is O; X.sup.3 is O or S; a is 0; b is 0; and c is 0.

8. A compound of formula I as defined in any of claims 1 to 7, which corresponds to the formula I-A, ##STR00015## wherein X.sup.1 is NH or O; and X.sup.3 is O or S; and the pharmaceutically acceptable salts thereof, or to the formula I-B, ##STR00016## wherein X.sup.1 is NH or O; and the pharmaceutically acceptable salts thereof, or to the formula I-C, ##STR00017## wherein X.sup.1 is NH or O; and X.sup.3 is O or S; and the pharmaceutically acceptable salts thereof, or to the formula I-D, ##STR00018## wherein R.sup.1 is hydrogen or methyl; and X.sup.1 is NH or O; and the pharmaceutically acceptable salts thereof.

9. The compound [2-(2-furylmethylcarbamoylamino)-2-oxo-ethyl] 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate and its pharmaceutically acceptable salts.

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

11. The compound [2-oxo-2-(2-pyridylmethylcarbamoylamino)ethyl] 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate and its pharmaceutically acceptable salts.

12. A compound of formula I as defined in claim 1, which is selected from N-[2-(2-furylmethylcarbamoylamino)-2-oxo-ethyl]-4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzamide, [2-oxo-2-(2-thienylmethylcarbamoylamino)ethyl] 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-benzoate, and 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-N-[2-oxo-2-(2-thienylmethylcarbamoylamino)ethyl]-benzamide, and the pharmaceutically acceptable salts thereof.

13. A compound of formula I as defined in claim 1, which is selected from (1-methyl-2-oxo-2-ureido-ethyl) 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate and [1-methyl-2-(methylcarbamoylamino)-2-oxo-ethyl] 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate, and the pharmaceutically acceptable salts thereof.

14. A pharmaceutical composition comprising at least one compound of the general formula I as defined in any of claims 1 to 13 or at least one pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.

15. The compound as claimed in any of claims 1 to 13 or a pharmaceutically acceptable salt thereof, for use as a medicament.

16. The compound as claimed in any of claims 1 to 13 or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of muscle wasting conditions.

17. The compound as claimed in any of claims 1 to 13 or a pharmaceutically acceptable salt thereof for use in the treatment or prophylaxis of skeletal or cardiac muscle atrophy resulting from one of the following diseases or conditions: congestive heart failure, chronic heart failure, cancer, cancer treatment with myotoxic and/or cardiotoxic substances such as doxorubicin, congenital myopathy, AIDS, chronic obstructive pulmonary disease (COPD), chronic renal diseases, renal failure, diabetes, severe burns, sarcopenia during aging, reduction in blood supply, temporary or long term immobilization, long term mechanical ventilation, denervation, prolonged weightlessness and malnutrition.

18. The compound as claimed in any of claims 1 to 13 or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of conditions which are associated with an increased Muscle RING Finger 1 (MuRF1) expression, in particular of myopathies which are associated with an increased Muscle RING Finger 1 (MuRF1) expression.

19. The compound for use as claimed in claim 18, where the myopathy is selected from critical illness myopathy, nemaline myopathy, inflammatory myopathy, myopathy from diabetes, myopathy from pulmonary hypertension, myopathy from chronic heart failure, myopathy from kidney failure and myopathy from tumor cachexia.

20. The compound as claimed in any of claims 1 to 13 or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of cardiac conditions associated with systolic or diastolic dysfunction.

21. The compound as claimed in any of claims 1 to 13 or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of diabetes.

22. The compound as claimed in any of claims 1 to 13 or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of skeletal or cardiac muscle atrophy resulting from or associated with heart failure with reduced ejection fraction (HF-rEF), heart failure with preserved ejection fraction (HF-pEF), hypertension or tumor cachexia; muscle atrophy and/or cardiac toxicity induced by Doxorubicin; sarcopenia and/or cardiomyopathy due to aging; muscle atrophy due to chronic renal disease; diaphragm weakness due to mechanical ventilation or congestive heart failure; congenital myopathy, in particular congenetic muscle atrophy; diabetes-induced muscle atrophy; and/or diabetes.

23. A method for treating or preventing a condition, as defined in any of claims 16 to 22, said method comprising the step of administering a therapeutically effective amount of a compound selected from compounds of the general formula I, as defined in any of claims 1 to 13, or a pharmaceutically acceptable salts thereof to a subject in need thereof.

Description

FIGURES

[0410] FIG. 1: Cytotoxicity test results based on the release of lactate dehydrogenase (LDH) activity in cultured myoblasts in the presence of increasing concentrations of the compounds MyoMed-946, MyoMed-946-5, MyoMed-946-8.

[0411] FIG. 2: Cytotoxicity test results based on the release of lactate dehydrogenase (LDH) activity in cultured myocytes in the presence of increasing concentrations of the compounds MyoMed-946, MyoMed-946-5, MyoMed-946-8.

[0412] FIGS. 3 to 7: Physical characteristics of sham (n=20) and mice treated with monocrotaline fed either normal chow (MCT; n=27) or the MyoMed-946 compound (MCT+MyoMed-946; n=27). The data confirm that MCT treatment induced cardiac cachexia independent of the chow administrated, as demonstrated by an impaired weight gain (FIG. 3), increased pulmonary congestion (FIG. 4) and increased heart weight over body weight (BW) (FIG. 5), and that right ventricular (RV) hypertrophy (FIG. 6), the latter visualized by representative H&E stained medial cross sections of the heart (FIG. 7), is attenuated. *P<0.01 vs. Sham.

[0413] FIGS. 8 to 12: Skeletal muscle wet-weights (normalized to tibia length; TL) for the extensor digitorum longus (EDL) (FIG. 8), soleus (FIG. 9), tibialis anterior (TA) (FIG. 10) for MCT-treated mice in the absence or in the presence of the MuRF1 inhibitor MyoMed-946. In addition the fiber cross sectional area (CSA) for the TA muscle is also presented (FIG. 11), the latter visualized by representative H&E stain (FIG. 12). *P<0.05 vs. sham; § P<0.01 vs. sham and MCT+MyoMed-946.

[0414] FIGS. 13 to 18: Physical characteristics of sham (n=10) and mice treated with monocrotaline fed either normal chow (MCT; n=10) or the 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<0.001 vs. begin; FIG. 14: *** p<0.001 vs. sham ** p<0.01 vs. sham; FIG. 15: *** p<0.001, ** p<001 vs. sham, §§ p<0.01, § p<0.05 vs. MCT; FIGS. 16 to 18: *P<0.01 vs. Sham. The data confirm that MCT treatment induced cardiac cachexia independent of the chow administrated, as demonstrated by an impaired weight gain (FIG. 13), increased pulmonary congestion (FIG. 14) and increased heart weight over body weight (BW) (FIG. 15), and that right ventricular (RV) hypertrophy is attenuated by the MuRF1 inhibitors MyoMed-946, MyoMed-203 and MyoMed-205 (FIGS. 16 to 18).

[0415] FIGS. 19 and 20: Skeletal muscle wet-weights (normalized to tibia length; TL) for the extensor digitorum longus (EDL) (FIG. 19) and tibialis anterior (TA) (FIG. 20) for MCT-treated mice in the absence (Sham) or in the presence of the MuRF1 inhibitors MyoMed-946, MyoMed-203 and MyoMed-205.

[0416] FIG. 21: Diaphragm maximum force for MCT-treated mice in the absence (Sham) or in the presence of the MuRF1 inhibitors MyoMed-946, MyoMed-203 and MyoMed-205.

[0417] FIGS. 22 and 23: Expression of MuRF1 (FIG. 22) and telethonin (FIG. 23) in the tibialis anterior muscle tissue of MTC-treated mice, in the absence (Sham) or in the presence of the MuRF1 inhibitors MyoMed-946, MyoMed-203 and MyoMed-205.

[0418] FIGS. 24 to 27: Diaphragm function, assessed during isometric contractions (FIG. 24) and also isotonic contractions (FIG. 25) of diaphragm myofiber bundles from mice suffering from chronic heart failure (CHF) with reduced ejection fraction (HFrEF) following myocardial infarction (MI), whereby diaphragm maximum force (FIG. 26) and diaphragm peak power (FIG. 27) were determined. Data are presented 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.

[0419] FIGS. 28 to 30: In vitro skeletal muscle contractile function, assessed during isometric contractions (FIG. 28) and also isotonic contractions, whereby shortening velocity (FIG. 29) and power (FIG. 30) were determined. MCT treated mice demonstrates impairments in shortening velocity and power by around 20% compared to shams. These impairments are essentially prevented in MCT mice fed the MyoMed-946 compound. *P<0.05 vs sham; § P<0.01 vs. sham and MCT+MyoMed-946.

[0420] FIG. 31: Muscle wet-weights of the tibialis anterior (TA) in B16F10 cell inoculated mice receiving regular chow (tumor) and B16F10 cell inoculated mice fed with the compounds MyoMed-946 (tumor+MyoMed-946) and MyoMed-205 (tumor+MyoMed-205).

[0421] FIG. 32: Wire-hang test of B16F10 cell inoculated mice (melanoma tumor cell model), 9d, 16d and 23d after inoculation. Tumor growth leads to a significant reduction in muscle function (tumor group) compared to mice of the control group (sham). This reduction of muscle function is attenuated in mice fed with the compounds MyoMed-946 (tumor+MyoMed-946) and MyoMed-205 (tumor+MyoMed-205).

[0422] FIG. 33 to 35: Expression levels of eIF2B subunit-delta (FIG. 33) and BAX (FIG. 34) for sham, MCT, and MCT+MyoMed-946 mice, determined by western blot analysis, with representative blots (FIG. 35). *P<0.05 vs sham; § P<0.01 vs. MCT.

[0423] FIG. 36 to 38: Protein expression levels and representative western blots for MuRF1 (FIG. 36), MAFBx (FIG. 37), and CARP (FIG. 38) for sham, MCT, and MCT+MyoMed-946 mice. The MCT treatment leads to an increase in MuRF1 and CARP expression, but this is attenuated in mice fed the MyoMed-946 compound. No changes are detected in levels of MAFBx. *P<0.05 vs. sham and MCT+MyoMed-946.

[0424] FIG. 39: Protein expression level and representative western blot for MRPS-5 in the diaphragm tissue of sham, CHF, and CHF+MyoMed-946 mice. Data are presented as mean±standard error of the mean.

[0425] FIGS. 40 to 42: Protein expression levels and representative western blots for MuRF1 (FIG. 40), MuRF2 (FIG. 41), and Telethonin (FIG. 42) in the diaphragm tissue of sham, CHF, and CHF+MyoMed-946 mice. CHF leads to an increase in MuRF1 and MuRF2 expression and to a decrease in telethonin expression, but these effects are attenuated in mice fed with the MyoMed-946 compound. Data are presented as mean standard error of the mean.

[0426] FIGS. 43 to 46: Protein expression levels of MuRF1 determined via western blot (WB) analysis (FIG. 43), Nox 2 (FIG. 44) and LC3 I/II (FIG. 46) as well as the level of the reactive oxygen species marker nitrotyrosine (FIG. 45) in muscle tissue of B16F10 cell inoculated mice fed with regular show (tumor) or fed with the compounds MyoMed-946 (tumor+MyoMed-946) or MyoMed-205 (tumor+MyoMed-205). Tumor growth leads to a significant increase of the protein expression level of MuRF1 and Nox 2 as well as of the nitrotyrosine level and to a significant decrease in the expression levels of LC3 I/II in the tumor group compared to mice of the control group (sham). These changes are attenuated in mice fed with the compounds MyoMed-946 (tumor+MyoMed-946) or MyoMed-205 (tumor+MyoMed-205). Data are presented as mean±standard error of the mean.

[0427] FIGS. 47 to 49: Enzyme activities of citrate synthase (FIG. 47), succinate dehydrogenase (FIG. 48) and mitochondrial complex I (FIG. 49) in diaphragm tissue samples from sham, chronic heart failure (CHF), and CHF+MyoMed-946 mice. The data reveal a significant down-regulation of citrate synthase, succinate dehydrogenase and mitochondrial complex I activity in CHF when compared with sham, but this is attenuated in mice fed with the compound MyoMed-946. Data are presented as mean standard error of the mean.

[0428] FIGS. 50 and 51: Protein expression levels of mitochondrial porin of the outer mitochondrial membrane (FIG. 50) and TOM-20 (FIG. 51) in diaphragm tissue samples from sham, chronic heart failure (CHF), and CHF+MyoMed-946 mice. The data reveal a significant down-regulation of porin and TOM-20 expression in CHF when compared with sham, but this is attenuated in mice fed the compound MyoMed-946. Data are presented as mean±standard error of the mean.

[0429] FIGS. 52 and 53: Enzyme activities of citrate synthase (FIG. 52) and mitochondrial complex I (FIG. 53) in muscle tissue samples from sham, B16F10 cell inoculated mice fed with regular show (tumor) and B16F10 cell inoculated mice fed with the compounds MyoMed-946 (tumor+MyoMed-946) or MyoMed-205 (tumor+MyoMed-205). The data reveal a significant down-regulation of citrate synthase and mitochondrial complex I activity in the tumor group when compared with sham, but this is attenuated in mice fed with the compounds MyoMed-946 or MyoMed-205. Data are presented as mean standard error of the mean.

[0430] FIG. 54: Expression of MuRF1 at the mRNA level in myotubes, following 24 h incubation with dexamethasone (DEX; 10 μmol/L). Displayed is the fold change of untreated cells (DEX), and cells which have been pre-treated for 2 h with the MyoMed-946 compound at 0.1 μmol/L and 10 μmol/L. The pre-treated cells show reduced MuRF1 mRNA levels. *P<0.05 vs. CON. § P<0.05 vs. DEX.

[0431] FIG. 55: Differential scanning fluorimetry (DSF) melting curves, plotted as the ratio of the fluorescence signal at 350 nm to the fluorescence signal at 330 nm against a thermal gradient, for MuRF1 central fragment in PBS-buffer (solid line), MuRF1 central fragment in PBS-buffer plus 1% DMSO (dashed line) and for MuRF1 central fragment in PBS-buffer plus 1% of a 10 mM stock of compound MyoMed-946 in DMSO (final concentration of MyoMed-946: 100 μM) (dotted line).

[0432] FIG. 56: Schematic drawing of the study design for a HFpEF rat model experiment.

[0433] FIG. 57: Results of myocardial function echocardiography and invasive hemodynamic measurements in HFpEF rat model experiment.

[0434] FIG. 58: Results of skeletal muscle mass and function measurements in HFpEF rat model experiment.

[0435] FIG. 59: Cachexia and body stress results in Doxorubicin-induced muscle atrophy and cardiac toxicity mouse model.

[0436] FIG. 60: Echocardiography results in Doxorubicin-induced muscle atrophy and cardiac toxicity mouse model.

[0437] FIG. 61: Wire hang test in obese mice with type 2 diabetes during diet-induced weight loss. Treatment with MyoMed-205 attenuated the loss of muscle function.

[0438] FIG. 62: Blood glucose levels after 6 h fasting in MyoMed-203-treated DIO mice as compared to DIO control mice.

[0439] FIGS. 63 and 64: Oral glucose tolerance test results at test days 14 and 28 of MyoMed-203-treated DIO mice as compared to DIO control mice.

[0440] FIG. 65: Insulin tolerance test results of MyoMed-203-treated DIO mice as compared to DIO control mice.

[0441] I. Synthesis of compounds I

1. Analytics

[0442] The compounds were characterized via .sup.1H NMR and eventually .sup.13C NMR in d6-dimethylsulfoxid (DMSO-d6), if not stated otherwise, on a 400 MHz NMR instrument (Bruker AVANCE III).

[0443] The magnetic nuclear resonance spectral properties (NMR) refer to the chemical shifts (6) expressed in parts per million (ppm). The relative area of the shifts in the 1H NMR spectrum corresponds to the number of hydrogen atoms for a particular functional type in the molecule. The nature of the shift, as regards multiplicity, is indicated as singlet (s), broad singlet (s. br.), doublet (d), broad doublet (d br.), triplet (t), broad triplet (t br.), quartet (q), quintet (quint.) and multiplet (m).

[0444] The compounds were further characterized by HPLC-MS and/or UPLC-MS in a fast gradient on C.sub.18-material (electrospray-ionisation (ESI) mode). If not stated otherwise, the ESI MS-data are recorded in positive mode. The MS-data refer to the protonated compounds (M+H).sup.+ given as mass over charge-ratio (m/z), where z is 1.

[0445] HPLC-MS Specifications:

[0446] HPLC-MS Instrument: Agilent 1100 Series LC/MSD system with DAD\ELSD and Agilent LC\MSD VL (G1956A), SL (G1956B) mass-spectrometer or Agilent 1200 Series LC/MSD system with DAD\ELSD and Agilent LC\MSD SL (G6130A), SL (G6140A) mass-spectrometer. All the LC/MS data were obtained using positive/negative mode switching.

[0447] Acquisition method: Column: Zorbax SB-C18 1.8 μm 4.6×15 mm Rapid Resolution cartridge (PN 821975-932). Mobile phase: A: acetonitrile plus 0.1% formic acid; B: water with 0.1% formic acid. Flow rate: 3 ml/min; Injection volume: 1 μl.

[0448] Solvent Gradient:

[0449] 100% B from 0 to 0.01 minutes;

[0450] 100% to 0% B from 0.01 to 1.5 minutes, linear gradient;

[0451] 0% B from 1.5 to 1.8 minutes;

[0452] 0% to 100% B from 1.8 to 1.81 minutes.

[0453] Ionization mode: atmospheric pressure chemical ionization (APCI);

[0454] Scan range: m/z 80-1000.

[0455] Uplc-Ms Specifications:

[0456] UPLC-MS instrument: Agilent Infinity 1290 with a Single Quadrupole, Electrospray Ionisation mass-spectrometer;

[0457] Acquisition method: Column: Acquity UPLC BEH C18; 1.7 μm; 2.1×50 mm; T=40° C. Mobile phase: A: Water plus 0.1% trifluoroacetic acid; B: MeCN plus 0.1% trifluoroacetic acid. Flow rate: 1 ml/min; inject volume 3 μl; runtime 3 min.

[0458] Solvent Gradients (3 Minute Gradient):

[0459] 5 to 100% B from 0 to 2.3 minutes, linear gradient;

[0460] 100% B from 2.3 to 2.5 minutes;

[0461] 100 to 5% B from 2.5 to 2.6 minutes, linear gradient;

[0462] 100% B from 2.6 to 3.0 minutes.

[0463] If not stated otherwise, the ESI MS-data are recorded in positive mode. The MS-data refer to the protonated compounds (M+H).sup.+ given as mass over charge-ratio (m/z), where z is 1.

2. Synthesis

2.1 Preparative HPLC-purification

[0464] Preparative HPLC-purification was performed using HPLC (H.sub.2O-MeOH or H.sub.2O—CH.sub.3CN; Agilent 1260 Infinity systems equipped with DAD and mass-detectors using a Waters SunFire C18 OBD Prep Column (100 Å, 5 μm, 19 mm×100 mm with SunFire C18 Prep Guard Cartridge, 100 Å, 10 μm, 19 mm×10 mm). The raw compounds were dissolved in 0.7 mL DMSO. Flow: 30 mL/min. Purity of the obtained fractions was checked via analytical LCMS. Spectra were recorded for each fraction as it was obtained straight after chromatography in the solution form. The solvent was evaporated in the flow of N.sub.2 at 80° C. The individual fractions were combined on the basis of post-chromatography LCMS analysis. Solid fractions were dissolved in 0.5 mL MeOH/CH.sub.3CN and transferred into pre-weighted marked vials. Obtained solutions were again evaporated in the flow of N.sub.2 at 80° C. After drying, products were finally characterized by LC-MS and .sup.1H-NMR.

2.2 Intermediates

2.2.1 4-[(4-Methyl-2-oxo-chromen-7-yl)oxymethyl]Acetic Acid

[0465] A mixture of 80.6 g (0.458 mol) 7-hydroxy-4-methylcoumarin, 110.1 g of methyl 4-(bromomethyl)benzoate (0.480 mol), and anhydrous K.sub.2CO.sub.3 95 g (0.686 mol) in anhydrous acetone (800 mL) was heated to reflux for 3 hrs. The mixture was then cooled, filtered, and the filtrate was concentrated under vacuum. The residue was dissolved in DMSO (450 mL) and aqueous solution of potassium hydroxide (200 mL, 20% KOH) was added. The resulting mixture was stirred at ambient temperature for 72 h. After completion of hydrolysis 1 L of water was added and the solution was acidified with 10% hydrochloric acid to pH=1-2. The formed precipitate was filtered and dried under vacuum to give a pure product. Yield of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]acetic acid was 75% (106 g).

[0466] HPLC-MS (positive mode): m/z 311 (M+H).sup.+; Retention time: 1.11 min.

[0467] .sup.1H NMR (400 MHz, DMSO-d6, ppm): δ=13.01 (br s, 1H), 8.0-7.97 (m, 2H), 7.71 (d, J=7.0 Hz, 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).

2.2.2 2-Amino-N-(2-furylmethylcarbamoyl)acetamide

[0468] A mixture of 1.46 g (6.74 mmol) 2-chloro-N-(2-furylmethyl-carbamoyl)acetamide and sodium azide 90 mg (NaN.sub.3, 2eq.) in 15 mL of ethanol was stirred at 70° C. overnight. After cooling to room temperature the solids were removed and obtained filtrate was concentrated in vacuo (⅓ of volume). The residue was portioned between EtOAc and water. Organic layer was washed with brine, dried over Na.sub.2SO.sub.4 and evaporated in vacuo. The residue was dissolved in MeOH, and Pd/C was added therein. The mixture was degassed, and was allowed to stir at RT under hydrogen atmosphere overnight. The catalyst was removed, and the filtrate was evaporated and dried in vacuo to afford crude amine, which was used in the next step without further purification. (X═O 0.8 g 60%; X═O 0.7 g 62% (from 2 steps)).

2.2.3 2-Amino-N-(2-thienylmethylcarbamoyl)acetamide

[0469] 2-Amino-N-(2-thienylmethylcarbamoyl)acetamide was prepared analogous to example 2.2.2, except that 1.26 g (5.41 mmol) 2-chloro-N-(2-thienylmethylcarbamoyl)-acetamide was used as the starting material instead of 2-chloro-N-(2-furylmethyl-carbamoyl)acetamide. The thus obtained crude 2-amino-N-(2-thienylmethyl-carbamoyl)acetamide was used in the next step without further purification. Yield: 0.7 g 62% (from 2 steps).

2.3 Compounds I

Example 1

Synthesis of [2-(2-furylmethylcarbamoylamino)-2-oxo-ethyl] 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate (MyoMed-946)

[0470] A mixture of 310 mg (1.0 mmol) 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-acetic acid, 228 mg (1.05 mmol) of 2-chloro-N-(2-furylmethylcarbamoyl)acetamide, 75 mg (0.5 mmol) of NaI, and DIPEA 155 mg (1.2 mmol) was dissolved in 6 mL of DMSO. The resulting slurry was stirred for 72 h at room temperature to completion of reaction; conversion was controlled by LC-MS. Then the reaction mixture was poured into 50 mL of water, the resulting precipitate was filtered and washed with additional portion of water, isopropyl alcohol, and hexane subsequently. The solid product was dried under vacuum. Yield of [2-(2-furylmethylcarbamoylamino)-2-oxo-ethyl] 4-[(4-methyl-2-oxo-chromen-7-yl)oxy-methyl]benzoate (MyoMed-946) was 61% (289 mg).

[0471] [2-(2-Furylmethylcarbamoylamino)-2-oxo-ethyl] 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate (MyoMed-946) showed no trace of decay over several months storage at room temperature (confirmed via NMR and LC-MS).

[0472] HPLC-MS (Positive mode): m/z 491/492 (M+H).sup.+; Retention time: 1.436 min.

[0473] .sup.1H NMR (400 MHz, DMSO-d6, ppm): δ=10.50 (br.s, 1H, NH), 7.85 (br.s, 1H, NH), 7.55 (d, J=8.0 Hz, 2H, CH+CH), 7.22 (d, J=8.4 Hz, 1H, CH), 7.15 (d, J=8.0 Hz, 2H, CH+CH), 7.09 (s, 1H, CH), 6.60 (m, 2H, CH+CH), 5.90 (m, 1H, CH), 5.78 (d, J=1.4 Hz, 1H, CH), 5.74 (s, 1H, CH), 4.87 (s, 2H, OCH.sub.2), 4.44 (s, 2H, OCH.sub.2), 3.88 (d, J=5.2 Hz, 2H, NCH.sub.2), 1.91 (s, 3H, CH.sub.3).

[0474] Further Analytical Characterization of MyoMed-946

[0475] The purity and identity of the compound was further assessed using 1D and 2D NMR and UPLC-MS as follows:

[0476] For NMR analysis, 2 mg of MyoMed-946 was dissolved in in 1 ml d.sub.6-dimethylsulfoxid with trace amounts of CCl.sub.4. .sup.1H- and .sup.13C-spectra were recorded as well as COSY and HSQC 2D-NMR spectra for .sup.1H-NMR peak assignment.

[0477] .sup.1H NMR (500 MHz, DMSO-d6+CCl.sub.4): δ=10.68 (s, 1H), 8.34 (s, 1H), 8.05 (d, J=7.37 Hz, 2H), 7.53-7.45 (m, 4H), 7.14-6.91 (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).

[0478] .sup.13C NMR (126 MHz, DMSO-d6+CCl4): δ=169.0, 165.1, 161.0, 160.0, 154.6, 153.3, 152.5, 151.8, 142.3 (2C), 129.6 (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.

[0479] For the UPLC-MS analysis, a small amount of MyoMed-946 was dissolved in acetonitrile (MeCN) and 3 μl of this solution was injected onto a C18 UPLC column (Acquity UPLC BEH C18; 1.7 μm; 2.1×50 mm). The UPLC-MS system and analytical method used was as described above.

[0480] UPLC-MS (positive mode): m/z=491.1 (M+H).sup.+; Retention time: 1.7 min.

Example 2

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

[0481] Synthesis was performed analogous to Example 1 except that N-carbamoyl-2-chloro-propanamide was used instead of 2-chloro-N-(2-furylmethyl-carbamoyl)acetamide.

[0482] HPLC-MS (Positive mode): m/z 439 (M+H).sup.+; Retention time 1.388 min.

[0483] .sup.1H NMR (400 MHz, 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).

Example 3

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

[0484] Synthesis was performed analogous to Example 1 except that 2-chloro-N-(methylcarbamoyl)propanamide was used instead of 2-chloro-N-(2-furylmethyl-carbamoyl)acetamide.

[0485] HPLC-MS (Positive mode): m/z 425 (M+H).sup.+; Retention time 1.327 min.

[0486] .sup.1H NMR (400 MHz, 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).

Example 4

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

[0487] To a cooled solution of 2-amino-N-(2-furylmethylcarbamoyl)acetamide obtained in example 2.2.2 (0.6 mmol), 558 mg (1.8 mmol) 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]-acetic acid, 1-hydroxy-7-azabenzotriazole 109 mg (HOAt, 0.8 mmol) in 2 mL of DMF, EDC 124 mg (0.8 mmol) was added dropwise, and the mixture was allowed to stir at room temperature for overnight. The formed precipitate was collected, washed with methanol, then water, again with methanol and dried to afford 110 mg of the title compound in. Yield: 37%.

[0488] .sup.1H NMR (400 MHz, DMSO-D.sub.6, ppm): δ=10.53 (s, 1H), 8.82 (t, J=5.7 Hz, 1H), 8.51 (br.s, 1H), 7.89 (d, J=8.0 Hz, 2H), 7.70 (d, J=8.6 Hz, 1H), 7.56 (m, 3H), 7.06 (m, 2H), 6.37 (s, 1H), 6.24 (d, J=2.5 Hz, 1H), 6.21 (s, 1H), 3.50 (s, 2H), 4.35 (d, J=5.7 Hz, 2H), 3.99 (d, J=5.0 Hz, 2H), 2.38 (s, 3H).

Example 5

Synthesis of [2-oxo-2-(2-thienylmethylcarbamoylamino)ethyl] 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate

[0489] A mixture of 310 mg (1.0 mmol) 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) DIPEA was dissolved in 6 mL of DMSO. The resulting slurry was stirred for 72 h at room temperature to completion of reaction; conversion was controlled by LC-MS spectra. Then the reaction mixture was poured into 50 mL of water, the formed precipitate was filtered and washed with additional portion of water, isopropyl alcohol and hexane subsequently. The product was dried under vacuum condition. Yield 61% (310 mg) of [2-oxo-2-(2-thienylmethylcarbamoyl-amino)ethyl] 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate.

[0490] HPLC-MS (Positive mode): m/z 507/508 (M+H).sup.+; Retention time 1.399 min.

[0491] .sup.1H NMR (400 MHz, 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).

Example 6

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

[0492] The title compound was prepared analogous to Example 3. 90 mg of the title compound was obtained. Yield: 30%.

[0493] .sup.1H NMR (400 MHz, DMSO-D.sub.6, ppm): δ=10.52 (s, 1H), 8.82 (t, J=5.7 Hz, 1H), 8.63 (br.s, 1H), 7.89 (d, J=8.0 Hz, 2H), 7.68 (d, J=8.7 Hz, 1H), 7.55 (d, J=8.0 Hz, 2H), 7.39 (d, J=4.2 Hz, 1H), 7.07 (m, 2H), 6.98 (s, 1H), 6.95 (m, 1H), 6.21 (s, 1H), 5.31 (s, 2H), 4.23 (d, J=5.2 Hz, 2H), 4.00 (d, J=5.7 Hz, 2H), 2.39 (s, 3H).

Example 7

Synthesis of [2-oxo-2-(2-pyridylmethylcarbamoylamino)ethyl] 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate (MyoMed-203)

Preparation of 2-chloro-N-(2-pyridylmethylcarbamoyl)acetamide

[0494] To a stirring solution of 2-pyridylmethanamine (7.84 g, 72.5 mmol) in anhydrous dichloromethane (100 mL) cooled to −10° C., chloroacetyl isocyanate (8.66 g, 72.5 mmol) was added and the reaction mixture was stirred for 2 h at r.t. The precipitated solid was collected by filtration, washed with dichloromethane (2×30 mL), and dried to obtain 12.0 g (52.7 mmol, yield: 73%) of 2-chloro-N-(2-pyridylmethylcarbamoyl)acetamide.

[0495] .sup.1H 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).

[0496] HPLC-MS (Negative mode) m/z 226 (M−H).sup.+; Retention time 0.565 min.

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

[0497] To a solution of 7-hydroxy-4-methyl-chromen-2-one (80.6 g, 458 mmol) in acetone (1000 mL) K.sub.2CO.sub.3 (94.9 g, 687 mmol) and methyl 4-(bromomethyl)benzoate (110 g, 480 mmol) were added and the reaction mixture was refluxed for 3 h. Then, it was cooled to r.t. and filtered. The filtrate was evaporated under reduced pressure and the residue was mixed with water (1000 mL). The insoluble solid was collected by filtration, washed with water, 2-propanol, and hexane, dried, and dissolved in DMSO (500 mL). 20% aqueous KOH (150 mL) was added to the obtained solution of methyl 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate and the mixture was left to stir overnight at r.t. After the reaction was completed, it was poured into water (3000 mL) and acidified until pH 1-2 with 10% hydrochloric acid. After 30 min of stirring, the precipitated solid was collected by filtration, washed with water, 2-propanol, and hexane, and dried to obtain 106 g (342 mmol, yield: 75%) of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid.

[0498] Methyl 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate:

[0499] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ=7.98 (d, J=8.1 Hz, 2H), 7.68 (d, J=8.5 Hz, 1H), 7.60 (d, J=8.1 Hz, 2H), 7.12-6.96 (m, 2H), 6.20 (s, 1H), 5.32 (s, 2H), 3.85 (s, 3H), 2.38 (s, 3H).

[0500] 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid:

[0501] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=12.99 (br s, 1H), 7.96 (d, J=7.2 Hz, 2H), 7.67 (d, J=8.4 Hz, 1H), 7.57 (d, J=7.2 Hz, 2H), 7.11-6.99 (m, 2H), 6.20 (s, 1H), 5.31 (s, 2H), 2.37 (s, 3H).

[0502] HPLC-MS (Positive mode) m/z 311 (M+H).sup.+; Retention time 1.242 min.

Preparation of [2-oxo-2-(2-pyridylmethylcarbamoylamino)ethyl] 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate

[0503] A mixture of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid (9.28 g, 29.9 mmol), 2-chloro-N-(2-pyridylmethylcarbamoyl)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 r.t. 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 obtain 13.8 g (27.5 mmol, yield: 92%) of [2-oxo-2-(2-pyridylmethylcarbamoyl-amino)ethyl] 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate (MyoMed-203).

[0504] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=10.65 (br s, 1H), 8.69 (br s, 1H), 8.47 (d, J=4.6 Hz, 1H), 8.01 (d, J=8.1 Hz, 2H), 7.73 (t, J=8.4 Hz, 1H), 7.67 (d, J=8.7 Hz, 1H), 7.61 (d, J=8.1 Hz, 2H), 7.28 (d, J=7.7 Hz, 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=5.3 Hz, 2H), 2.36 (s, 3H).

[0505] HPLC-MS (Negative mode) m/z 502 (M−H).sup.+; Retention time 1.176 min.

Example 8

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

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

[0506] To a suspension of CDI (42.8 g, 264 mmol) in anhydrous acetonitrile (400 mL) 2-thienylmethanamine (14.9 g, 132 mmol) was added and the reaction mixture was maintained in an ultrasonic bath for 1 h at r.t. Then, water (2.5 mL) was added and the mixture was maintained in an ultrasonic bath for further 30 min. After the degassing of the solution, N-boc-ethylenediamine (21.1 g, 132 mmol) was added and the reaction was stirred for 2 h at 50° C. Then, the mixture was cooled to r.t. and evaporated under reduced pressure. The residue was triturated with water (100 mL), filtered, and dried to obtain 35.2 g (118 mmol, 89%) of tert-butyl N-[2-(2-thienylmethylcarbamoylamino)-ethyl]carbamate.

[0507] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=7.35 (d, J=5.8 Hz, 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=5.9 Hz, 2H), 3.11-3.00 (m, 2H), 2.99-2.88 (m, 2H), 1.37 (s, 9H).

[0508] HPLC-MS (Positive mode) m/z 300 (M+H).sup.+; Retention time 1.156 min.

Preparation of 2-(2-thienylmethylcarbamoylamino)ethylammonium chloride

[0509] To a solution of tert-butyl N-[2-(2-thienylmethylcarbamoylamino)-ethyl]carbamate (19.5 g, 65.1 mmol) in anhydrous dichloromethane (200 mL) 10% dioxane/HCl (50 mL) was added and the reaction mass was stirred for 2 h at r.t. The precipitated solid was collected by filtration and dried under vacuum to obtain 14.3 g (60.6 mmol, 95%) of 2-(2-thienylmethylcarbamoylamino)ethylammonium chloride.

[0510] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ=8.08 (br s, 3H), 7.35 (s, 1H), 6.93 (s, 2H), 6.78 (br s, 1H), 6.51 (br s, 1H), 4.38-4.29 (m, 2H), 3.31-3.19 (m, 2H), 2.88-2.74 (m, 2H).

[0511] HPLC-MS (Positive mode) m/z 200 (M+H).sup.+; Retention time 0.428 min.

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

[0512] To a solution of 7-hydroxy-4-methyl-chromen-2-one (80.6 g, 458 mmol) in acetone (1000 mL) K.sub.2CO.sub.3 (94.9 g, 687 mmol) and methyl 4-(bromomethyl)benzoate (110 g, 480 mmol) were added and the reaction mixture was refluxed for 3 h. Then, it was cooled to r.t. and filtered. The filtrate was evaporated under reduced pressure and the residue was mixed with water (1000 mL). The insoluble solid was collected by filtration, washed with water, 2-propanol, and hexane, dried, and dissolved in DMSO (500 mL). 20% aqueous KOH (150 mL) was added to the obtained solution of methyl 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate and the mixture was left to stir overnight at r.t. After the reaction was completed, it was poured into water (3000 mL) and acidified until pH 1-2 with 10% hydrochloric acid. After 30 min of stirring, the precipitated solid was collected by filtration, washed with water, 2-propanol, and hexane, and dried to obtain 106 g (342 mmol, yield: 75%) of 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid.

[0513] Methyl 4-[(4-methyl-2-oxo-chromen-7-yl)oxymethyl]benzoate:

[0514] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ=7.98 (d, J=8.1 Hz, 2H), 7.68 (d, J=8.5 Hz, 1H), 7.60 (d, J=8.1 Hz, 2H), 7.12-6.96 (m, 2H), 6.20 (s, 1H), 5.32 (s, 2H), 3.85 (s, 3H), 2.38 (s, 3H).

[0515] 4-[(4-Methyl-2-oxo-chromen-7-yl)oxymethyl]benzoic acid:

[0516] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=12.99 (br s, 1H), 7.96 (d, J=7.2 Hz, 2H), 7.67 (d, J=8.4 Hz, 1H), 7.57 (d, J=7.2 Hz, 2H), 7.11-6.99 (m, 2H), 6.20 (s, 1H), 5.31 (s, 2H), 2.37 (s, 3H).

[0517] HPLC-MS (Positive mode) m/z 311 (M+H).sup.+; Retention time 1.242 min.

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

[0518] 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) CDI (7.26 g, 44.8 mmol) was added and the mixture was stirred for 30 min at r.t. 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 mass was stirred at 50° C. for 16 h. After the mixture cooled down to r.t., water (600 mL) was added. The precipitated solid was collected by filtration, washed with water, 2-propanol, and hexane, and dried to obtain 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).

[0519] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=8.61-8.47 (m, 1H), 7.86 (d, J=7.9 Hz, 2H), 7.70 (d, J=8.7 Hz, 1H), 7.55 (d, J=7.9 Hz, 2H), 7.38-7.27 (m, 1H), 7.12-7.02 (m, 2H), 6.51 (t, J=6.0 Hz, 1H), 6.22 (s, 1H), 6.17-6.06 (m, 1H), 5.30 (s, 2H), 4.36 (d, J=5.9 Hz, 2H), 3.32-3.17 (m, 4H), 2.39 (s, 3H).

[0520] HPLC-MS (Positive mode) m/z 492 (M+H).sup.+; Retention time 1.249 min.

II. Biochemical Assay

[0521] 1. MuRF1—Titin Interaction Assay:
Small molecule screens were performed using an ALPHA screen to identify compounds that inhibit interaction between MuRF1 and titin. This screen is based on MuRF1 and titin interaction studies that identified the MuRF1 B-box-coiled domain to interact with titin A169 (see for example Mrosek et al., Biochemistry 2008, 47, 10722-10730). A prototype of this ALPHA screen assay is described in WO 2009/077618.

Procedure:

[0522] These interacting fragments were expressed as GST and biotin fusion proteins so that complex formation could be monitored with glutathione acceptor and avidin donor beads, respectively, as described in WO 2009/077618. A survey of 280,000 compounds (in-house library, EMBL chemical core facility) identified a total of 40 molecules with Ki values of 5-25 μmol/L for the MuRF1-titin interaction. [0523] 2. Determination of the Inhibition of MuRF1 E3 Ligase Activity:
Compounds were then assessed for effects on MuRF1 E3 ligase activity directed to titin or to MuRF1 itself (self-ubiquitination) 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 respective compounds. Reactions were started by addition of 220 nmol/L MuRF1, followed by 1 h at 37° C., SDS PAGE and Western blot analysis with MuRF1 and titin specific antibodies. All reactions also included 5% DMSO. MyoMed-946, MyoMed-946-5, and MyoMed-946-8 were tested and could be identified to significantly inhibit MuRF1 E3 ligase activity, based on ubiquitination patterns. [0524] 3. Differential Scanning Fluorimetry (DSF) of MuRF1 Central Fragment in the Absence and Presence of the Compound MyoMed-946:

[0525] The effect of compound MyoMed-946 on MuRF1 protein stability was determined in vitro by differential scanning fluorimetry (DSF).

[0526] Method:

[0527] “MuRF1 central fragment” was expressed as previously described (Mrosek M et al., FASEB J, 2007, 21, 1383-1392) and used in DSF experiments at 75 μM final concentration. Compound MyoMed-946 was diluted from a 10 mM stock in DMSO to 100 μM in PBS as DSF assay buffer, resulting in a final concentration of 1% DMSO. After 1 hour pre-incubation at room temperature, the aqueous protein solutions were soaked into capillaries and placed into a Prometheus NT.48 nanoDSF device (NanoTemper Technologies, Munich, Germany). Changes in the intrinsic tryptophan or tyrosine fluorescence that occurred after LED laser excitement upon protein unfolding in a thermal gradient was detected at 330 nm and 350 nm, respectively. Changes in the intrinsic protein fluorescence in a thermal gradient were monitored at 350 nm and 330 nm, respectively. The first derivative of the fluorescence wavelength ratio of 350/330 nm upon thermal protein unfolding was used to calculate transition midpoint (Tm) of single and multiple transition states.

[0528] Results:

[0529] As can be seen from FIG. 55, Tm of MuRF1 central fragment in PBS was 65.2° C. (solid line) and was only negligibly changed to 65.8° C. by the addition of 1% DMSO (dashed line). In contrast, a strong effect on the thermal unfolding of MuRF1 was observed by the addition of compound MyoMed-946 (dotted line). Compound MyoMed-946 destabilized MuRF1 as indicated by the significantly reduced main Tm of 52.5° C.

III. Biological Investigations

[0530] 1. Cell Culture Experiments
Murine C2C12 myoblasts (CRL-1772, ATCC) were cultured in DMEM (Lonza; Basel, Switzerland) supplemented with 10% fetal calf serum (FCS; Gibco®Invitrogen, Carlsbad, Calif.). For induction of differentiation into myotubes, subconfluent cultures were switched to DMEM containing 2% horse serum (Sigma-Aldrich; Seelze, Germany). Myotubes were subsequently pre-treated for 2 h with increasing compound concentrations (0.1 to 10 μmol/L, dissolved in DMSO) or with an equal volume of DMSO before treatment for 24 h with 10 μmol/L dexamethasone (DEX; Sigma-Aldrich; Seelze, Germany). Myotube diameter was then evaluated by image analysis software (Analysis 3.0, Olympus Soft Imaging Solutions GmbH, Munster, Germany). To determine cytotoxicity of selected compounds, myoblasts or myotubes were incubated with increasing concentrations for 24 h. Subsequently, the concentration of lactate dehydrogenase (LDH) activity was quantified in the cell culture supernatant as a measure for cell destruction as described in Bellocci et al., Anal Biochem, 2008, 374, 48-55. As can be seen from the FIGS. 1 and 2, the compound MyoMed-946 shows low toxicity in both, myoblasts and myotubes (myocytes). [0531] 2. Animal Experiments [0532] 2.1 Pulmonary Hypertension Mouse Model: [0533] 2.1.1 Test Series 1 with MuRF1 Inhibitor MyoMed-946:
The animal experiments were approved by the Regierungspräsidium Karlsruhe (35-9185.81/G-141/13) and the Regierungspräsidium Leipzig (TVV 40/16). Three groups of mice were included in this study, including: 1) saline-treated (sham; n=20); 2) monocrotaline (MCT)-treated fed normal chow (MCT; n=27); and 3) MCT-treated fed MuRF1 inhibitor chow (MCT+compound; n=27). Briefly, C57BL/6 mice (aged 8 weeks) were subcutaneously injected weekly with either MCT (600 mg/kg) or a matched volume of saline for 6 weeks, a time period where MCT is known to induce cardiac cachexia due to pulmonary hypertension and subsequent RV dysfunction rather than anorexia, as described by Ahn et al., PLoS One, 2013, 8:e62702. Mice were exposed to identical conditions under a 12:12 h light/dark cycle with food and water provided ad libitum. The MCT+compound group started receiving the inhibitor chow 1 week prior to the MCT injections, whereas the sham and MCT groups were fed an identical chow but without the addition of the selected compound. Body weight was recorded every week for each mouse. Mice were sacrificed following deep anesthetization with i.p. administration of fentanyl (0.05 mg/kg), medetomidine (0.5 mg/kg), midazolam (5 mg/kg) and ketamine (100 mg/kg). At sacrifice, the heart and lungs were dissected, cleaned, blotted dry and weighed, with the heart fixed in 4% PBS-buffered formalin. The left tibialis anterior (TA), soleus, extensor digitorum longus (EDL), and section of costal diaphragm were also dissected, weighed and fixed in 4% PBS buffered formalin, while the remaining muscle portions were immediately frozen in liquid N.sub.2 for molecular analysis.

[0534] For histological evaluation, paraffin-embedded TA muscle sections (3 μm) were stained with H&E and fiber cross-sectional area (CSA) and then evaluated by imaging software (Analysis 3.0, Olympus Soft Imaging Solutions GmbH, Munster, Germany). In addition, medial cross sections (3 m) of the heart were mounted on glass slides and subsequently stained with H&E to assess RV wall thickness.

As can be seen from FIGS. 3 to 5, the weight gain, lung weight and heart weight were near identical between the MCT and MCT+MyoMed-946 fed mice, which suggests that the disease progressed in both groups independent of compound feeding. Importantly, as can be seen from FIGS. 6 and 7, the MuRF1 inhibitor MyoMed-946 attenuates the development of right ventricle hypertrophy. Furthermore, as can be seen from FIGS. 8 to 12, while MCT treated mice showed a progressive loss of skeletal muscle mass, the MCT+compound fed mice did not follow this trend and were protected—the most obvious effect seen in the TA muscle. [0535] 2.1.2 Test Series 2 with MuRF1 Inhibitors MyoMed-946, MyoMed-203 and MyoMed-205:

[0536] The test series 2 were performed analogous to test series 1. Briefly, Three groups of mice were included in this study: 1) saline-treated mice (sham; n=10); 2) monocrotaline (MCT)-treated mice fed with normal chow (MCT; n=10); and 3) MCT-treated mice fed with MuRF1 inhibitor chow (MCT+compound; n=10 for each compound). 8 weeks old C57BL/6 mice were subcutaneously injected weekly with either MCT (600 mg/kg) or a matched volume of saline for 8 weeks. The MCT+compound group started receiving the inhibitor chow 1 week prior to the MCT injections, whereas the sham and MCT groups were fed an identical chow but without the addition of the selected compound. The compound concentration in the chow was 0.1 weight-%, resulting in a daily compound intake per mouse of about 3 mg. Body weight was recorded every week for each mouse. Mice were sacrificed after 8 weeks treatment. At sacrifice, examination and tissue analysis were performed as described in test series 1.

[0537] As can be seen from FIGS. 13 to 15, the weight gain, lung weight and heart weight were similar between the MCT and MCT+compound fed mice, which suggest that the disease progressed in both groups independent of compound feeding. Also in this test series, as can be seen from FIGS. 16 to 18, the MuRF1 inhibitors MyoMed-946, MyoMed-203 and MyoMed-205 attenuate the development of right ventricle hypertrophy. Furthermore, as can be seen from FIGS. 19 to 21, while MCT treated mice showed a progressive loss of skeletal muscle mass, the MCT+compound fed mice did not follow this trend and were protected—the most pronounced effect seen for the compound MyoMed-203. Furthermore, the MuRF1-expression level in the TA muscle tissue is clearly reduced in MCT+compound fed mice, where the most pronounced reduction is seen for the compound MyoMed-205, as can be seen from FIG. 22. Also the expression level of telethonin, a MuRF1 target protein, is essentially normalized in the MCT+compound fed mice, as can be seen from FIG. 23. [0538] 2.2 Myocardial Infarction LAD Mouse Model:

[0539] A myocardial infarction mouse model suffering from a heart failure with reduced ejection fraction (HFrEF) was generated by ligating the left anterior dexterior coronary artery (LAD) as described below to induce an acute myocardial infarction (MI) followed by the development of a chronic (systolic) heart failure (CHF).

[0540] LAD Ligation Procedure: A small animal surgery was carried out on C57/BL6 mice at 12 weeks of age in the Leipzig Heart Center following established and well known procedures in the field (see for example Bowen et al., J Appl Physiol, 2015, 118, 11-19; Mangner et al., J Cachexia Sarcopenia Muscle, 2015, 6, 381-390). Briefly, mice from the LAD group were anesthetized by i.p. injection of MMF; Medetomidin (0.5 mg/kg body weight), Midazolam (5.0 mg/kg body weight), Fentanyl (0.05 mg/kg body weight). Unconscious mice were fixed on an operation table. Ventral parts of the thorax were shaved, washed and sterilized. Unconscious mice were then 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 operation, the thorax was opened about 1 cm above the Processus xiphoideus and about 1 cm left parasternally by dissecting the skin here. The below located M. pectoralis was moved to the sides without further injury to access the thorax wall. The intercostal muscles between two ribs were moved to the side without breaking the ribs. The thereby created intrathoracic access was widened by a surgical spreader inserted above the pericardium. The pericardium was opened surgically stump by using two anatomical forceps. The thymus, if present in the surgical field, was pushed to the side with a surgical swab to gain access to the aortic root. The heart was carefully moved out of the pericardium using a hook, and the left atrium was put to the side with a surgical swab. The LAD was then ligated with a 5.0 Prolene suture (Ethicon, see http://www.ethicon.com/healthcare-professionals/products/wound-closure/non-absorbable-sutures/prolene-polypropylene). The ligation was made close to the aortic root, and the suture was tightened until paleness of the coronary artery anterior wall was noted. After ligation, the thorax wall and then the skin were closed by a single button seam using a 4.0 Prolene suture (company: Ethicon).

[0541] As a control, sham operations were performed. For the sham control group, the procedure followed was exactly the same as described above, except that the 5.0 prolene suture was only loosely out around the LAD without tightening.

[0542] The operation is ended by extubation, and antagonizing the anesthesia by i.p. injection of Atipamezol (2.5 mg/kg body weight) and Flumazenil (0.5 mg/kg body weight). Mice were put onto a warming mate until waking up, and then were transferred back to animal cages. The time for the whole procedure above by expert staff was around 30 min.

[0543] One week after LAD ligation, echocardiography was performed in M-mode to confirm MI, i.e. the left ventricular end-diastolic (LVEDD) and systolic (LVESD) diameters were assessed to allow calculation of left ventricular (LV) fractional shortening (LVFS=[LVEDD_LVESDLVEDD]×100). Only mice with a large infarct (left ventricular ejection fraction (LVEF)<20%) were subsequently randomized into two groups, i.e. one group receiving normal chow (CHF, n=11) and a second group receiving chow supplemented with compound (0.1% of compound MyoMed-946, CHF+MyoMed-946, n=12). Sham operated animals received only normal chow (n=15). Nine weeks later, echocardiography was performed again.

TABLE-US-00001 TABLE 1 Animal characteristics after 10 weeks of intervention CHF + Sham CHF MyoMed-946 (n=15) (n = 11) (n = 12) Physical Body weight (g) 22.6 ± 0.5 22.4 ± 0.6 23.8 ± 0.4 Heart-to-body weight (g/mg) 5.03 ± 0.11 7.87 ± 0.55*** 7.52 ± 0.57*** Lung weight (wet/dry) 4.16 ± 0.07 4.48 ± 0.06* 4.47 ± 0.10* Histology LV infarct size (%) — 30.5 ± 4.6 27.1 ± 2.9 Echocardiography LVEDD (mm)  3.8 ± 0.1  6.2 ± 0.2***  5.9 ± 0.2*** LVESD (mm)  2.6 ± 0.1  5.8 ± 0.3***  5.3 ± 0.2*** LVEF (%) 59.6 ± 2.7 16.1 ± 2.9*** 21.1 ± 2.5*** LVFS (%) 31.8 ± 1.9  7.5 ± 1.4***  9.9 ± 1.2*** *P < 0.05 vs. sham; ***P < 0.001 vs. sham.

[0544] The animals were sacrificed to collect tissues, in particular diaphragm tissue, for functional and molecular characterization. All experiments and procedures were approved by the local Animal Research Council, University of Leipzig, and the Landesbehörde Sachsen (TVV 36/15). [0545] 2.3 Contractile Function:

[0546] To provide a direct functional assessment, the contractility in skeletal muscle fiber bundles, i.e. fibre bundle from the diaphragm of MCT mice and CIF mice, was measured as follows. A fiber bundle from the diaphragm was isolated to allow in vitro contractile function to be assessed using a length-controlled lever system (301B, Aurora Scientific Inc., Aurora, Canada), as described by Bowen et al., FASEB J, 2017, 31. Briefly, a muscle bundle was mounted vertically in a buffer-filled organ bath (˜22° C.), set at optimal length, and after 15 min was stimulated over a force-frequency protocol between 1-300 Hz (600 mA; 500 ms train duration; 0.25 ms pulse width). The muscle then underwent a force-velocity protocol whereby the muscle was allowed to shorten against external loads (80-10% of the maximal tetanic force; each separated by 1 min) after being stimulated at 150 Hz for 300 ms. Shortening velocity was determined 10 ms after the first change in length and on the linear section of the transient (DMA software, Aurora Scientific). Force (N) was normalized to muscle cross-sectional area (CSA; cm.sup.2) by dividing muscle mass (g) by the product of L.sub.o (cm) and estimated muscle density (1.06), which allowed specific force in N/cm.sup.2 to be calculated. Shortening velocity was normalized to optimal muscle length (in L.sub.o/s), while power was calculated for each load as the product of shortening velocity and specific force (in W/cm.sup.2).

[0547] As can be seen from FIGS. 24 to 27, the diaphragm myofiber bundles from chronic heart failure mice (CHF mice) developed less force during electric stimulation (FIG. 24), and had also reduced maximal force (FIGS. 25 and 26) and peak power (FIG. 27). Thus, compared with sham animals, mice with CHF on control chow developed a diaphragm myopathy at week 10. Feeding with compound MyoMed-946 protected mice from such a post-infarct diaphragm weakness. The loss of diaphragm function and diaphragm maximal force due to chronic heart failure can be significantly reduced in mice fed with the compound MyoMed-946 compared to the untreated group of CHF mice. This indicates that the selective inhibition of MuRF1 by the compound MyoMed-946 mediates a benefit to diaphragm function after heart failure with reduced ejection fraction (HFrEF) induced by myocardial infarction.

[0548] Likewise, as can be seen from FIGS. 28 to 30 the contractile dysfunction of the diaphragm (in terms of shortening velocity and power) in mice suffering from MCT-induced pulmonary hypertension was also essentially prevented when the MCT mice were fed with the compound MyoMed-946 (MCT+MyoMed-946).

[0549] Collectively, therefore, the above findings suggest that the selective inhibition of MuRF1 by the compound MyoMed-946 mediates a benefit to both skeletal muscle quantity (i.e., mass) and quality (i.e. contractile function) in chronic heart failure and in cardiac cachexia. [0550] 2.4 Tumor Mouse Model:

[0551] 8 weeks old female C57BL/6N mice were inoculated with B16F10 melanoma cells (9×10.sup.5 cells) or saline (sham, n=10). Within the first 3 days after tumor inoculation, the B16F10 mice as well as the sham mice received regular chow. Then the B16F10 mice were randomly divided in three groups, where the first group received MyoMed-946 chow (Tumor+MyoMed-946, n=10), the second group received MyoMed-205 chow (Tumor+MyoMed-205, n=10) and the third group received an identical chow but without the addition of the selected compounds (Tumor, n=10). The compound concentration in the chow was 0.1 weight-%, resulting in a daily compound intake per mouse of about 3 mg. Also the sham group was fed with an identical chow but without the addition of the selected compound. Wire-hang tests (muscle function) were performed with each mouse 9, 16 and 23 days after tumor inoculation. For this purpose, a standard wire hang construction was used with a wire (length: of 40 cm, diameter: 2.5 mm) at a height of 70 cm above the floor. Under the center of the wire a large box with sawdust was placed. For testing, the mouse was hung to the wire with the two limbs and hang time was recorded. Each animal had three attempts for 180 sec. max each. After three attempts, the maximal holding impulse was calculated (hanging time×body weight).

[0552] Mice were sacrificed 25 days after tumor inoculation. At sacrifice, examination and tissue analysis were performed as described above.

[0553] As can be seen from FIG. 31, tumor induced skeletal muscle atrophy (based on TA muscle weight) could be attenuated in the tumor+MyoMed-946 mice and the tumor+MyoMed-205 mice at least to certain extent when compared to the tumor mice. This effect is more pronounced in the wire-hang test (FIG. 32), where the administration of compounds MyoMed-946 and MyoMed-205 (tumor+MyoMed-946 mice and the tumor+MyoMed-205 mice) significantly attenuates the loss of muscle function compared to the tumor mice on normal diet.

2.5 Myocardial and Skeletal Muscle Alterations in Heart Failure with Preserved Ejection Fraction (HFpEF)—Rat Model

[0554] A schematic drawing of the study design is shown in FIG. 56. For this test, ZSF1 rats were used. After 20 weeks of age, ZSF1 rats lose diastolic compliance, leading to increased end-diastolic volumes, and thus mimic human HFpEF (also known as diastolic heart failure.
Female ZSF1 lean (control, n=25) and ZSF1 obese (n=40) rats were included into the study. At the age of 20 weeks the development of HFpEF was confirmed by echocardiography/invasive hemodynamic measurements, and tissue material from a subset of animals were collected (control n=10; ZSF1 obese n=10). The remaining control rats (control, n=15) were kept sedentary for another 12 weeks whereas the remaining ZSF1 obese animals (n=30) were randomized into the following groups: (1) rats receiving normal chow (HFpEF group, n=15) or chow supplemented with compound MyoMed-205 (0.1% of MyoMed-205, HFpEF+MyoMed-205, n=15). Rats were exposed to identical conditions in a 12 h light/dark cycle, with food and water provided ad libitum. Twelve weeks after randomization, echocardiography and invasive hemodynamic measurements were performed to elucidate the degree of diastolic dysfunction. Rats were subsequently sacrificed (opening of the chest in deep anesthesia), and skeletal muscle and myocardial tissue was harvested for functional and molecular analyses (formalin fixation and snap frozen in liquid nitrogen). All experiments and procedures were approved by the local animal research council, TU Dresden and the Landesbehörde Sachsen (TVV 42/2018).

Echocardiography

[0555] Rats were anesthetized by 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 as previously described (T. S. Bowen et al., Eur. J. Heart Fail., 2015, 17, 263-272). For systolic function, B- and M-Mode of parasternal long- and short axis were measured at the level of the papillary muscles. Diastolic function was assessed in the apical 4-chamber view using pulse wave Doppler (for measurement of early (E) and atrial (A) waves of the mitral valve velocity) and tissue Doppler (for measurement of myocardial velocity (E′ and A′)) at the level of the basal septal segment in the septal wall of the left ventricle. Functional parameters (i.e. LV ejection fraction (LVEF) and stroke volume (SV)) and ratios of [E/E′] and [E/A]) were obtained using the Vevo LAB 2.1.0 software.

Invasive Hemodynamic Measurements

[0556] Invasive hemodynamic pressure measurements were performed as the terminal procedure. In anesthetized (ketamine, xylazine) but spontaneous breathing rats the right carotid artery was cannulated with a Rat PV catheter (SPR-838, ADInstruments Limited) which was gently placed in the middle of the left ventricle. The LV end-diastolic and end-systolic pressure, maximum rate of pressure rise (dP/dtmax), maximum rate of pressure fall (dP/dtmin), and time constant (τ) for LV relaxation, after which withdrawal of the catheter into the aorta followed, and phasic and mean arterial pressures were measured. Mean arterial pressure was measured in the ascending aorta. Data were recorded in LabChart8 software (ADInstruments).

Skeletal Muscle Function

[0557] The right EDL and the left soleus were dissected and mounted vertically in a Krebs-Henseleit buffer-filled organ bath between a hook and force transducer, with the output continuously recorded and digitized (1205A: Isolated Muscle System—Rat, Aurora Scientific Inc., Ontario, Canada). In vitro muscle function was assessed by platinum electrodes stimulating the muscle with a supra-maximal current (700 mA, 500 ms train duration, 0.25 ms pulse width) from a high-power bipolar stimulator (701C; Aurora Scientific Inc., Ontario, Canada). The muscle bundle was set at an optimal length (Lo) equivalent to the maximal twitch force produced, after which bath temperature was increased to 25° C. and a 15-minute thermos equilibration period followed. A force-frequency protocol was then performed at 1, 15, 30, 50, 80, and 120 Hz, separated by 1-minute rest intervals. After a 5-minute rest period, the muscles then under-went a fatigue protocol over 5 minutes (40 Hz every 2 seconds). Forces generated during the fatigue protocol were normalized to the initial force generated to provide a relative assessment of fatigability.

Results:

Animal Characteristics at 20 Weeks (Time Point of Randomization)

[0558] To verify the development of HFpEF before the animals were randomized into the different treatment groups, 10 ZSF1-lean and 10 ZSF1-obese animals were analyzed by echocardiography, invasive hemodynamic measurements and measurements of skeletal muscle function. The ZSF1-obese animals exhibited an increased body weight (sign of obesity), and signs of myocardial hypertrophy (increased heart weight when normalized to tibia length) were evident. With respect to markers for diastolic function the ratio E/6 and the left ventricular end-diastolic pressure (LVEDP) were significantly increased in the ZSF1-obese animals. Despite of a disturbance of diastolic function, left ventricular ejection fraction (LVEF) was normal (>60%) and even a little bit higher when compared to the lean control rats. With respect to mean arterial blood pressure (MABP) a significant elevation was seen in the ZSF1-obese animals. This increase in MABP is a sign of a hypertonic state in the animals, a feature which is well known from HFpEF patients. With respect to the peripheral skeletal muscle, the ZSF1-obese animals developed muscle atrophy and skeletal muscle dysfunction. In summary, the animals at an age of 20 week developed features which are in accordance with the diagnosis of HFpEF.

Impact of MvoMed-205 Treatment on Cardiac Parameters in HFpEF

[0559] To evaluate the impact of MyoMed-205 on myocardial function echocardiography and invasive hemodynamic measurements were performed. As shown in FIG. 57, the left ventricular ejection fraction (LVEF) (FIG. 57A) was significantly reduced in the non-treated HFpEF animals when compared to the control ZSF1-lean animals. This reduction is significantly attenuated by the 12 week treatment with MyoMed-205. With respect to the parameters for diastolic function, the ratio E/6 (FIG. 57B) and LVEDP (FIG. 57C), the treatment with MyoMed-205 attenuated the increase seen in the ZSF1-obese untreated animals. No treatment effect of MyoMed-205 was seen MABP (FIG. 57D). Taken together, these results show that the treatment with MyoMed-205 improved systolic as well as diastolic function significantly and this effect is not mediated by modulating the blood pressure.

Impact of MvoMed-205 Treatment on Skeletal Muscle Mass And Function

[0560] The skeletal mass and skeletal muscle function was already impaired in the ZSF1-obese animals at the time point of randomization into the different treatment groups. The present test examined whether MyoMed-205 was able to modulate skeletal muscle mass and function. The results can be seen in FIG. 58. Analysis of the muscle weight of the tibialis anterior muscle (TA) (FIG. 58A) revealed a significant drop in muscle wet weight in the HFpEF untreated animals. This muscle atrophy was attenuated by MyoMed-205. With respect to the EDL (FIG. 58C) and soleus muscle (FIG. 58B) the development of HFpEF had no impact on muscle wet weight. However, in the EDL muscle the treatment with MyoMed-205 resulted in a small but significant increase in muscle weight (FIG. 58C).

[0561] Besides measuring muscle weight for the development of muscle atrophy, the assessment of muscle function is very important. As shown in FIG. 58D, ZSF1-obese animals which were not treated develop a skeletal muscle dysfunction when compared to the ZSF1-lean control group. This drop in muscle force is evident in the soleus muscle (FIG. 58D) when measuring the absolute specific muscle force. Treating the HFpEF animals for 12 weeks with MyoMed-205 resulted in an attenuation of functional loss.

Conclusion:

[0562] The results show that in HFpEF MyoMed-205 attenuates the development of myocardial diastolic dysfunction and attenuates skeletal muscle atrophy and skeletal muscle dysfunction. [0563] 2.6 Doxorubicin-Induced Muscle Atrophy and Cardiac Toxicity
Doxorubicin (DOX) is an efficient chemotherapeutic drug used in various cancer treatments. However, its use is associated with early and chronic cardiotoxicity and myotoxicity. In rodents, a single injection of Doxorubicin is capable of reducing heart and skeletal muscle mass, followed by a marked impairment of function.
The effects of MyoMed-205 enriched food in a mouse model that was treated with Doxorubicin was evaluated. C57bl/6 mice were acclimated at the vivarium for three days, then randomly sorted into four groups, as follows: 1. Control (placebo food+0.9% saline i.p. injections); 2. MyoMed-205 (MyoMed-205-food, food supplemented with 1 g/kg MyoMed-205; +0.9% saline injections); 3. DOX (placebo food+DOX injections): 4. DOX+MyoMed-205 (MyoMed-205 food+DOX injections). Animals were pre-fed with either MyoMed-205 food or placebo for seven days before the first DOX injection. DOX treatment was at a total dosage of 25 mg/kg, given in five injections at days 10, 12, 16, 25, and 28, respectively. Animals' weight and food intake were measured daily at the same hour of the day to assess food consumption and weight gain. Cardiac functions were evaluated at days 19 and 42 by echocardiography. As shown in FIG. 59, feeding with MyoMed-205 was capable to reduce features of cachexia wasting and body stress markedly by day 43: MyoMed-205-fed mice had higher lean mass (FIG. 59A), retained more body fat (FIG. 59B), and had about two-fold less interstitial edema free liquids (FIG. 59C). * means p<0.05 v. control; $ means p<0.05 vs. MyoMed-205; § means p<0.05 vs. DOX+MyoMed-205 (Tukey post-test).
Echocardiography at day-15 and after 25 mg/Kg accumulated DOX dosage indicated cardiac toxicity and failure, as can be seen from FIG. 60, i.e. a decreased heart weight (corrected by tibial length) (FIG. 60C), reduced ejection fractions (calculated from long axis plan) (FIG. 60A), and reduced fractional shortening (calculated from short axis plan) (FIG. 60B), respectively. MyoMed-205 treatment until day 43 prevented this. In summary, administration of MyoMed-205 during DOX chemotherapy is useful to protect the heart. * means p<0.05 v. control; $ means p<0.05 vs. MyoMed-205; § means p<0.05 vs. DOX+MyoMed-205 (Tukey post-test). [0564] 2.7 Muscle Function in Obese Mice with Type 2 Diabetes During Diet-Induced Weight Loss (DIO Mouse Model: Diabetes with Insulin Resistance and Obesity)
The effects of compound-feeding in a mouse model for diabetes that also develops myopathy during the progression of diabetes were tested. Diabetes was induced by a high-fructose high-fat diet (HFD) for 4 months in DIO mice. The body weight gain was 22.2%. The mice developed increased fasting glucose and insulin resistance. Control group animals received a normal rodent diet (regular diet). Weight loss was then induced during a 30 days test period. In a first stage, the animals were fed normal rodent diet for 16 days. Then in a second stage the animals received a low-calory diet for 14 days. The body weight decreased by 12.4% on average at the endpoint. Two days after start of the first stage, the diet was supplemented with MyoMed-205 (at 1 g compound/1 kg food). At start of the first stage and then at days 3, 7, 14, 21 and 28 of the test period, compound-fed and control mice were compared for their muscle strength by wire hang tests.
Test design: ICR-DIO male mice were randomly assigned to groups of 8-10 animals each. Three control groups were included: Control group of mice without obesity (Control; regular diet (RD); n=10), obese mice fed with high-fructose high-fat diet during the entire study period (DIO HFD; n=8); and obese mice with initiated weight loss (DIO control; n=8).
Wire hang test: For the wire hang tests (WHT), a standard wire hang construction was used with a wire (length: of 40 cm, diameter: 2.5 mm) at a height of 70 cm above the floor. Under the center of the wire a large box with sawdust was placed. For testing, the mouse was hung to the wire with the two limbs and the time that mice were able to hold onto the wire before falling off was recorded. Each animal had three attempts for a maximum of 180 sec. After the three attempts, the maximal holding impulse was calculated (hanging time×body weight).

Results:

[0565]

TABLE-US-00002 Observed effects on muscle strength in wire Groups hang test (WHT) 1 Control (RD) No effect on WHT, control group 2 DIO control Decreased WHT performance, compared to group 1 3 DIO HFD Decreased WHT performance, compared to groups 1 and 2 5 MyoMed-205-treated DIO Significant improvement of muscle strength in WHT by week 4 compared to groups 2 and 3
FIG. 61 is the graphical illustration of the results of the WHT of MyoMed-205-treated DIO mice as compared to DIO control mice. As can be seen, MyoMed-205 significantly improved the muscle strength as compared to DIO control mice by week 4 (28 days). The muscle strength was also improved significantly as compared to DIO HFD mice and did not differ significantly from the control (RD) mice group by week 4 (not shown in FIG. 61). [0566] 2.8 Glucose and Insulin Regulation in Obese Mice with Type 2 Diabetes During Diet-Induced Weight Loss (DIO Mouse Model: Diabetes with Insulin Resistance and Obesity)
Analogously to example 2.7, diabetes was induced by a high-fructose high-fat diet (HFD) for 4 months in DIO mice. The mice developed increased fasting glucose and insulin resistance. Control group animals received a normal rodent diet (regular diet). Weight loss was then induced during a 30 days test period. In a first stage, the animals were fed normal rodent diet for 16 days. Then in a second stage the animals received a low-calory diet for 14 days. Two days after start of the first stage, the diet was supplemented with MyoMed-203 (at 1 g compound/1 kg food).
Test design: ICR-DIO male mice were randomly assigned to groups of 8-10 animals each. Three control groups were included: Control group of mice without obesity (control; regular diet (RD); n=10), obese mice fed with high-fructose high-fat diet during the entire study period (DIO HFD; n=8); and obese mice with initiated weight loss (DIO control; n=8).

Determination of Blood Glucose Level

[0567] Determining the level of blood glucose was performed after 6-hour fasting. An On Call Plus glucometer (Acon Laboratories, Inc., USA) and specific test strips (REF G133-111) were used for determination of glucose levels. Blood was obtained from the tail vein by incision of the tail tip. 5-6 μl of blood were used for each assay.
In MyoMed-203-treated animals, the blood glucose level was significantly lower compared with the DIO control group throughout the treatment period where the fasting blood glucose value significantly changed on the 2.sup.nd and 21.sup.st day of the study. FIG. 62 shows the comparison of MyoMed-203-treated DIO mice with DIO control mice.

Glucose Tolerance Test

[0568] For oral glucose tolerance test (OGTT), mice were orally treated with glucose at dose of 2 g/kg at the dose volume of 10 ml/kg after a 6-hour fasting. Glucose measurements were performed immediately before treatment and at 15, 30, 60 and 120 min after glucose administration. The AUC describing the rate of blood glucose output in each mouse during the test was calculated using the formula:

[00001]$S=\underset{a}{\stackrel{b}{\int }}f\left(x\right)dx$

MyoMed-203 showed mild hypoglycemic effect in obese mice after 14 days of weight loss and treatment according to OGTT data. Blood glucose levels in the animals of this group tended to be lower at 30 minutes of OGTT test compared to DIO control group. The same is true for the calculated AUCs describing glucose elimination. A similar trend continued in the 28.sup.th day of the study: significant decrease in the glucose level at 120 min was observed in MyoMed-203 treated group compared with all control groups; the calculated area under the glucose elimination curve tended to be lower as well. FIG. 63 and FIG. 64 show the OGTT data at days 14 and 28, respectively, of MyoMed-203-treated DIO mice and DIO control mice.

Test of Tolerance to Insulin Action

[0569] For insulin tolerance test (ITT), mice were injected intraperitoneally with recombinant human insulin solution (Lilly, France; REF C620001K) at the dose of 0.75 or 0.60 IU/g of body weight in the volume of 5 ml/kg after 6-hour fasting. Glucose measurements were performed immediately before treatment and at 15, 30, 60 and 120 min after insulin administration. The results of the rate of blood glucose output were expressed as area under the curve (AUC).
A significant decrease of glucose level was observed in MyoMed-203-treated mice at 120 minutes after insulin injection compared with all control groups. FIG. 65 shows the ITT results in MyoMed-203-treated DIO mice and DIO control mice. [0570] 3. Tissue Analyses [0571] 3.1 Proteomic and Western Blot Analysis: [0572] 3.1.1 MCT Mice:

[0573] Proteomic Analysis:

[0574] Proteins from frozen diaphragm samples in sham, MCT, and MCT+compound mice (n=3 per group) were powdered under liquid N.sub.2. Mass spectrometry was then performed at the DZHK mass spectrometry core facility at Bad Nauheim, as described in Konzer et al., Methods in molecular biology, 2013, 1005, 39-52. The relative ratios for MCT/sham and MCT+compound/MCT were determined, with only hits deemed to be differentially expressed and highly significant (P<0.01) further studied by Western blot.

[0575] Western Blot Analysis:

[0576] The Western blot analyses consisted of frozen TA muscle samples being homogenized in RIPA buffer (50 mmol/L Tris, 150 mmol/L sodium chloride, 1 mmol/L EDTA, 1% NP-40, 0.25% sodium-deoxycholate, 0.1% SDS, 1% Triton X-100; pH 7.4) or relax buffer (90 mmol/L HEPES, 126 mmol/L potassium chloride, 1 mmol/L MgCl, 50 mmol/L EGTA, 8 mmol/L ATP, 10 mmol/1 Creatinephosphate; pH 7.4) containing a protease inhibitor mix (Inhibitor mix M, Serva, Heidelberg, Germany), sonicated, and centrifuged at 16,000×g for 5 min. Protein concentration of the supernatant was determined (BCA assay, Pierce, Bonn, Germany) and aliquots (5-20 μg) were separated by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene fluoride membrane (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). Membranes were subsequently incubated with a horseradish peroxidase-conjugated secondary antibody and specific bands visualized by enzymatic chemiluminescence (Super Signal West Pico, Thermo Fisher Scientific Inc., Bonn, Germany) and densitometry quantified using a 1D scan software package (Scanalytics Inc., Rockville, USA). Blots were then normalized to the loading control GAPDH ( 1/30000; HyTest Ltd, Turku, Finland). All data are presented as fold change relative to sham.

[0577] The expression levels of 5 proteins were found to specifically respond to the compound, as indicated by comparison of the MCT and MCT+MyoMed-946 proteomes. The expression levels of these proteins are summarized in Table 2.

TABLE-US-00003 TABLE 2 Protein −log.sub.10 Gen Function Group P-value Ratio (log.sub.2) P value eIF2B4 protein synthesis MCT/sham 0.005 −0.46 2.29 MCT + MyoMed- 0.005 0.32 2.28 946/MCT AS3MT methylation MCT/sham 0.008 0.16 2.09 MCT + MyoMed- 0.009 −0.31 2.05 946/MCT ATPAF1 oxidative MCT/sham 0.006 0.27 2.26 phosphorylation MCT + MyoMed- 0.001 −0.68 3.69 946/MCT GHDC unknown MCT/sham 0.005 0.54 2.32 MCT + MyoMed- 0.005 −0.39 2.33 946/MCT BAX apoptosis MCT/sham 0.005 0.97 2.28 MCT + MyoMed- 0.006 −0.73 2.25 946/MCT

[0578] This included an upregulation of eIF2B (delta subunit) and downregulation of BAX, which was subsequently confirmed by immunoblotting, as can be seen from FIGS. 33 to 35. The eIF2B pathway is a known translational regulator of protein synthesis. elF2B was previously identified as a MuRF1 interacting factor, suggesting that MuRF1-mediated a depletion of the translation initiation factor elF2B under MCT stress, but this was relieved by the compound. In addition, the compound also modulated the pro-apoptotic regulator BAX, with this protein upregulated in MCT mice compared to shams and normalized in the MCT+MyoMed-946 group. In human patients with chronic heart failure, apoptosis is increased in skeletal muscle and is closely correlated 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 noted before that BAX is elevated in cardiac cachexia and associated with an increased MuRF1 expression (see for example Dalla Libera et al., Am J Physiol Cell Physiol, 2004, 286, C138-144 and Rezk et al., PLoS One, 2012, 7, e30276).

[0579] As can be further seen from FIGS. 36 to 38, the proteomic analysis confirmed that the increased protein expression of MuRF1 in MCT mice is prevented by the compound MyoMed-946 yet no effects were observed for MAFBx (another key atrogin E3 ligase). This indicates the underlying mechanism of the compound appears to be MuRF1 specific, which would be expected based on the preliminary in vitro studies. MuRF1 is also known to interact with numerous substrates, with one in particular being CARP (a member of the muscle ankyrin repeat proteins (MARP) family), which is a purported nuclear- and sarcomere(titin)-based protein with transcriptional functions (see for example Miller et al., J Mol Biol, 2003, 333, 951-964). CARP is known to be upregulated in stress-related conditions and is associated with contractile dysfunction and muscle atrophy (see for example Laure et al., The FEBS journal, 2009, 276, 669-684 and Moulik et al., J Am Coll Cardiol, 2009, 54, 325-333). In line with such evidence, an increase in CARP expression in MCT-stressed mice is observed, while this effect is abolished in the MyoMed-946 fed mice, suggesting that this compound may blunt CARP expression via its inhibition on MuRF1, which in turn may contribute to maintenance of muscle mass and function. [0580] 3.1.2 CHF Mice:

[0581] To analyze the molecular mechanisms underlying the observed physiological changes and benefits of MyoMed-946-treated mice suffering from CHF, comparative quantitative proteomic analysis as well as Western blot analysis of diaphragm tissue from sham, CHF, and CHF+MyoMed-946-treated mice were performed.

[0582] Proteomic Analysis:

[0583] Mass spectrometry-based proteomic analysis was performed at the DZHK Core Facility, Bad Nauheim, Germany. Obtained MS raw data were processed by MaxQuant (1.6.0.1) using the Andromeda search engine and the Uniprot database for Mus musculus (as of 20 Apr. 2017). At a false discovery rate of 1% (both peptide and protein levels), >2600 protein groups were identified. The reductive dimethylation protocol employed (see Boersema et al., Nat Protoc, 2009, 4, 484-494) yielded pairwise relative comparative quantitation (ratios) between proteins from CHF+MyoMed-946, CHF, and sham conditions, which were statistically queried for significant differences using standard statistical tests. Several proteins were identified to be statistically different (e.g. TNNT3, Timm9, Ccdc5, Adi1, Ptges3, and Ndufa3). After applying multiple hypothesis testing (Benjamini-Hochberg; corrected P<0.05) for comparison of CHF mice with and without compound feeding, only Mrps5 (mitochondrial ribosomal protein 5), a mitochondrial-cytosolic shuttle protein in charge of protein initiation and elongation in the mitochondrial ribosome, remained as a significantly up-regulated protein (P=0.02; FIG. 4A) and was further studied by Western blot analysis as described below.

[0584] As can be seen from FIG. 39, Western blot analysis of the diaphragm of all animals included into the CHF study reveals a significant reduction of Mrps5 expression in the CHF group in comparison with the sham control group, which was reversed by compound MyoMed-946 feeding. Data are presented as mean±standard error of the mean.

[0585] Western Blot Analysis:

[0586] For western blot analyses, frozen diaphragm was homogenized in relaxing buffer (90 mmol/L HEPES, 126 mmol/L potassium chloride, 36 mmol/L sodium chloride, 1 mmol/L magnesium chloride, 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. Protein concentration of the supernatant was determined (bicinchoninic acid assay, Pierce, Bonn, Germany), and aliquots (5-20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene fluoride membrane and incubated overnight at 4° C. using the following primary antibodies: porin and telethonin (both 1/1000, Abcam, Cambridge, UK), MRPS-5 ( 1/500, Thermo Fisher, Rockford, Ill., USA), MuRF1 and MuRF2 (both 1/1000; commercially available from Myomedix, Neckargemünd, Germany), and Tom20 (1:200, Santa Cruz Biotechnologies, Heidelberg, Germany). Membranes were subsequently incubated with a horseradish peroxidaseconjugated secondary antibody, specific bands were visualized by enzymatic chemiluminescence (Super Signal West Pico, Thermo Fisher Scientific Inc., Bonn, Germany), and densitometry was quantified using a one-dimensional scan software package (Scanalytics Inc., Rockville, Md., USA). Measurements were normalized to the loading control GAPDH ( 1/30000; HyTest Ltd, Turku, Finland) or α-tubulin (1:1000, Santa Cruz Biotechnologies). All data are presented as fold change relative to sham.

[0587] As can be seen from FIGS. 40 to 42, MuRF1 and MuRF2 expression is significantly up-regulated in the CHF group, and this was prevented by treatment with compound MyoMed-946 (FIGS. 40 and 41). Quantifying the expression of telethonin, a MuRF1 target protein, a trend (P=0.08) towards a reduced expression in the CHF group was observed, which was not evident in the compound MyoMed-946-treated group (FIG. 42). [0588] 3.1.3 B16F10 Mice (Tumor Mice):

[0589] Protein expression of MuRF1, Nox 2 and LC3 I/II in muscle tissue of B16F10 mice was determined as described above for the MCT and CHF mice. In addition, the level of the reactive oxygen species marker nitrotyrosine was determined.

[0590] As can be seen from FIGS. 43 to 46, MuRF1 expression is significantly up-regulated in the tumor group, and this was prevented by treatment with compounds MyoMed-946 and MyoMed-205 (FIG. 43). Furthermore, the expression of Nox 2, which is a known endogenous reactive oxygen species (ROS), as well as the tissue level of nitrotyrosine is also significantly up-regulated in the tumor mice group, whereas in the tumor+MyoMed-946 and in the tumor+MyoMed-205 mice groups the amounts of these species are significantly reduced in the muscle tissue (FIGS. 44 and 45). On the other hand, the expression levels of LC3 I/II, which are key proteins involved in autophagocytosis, are downregulated in the tumor group, which is not observed in the the tumor+MyoMed-946 and in the tumor+MyoMed-205 groups (FIG. 46). [0591] 3.2 Enzyme activity measurements: [0592] 3.2.1 CHF mice:

[0593] Since proteomic profiling data suggests an impaired mitochondrial homeostasis, the enzymatic activities of key mitochondrial enzymes in the diaphragm tissue of CHF mice were measured.

[0594] Diaphragmatic tissue was homogenized in relaxing buffer, and aliquots were used for enzyme activity measurements. Enzyme activities for 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), O-hydroxyacyl-COA dehydrogenase (EC 1.1.1.35), and mitochondrial complex I were measured spectrophotometrically as previously described in detail (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 Physiol, 2014, 592, 3767-3782). Enzyme activity data are presented as the fold change relative to sham.

[0595] As can be seen from FIGS. 47 to 51 the enzyme activity of mitochondrial enzymes including citrate synthase (FIG. 47), succinate dehydrogenase (FIG. 48), and mitochondrial complex I (FIG. 49) is significantly reduced by 21, 28, and 27%, respectively, in the diaphragm of CHF animals when compared with sham. No difference was noted for creatine kinase. The amount of mitochondria in diaphragm tissue, as assessed by the protein expression of the mitochondrial porin expression (FIG. 50) and TOM-20 (FIG. 51), was also significantly reduced in CHF mice. Consistent with effects on mitochrondrial functions, treatment with compound MyoMed-946 partially improved citrate synthase, succinate dehydrogenase, and mitochondrial complex I enzyme activity (FIGS. 47 to 49) and resulted in near-normal porin and a modest, but statistically significant, improvement in TOM-20 expression (FIGS. 50 and 51).

[0596] In contrast, when assessing cytoplasmic enzymes for glycolysis and fatty acid metabolism (glycolysis: pyruvate kinase and lactate dehydrogenase; fatty acid metabolism: β-hydroxyacyl-COA dehydrogenase), no difference was detected between these three groups. [0597] 3.2.2 B16F10 Mice (Tumor Mice):

[0598] The enzyme activities of citrate synthase and mitochondrial complex I in muscle tissue of B16F10 inoculated mice were determined as described above for the CIF mice.

[0599] As can be seen from FIGS. 52 and 53, the enzyme activities of the mitochondrial enzymes citrate synthase (FIG. 52) and mitochondrial complex I (FIG. 53) are significantly reduced in the muscle tissue of the tumor group when compared with sham. Treatment with compounds MyoMed-946 and MyoMed-205 partially or complete restores the citrate synthase and mitochondrial complex I enzyme activities in the muscle tissue of B16F10 inoculated mice (FIGS. 52 and 53). [0600] 4. C2C12 Myotube Cell Culture, Reverse Transcription PCR

[0601] C2C12 myotubes were incubated with or without compound MyoMed-946 for 20 min at a final concentration of 10 μmol/L. After the incubation period, Total RNA was isolated from C2C12 cells and reverse transcribed into cDNA using random hexamers and Sensiscript reverse transcriptase (Qiagen, Hilden, Germany). An aliquot of the cDNA was used for quantitative RT-PCR, applying the Light Cycler system (Roche Diagnostics, Mannheim, Germany). The expression of specific genes was normalized to the expression of hypoxanthin-phosphoribosyl-transferase (HPRT)-mRNA. For quantification of MuRF-1 expression fluorescence resonance energy transfer (FRET) technology was applied using the following primers (TIB MolBiol, Berlin, Germany) and conditions: HPRT: 5′-CTCATggACTgATTATggACAggAC-3′ (SEQ IN NO:1) and 5′-gCAggTCAgCAAAgAACTTATAgCC-3′ (SEQ ID NO:2), 60° C. annealing; 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),

[0602] annealing 58° C. LC640 stands for LC Red 640, a fluorescent dye. FL stands for fluorescein. PH stands for a phosphate group (blocks the free 3-hydroxyl group against undesired extension by the polymerase).

[0603] As can be seen from FIG. 54, the increased protein expression of MuRF1 in MCT mice was prevented by the compound MyoMed-946. [0604] 5. Statistical analysis:

[0605] Data are presented 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 assess contractile function (GraphPad Prism). Significance was accepted as P<0.05.