WO2013137832A1 - Myostatin inhibitors - Google Patents

Abstract

The invention relates to myostatin inhibitors, and in particular, to myostatin inhibitors comprising small molecules, and pharmaceutical compositions comprising the myostatin inhibitors. The invention also relates to methods for treating or preventing a myostatin-related disease or disorder, or methods for increasing muscle growth or muscle regeneration in a subject, or methods for antagonizing myostatin activity in a cell. The invention further relates to methods of identifying compounds that can bind to myostatin.

Description

MYOSTATIN INHIBITORS

Cross-Reference to Related Application

[0001] This application claims the benefit of priority of United States of America Provisional Patent Application No. 61/61 1,688, filed March 16, 2012, the contents of which being hereby incorporated by reference in its entirety for all purposes.

Technical Field

[0002] The invention relates to myostatin inhibitors, and in particular, to myostatin inhibitors comprising small molecules, and pharmaceutical compositions comprising the myostatin inhibitors. The invention also relates to methods for treating or preventing a myostatin-related disease or disorder, or methods for increasing muscle growth or muscle regeneration in a subject, or methods for antagonizing myostatin activity in a cell. The invention further relates to methods of identifying compounds that can bind to myostatin.

Background

[0003] Myostatin (Mstn) is a secreted growth and differentiating factor belonging to the transforming growth factor-beta (TGF-/3) super-family. Myostatin is predominantly expressed in skeletal muscle, with low level expression observed in the heart, adipose tissue and mammary glands. Naturally occurring mutations in bovine, ovine, canine and human myostatin genes lead to an increase in muscle mass due to hyperplasia, and knock-out of the murine myostatin gene yields a similar phenotype.

[0004] Myostatin has been shown to regulate muscle growth not only by controlling myoblast proliferation and differentiation during fetal myogenesis, but also by regulating postnatal satellite cell activation and self-renewal. Consistent with genetic studies, injections of several myostatin inhibitors including follistatin, myostatin antibodies and the prodomain of myostatin have all been independently shown to increase muscle regeneration and growth in muscular dystrophy mouse models that exhibit muscle wasting.

[0005] Furthermore, prolonged absence of myostatin in mice has also been shown to reduce sarcopenic muscle loss through efficient satellite cell activation and regeneration of skeletal muscle in aged mice. Similarly, treatment of aged mice with Mstn-antl increases satellite cell activation and enhances the efficiency of muscle regeneration. Given that antagonism of myostatin leads to significant increases in postnatal muscle growth, it is proposed that myostatin antagonists have significant therapeutic value in alleviating muscle wasting conditions seen in human diseases such as muscular dystrophy, cachexia and sarcopenia.

[0006] In addition to its function in skeletal muscle, myostatin also has been shown to regulate lipid metabolism. A number of murine studies report significant decreases in the amount of adipose tissue in association with loss of myostatin function. For example, a decrease in fat pad weight and total lipid content by 12- weeks of age were observed in Mstn-/- mice in comparison to wild-type (WT) mice. In agreement, fat pads in WT mice were reported to weigh approximately 2-4 times those of Mstn-/- mice at 5- to 6-months of age. With advancing age, WT fat pads continue to increase in size while fat pads from Mstn-/- mice do not. Moreover, mean total body fat is significantly reduced in Mstn-/- mice, and serum leptin levels are also significantly lower. Despite having a normal food intake, body temperature and a reduced metabolic rate, the gonadal fat pads in Mstn-/- mice have approximately 25% fewer cells and reduced fat cell size. Furthermore, loss of Mstn in genetic models of obesity (agouti lethal yellow and Leptin-deficient [ob/ob] mice) leads to suppression of body fat accumulation.

[0007] These results suggest that loss of functional Mstn not only increases muscle mass but also decreases body fat accumulation. In addition, Mstn prodomain over-expressing transgenic mice (in which Mstn function is blocked) demonstrate a significant decrease in epididymal fat pad weight. Furthermore, neither Mstn-/- mice nor transgenic mice that overexpress Mstn prodomain become obese when fed a high- fat diet. These findings establish that a lack (or inactivation) of myostatin results in reduced fat accumulation. In addition, myostatin also appears to regulate insulin sensitivity, as there is increased insulin sensitivity in myostatin null mice. Molecular analysis indicates that a lack of myostatin results in increased expression of AMP kinase, which in turn increases the expression a major glucose transporter (Glu4 receptor) which results in increased glucose uptake in skeletal muscle. Thus, myostatin antagonists may not only have utility in increasing muscle growth, they may also ameliorate obesity and type II diabetes.

[0008] Several biologies and naturally existing myostatin antagonists are being developed commercially for treatment of cachexia and Duchenne muscular dystrophy. Examples of such biologies and naturally existing myostatin antagonists include anti-myo statin monoclonal antibodies, soluble receptor (Act IIB) of myostatin, follistatin, myostatin pro-peptide, and small peptide molecules derived from mature myostatin.

[0009] To-date, few, if not no, small molecule inhibitors of myostatin function have been developed.

Summary

[0010] In a first aspect, there is provided a compound of formula I or formula II

Formula I

. Formula II

wherein

R¹, R², R³, R⁴, R⁵, R⁸, R⁹, R¹⁰ and R¹¹ are independently selected from the group consisting of H, halo, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, -C(0)-R, -NRR', -OR, -SR, -COOR,

-CN, -NO₂, -C(0)-NRR', -NR'-C(0)-R, -S0₂-R and -(S0₂)-OR;

R⁶, R⁷, R¹² and R^12' are independently selected from H, -OR, -NRR' and C1-C4 alkyl;

X is -C(O)-, -S0₂-, -NH- or is missing;

Y is -NH-, -(CRR'h -C(O)- or is missing;

Z is -NH-, -(CRR')- or is missing;

HAr is a substituted or unsubstituted 5-10-membered heteroaryl or heteroalicyclic ring comprising 1 to 4 heteroatoms selected from N, O and S, provided that at least one of the heteroatoms is nitrogen;

R and R' are independently selected from H and C1-C4 alkyl;

m is 0 or 1 ; n is 1 or 2;

wherein when HAr is substituted, the substituent may be 1 to 9 groups independently selected from of oxo, halo, -C(0)-R¹³, -NR¹³R¹⁴, -OR¹³, -SR¹³, -COOR¹³, -CN, -N0₂, -C(0)-NR^,3R¹⁴, -NR¹⁴-C(0)-R¹³, -S0₂-R¹³, -(S0₂)-OR¹³ , -(CH₂)_P-R¹⁵, 5-14-membered aryl, 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S, 5-14-membered cycloalkyl, and 5-14-membered heterocycloalkyl comprising 1 to 4 heteroatoms selected from N, O and S;

R¹³ and R¹⁴ are independently selected from H, C1-C4 alkyl, 5-14-membered aryl, 5-14- membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S, 5-14- membered cycloalkyl, and 5-14-membered heterocycloalkyl comprising 1 to 4 heteroatoms selected from N, O and S;

R^1S is selected from H, -C(0)-R, -NRR', -OR, -SR, -COOR, -CN, -N0₂, -C(0)-NRR', -NR'- C(0)-R, -S0₂-R and -(S0₂)-OR, 5-14-membered aryl, 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S, 5-14-membered cycloalkyl and 5- 14-membered heterocycloalkyl comprising 1 to 4 heteroatoms selected from N, O and S; and p is an integer from 1 to 4.

] In various embodiments, the compound is selected from:

D29, E746-0699 D33, G271-0164

[0012] In a second aspect, a pharmaceutical composition comprising a compound of the first aspect and a pharmaceutically acceptable excipient is disclosed herein.

[0013] In a third aspect, a method for the treatment or prevention of a myostatin-related disease or disorder in a subject is provided. The method comprises administering a therapeutically or prophylactically effective amount of a compound of the first aspect to said subject.

[0014] In various embodiments, the myostatin-related disease or disorder is selected from the group consisting of cachexia, sarcopenia, obesity, insulin resistance and dystrophic muscle loss.

[0015] In a fourth aspect, a method for increasing muscle growth or muscle regeneration in a subject is disclosed. The method comprises administering a therapeutically or prophylactically effective amount of a compound of the first aspect to said subject. [0016] In a fifth aspect, a method for antagonizing myostatin activity in a cell, comprising contacting said cell with a compound of the first aspect is provided.

[0017] In a sixth aspect, a method of identifying compounds that can bind to myostatin by comparing the 3-D structure of candidate compounds with the 3-D molecular model of myostatin shown in Figure 1 is provided, the method comprising the steps:

(a) calculating the distances between hydrogen bonding moieties of different candidate compounds and the amino acid residues that form the binding site of myostatin in the 3-D molecular model, wherein the binding site is defined by amino acid residues CI 6, R17, Y18, N41 and Y55 of myostatin and wherein the main chain and one side chain guanidinium nitrogen of R17 act as a hydrogen bond donor and hydrogen bond acceptor, respectively, and the side chain of C 16 provides for a hydrophobic interaction site, to identify compounds that can bind to the binding site of myostatin; and

(b) including size exclusion volume spheres around 10 A of the binding site region to discriminate against candidate compounds that spatially clash with protein atoms.

Brief Description of the Drawings

[0018] In the drawings, like reference characters generally refer to the same parts throughout the different views. The drawings are not necessarily drawn to scale, emphasis instead generally being placed upon illustrating the principles of various embodiments. In the following description, various embodiments of the invention are described with reference to the following drawings.

[0019] Fig. 1A shows a structure-based pharmacophore model comprising a minimum of three features: an acceptor (HBA), a donor (HBD), and a hydrophobic (Hy) feature mainly targeting the R17, C16 residues; Fig. IB shows a structure-based pharmacophore model along with exclusion volume spheres added around lOA region of active site to improve the binding by preventing clashes with proteins atoms.

[0020] Fig. 2 shows various embodiments of present small molecules obtained from myoblast proliferation assays.

[0021] Figs. 3A, 3B, and 3C show various predicted mode of binding of active hits: (A)

Indazole moiety positions into the active pocket lined by Y55, C16, R17 residues, Nl, N2 atoms of indazole form hydrogen bonding interactions with R17, while the o-methyl -phenyl makes stacking interactions with Y18; (B) D25, piperazine moiety fits into active pockets making bonding contacts with C16 and R17 while the phenyl group makes stacking interactions with Y18; (C) Overlay of four actives (D20, D21, D25, and D33) at the binding active pocket.

[0022] Fig. 4 shows the effect on proliferation rate of myoblasts by increasing the

concentrations of present small molecules to growth media of C2C12 myoblasts. [0023] Fig. 5 shows the effect on luciferase activity (as compared to control untreated cells) when C2C12 cells harboring the SBE4-Luc vector were treated with myostatin.

[0024] Fig. 6 shows the results of using surface plasmon resonance to measure the binding of small molecule D20 to myostatin and the binding ability of myostatin to its receptor Act IIB. The results show that small molecule D20 binds to myostatin at Kd of 662.5 ± 24.7μΜ.

[0025] Fig. 7 shows the results of an inhibition study performed on surface resonance plasma, suggesting that in the presence of increased concentration of small molecule D20, there is a reduced level of myostatin binding to Act IIB receptor.

Description

[0026] The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the invention may be practised. These embodiments are described in sufficient detail to enable those skilled in the art to practise the invention. Other embodiments may be utilized and structural or other changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0027] The present invention is based on the finding that the herein-described small molecules or compounds can effectively and efficiently inhibit myostatin signaling and functions by physically interacting with myostatin (also herein termed antagonists or inhibitors), thereby improving muscle growth and insulin sensitivity. Such small molecules show promises to have significant commercial value for use, for example, as drugs for increasing muscle mass in all muscle wasting conditions and metabolic disorders.

[0028] Myostatin function is highly conserved as inactivation of myostatin leads to increased muscle growth in all vertebrates. Therefore, the presently disclosed compounds can also be used to increase muscle growth in farm animals such as chicken, fish, pigs, cattle, deer, sheep, goat, dogs, and horses.

[0029] In addition, since it has shown that loss of myostatin functioning increases

performance, in an alternative use, the present compounds can be injected to enhance performance in dogs, horses and all other animals that participate in competitive environment.

[0030] Similarly, a lack of myostatin functioning also increases muscle mass in humans.

Therefore, the present compounds have utility in increasing muscle mass in muscle wasting conditions like sarcopenia, cachexia, atrophy and dystrophy. Alternatively or additionally, these compounds can also be used to increase muscle mass in chronic obstruction pulmonary disorder or any condition that reduces muscle mass or weakens muscle mass in humans.

[0031] Accordingly, a first aspect of the present disclosure provides a compound of formula I or formula II R⁶ R⁷

HAr

Formula I

Formula II

wherein

R¹, R², R³, R⁴, R⁵, R⁸, R⁹, R¹⁰ and R¹¹ are independently selected from the group consisting of H, halo, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, -C(0)-R, -NRR', -OR, -SR, -COOR,

-CN, -NO₂, -C(0)-NRR\ -NR'-C(0)-R, -S0₂-R and -(S0₂)-OR;

R°, R⁷, R¹² and R¹² are independently selected from H, -OR, -NRR' and C1-C4 alkyl;

-C(O)-, -SO₂-, -NH- or is missin;

-NH-, -(CRR')-, -C(O)- or is missinj

-NH-, -(CRR')- or is missing; HAr is a substituted or unsubstituted 5-10-membered heteroaryl or heteroalicyclic ring comprising 1 to 4 heteroatoms selected from N, O and S, provided that at least one of the heteroatoms is nitrogen;

R and R' are independently selected from H and C1-C4 alkyl;

m is 0 or 1 ;

n is 1 or 2;

wherein when HAr is substituted, the substituent may be 1 to 9 groups independently selected from of oxo, halo, -C(0)-R¹³, -NR¹³R¹⁴, -OR¹³, -SR¹³, -COOR¹³, -CN, -N0₂, -C(0)-NR¹³R¹⁴, -NR¹⁴-C(0)-R¹³, -S0₂-R¹³, -(S0₂)-OR¹³ , -(CH₂)_P-R¹⁵, 5- 14-membered aryl, 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S, 5- 14-membered cycloalkyl, and 5-14-membered heterocycloalkyl comprising 1 to 4 heteroatoms selected from N, O and S;

R¹³ and R¹⁴ are independently selected from H, C1-C4 alkyl, 5-14-membered aryl, 5-14r membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S, 5-14- membered cycloalkyl, and 5- 14-membered heterocycloalkyl comprising 1 to 4 heteroatoms selected from N, O and S;

R¹⁵ is selected from H, -C(0)-R, -NRR', -OR, -SR, -COOR, -CN, -N0₂, -C(0)-NRR', -NR'-

C(0)-R, -S0₂-R and -(S0₂)-OR, 5-14-membered aryl, 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S, 5-14-membered cycloalkyl and 5- 14-membered heterocycloalkyl comprising 1 to 4 heteroatoms selected from , O and S; and p is an integer from 1 to 4.

[0032] In the present context, the term "optionally substituted" or "substituted or

unsubstituted" refers to a group in which none, one, or more than one of the hydrogen atoms have been replaced with one or more groups such as, but are not limited to, alkyl, heteroalkyl, haloalkyl, heteroholoalkyl, cycloalkyl, aryl, arylalkyl, heteroaryl, non-aromatic heterocycle, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N- carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C- carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino, including mono- and di-substituted amino groups. In embodiments in which two or more hydrogen atoms have been substituted, the substituent groups may be linked to form a ring.

[0033] In the present context, the term "aliphatic", alone or in combination, refers to a straight chain or branched chain hydrocarbon comprising at least one carbon atom. Aliphatics include alkyls, alkenyls, and alkynyls. Aliphatics include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert. -butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, ethynyl, butynyl, propynyl, and the like, each of which may be optionally substituted. [0034] In the present context, the term "alkyl", alone or in combination, refers to a fully saturated aliphatic hydrocarbon. In certain embodiments, alkyls are optionally substituted. In certain embodiments, an alkyl comprises 1 to 10 carbon atoms, for example 1 to 4 carbon atoms, wherein (whenever it appears herein in any of the definitions given below) a numerical range, such as "1 to 4" or "C1-C4", refers to each integer in the given range, e.g. "C1-C4 alkyl" means that an alkyl group comprising only 1 carbon atom, 2 carbon atoms, 3 carbon atoms, or 4 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, sec-butyl, tert-butyl, tert-amyl, pentyl, hexyl, heptyl, octyl and the like.

[0035] In the present context, the term "alkenyl", alone or in combination, refers to an aliphatic hydrocarbon having one or more carbon-carbon double-bonds, such as two or three carbon-carbon double-bonds. In certain embodiments, alkenyls are optionally substituted, i.e. substituted or unsubstituted. In certain embodiments, an alkenyl comprises 2 to 15 carbon atoms, for example 2 to 4 carbon atoms. "C2-C4 alkenyl" means that an alkenyl group comprising only 2 carbon atoms, 3 carbon atoms, or 4 carbon atoms. Examples of alkenyls include, but are not limited to, ethenyl, propenyl, butenyl, 1,4-butadienyl, pentenyl, hexenyl, 4-methylhex-l-enyl, 4-ethyl-2- methylhex- 1 -enyl and the like.

[0036] In the present context, the term "alkynyl", alone or in combination, refers to an aliphatic hydrocarbon having one or more carbon-carbon triple-bonds, such as two or three carbon- carbon triple-bonds. In certain embodiments, alkynyls are optionally substituted, i.e. substituted or unsubstituted. In certain embodiments, an alkynyl comprises 2 to 15 carbon atoms, for example 2 to 4 carbon atoms. "C2-C4 alkynyl" means that an alkynyl group comprising only 2 carbon atoms, 3 carbon atoms, or 4 carbon atoms. Examples of alkynyls include, but are not limited to, ethynyl, propynyl, butynyl, and the like.

[0037] In the present context, the term "halogen", or "halo" for short, refers to fluorine (F), chlorine (CI), bromine (Br) or iodine (I).

[0038] In the present context, the term "heteroatom" refers to an atom other than carbon or hydrogen. Heteroatoms are typically independently selected from oxygen (O), sulfur (S), nitrogen

(N), and phosphorus (P), but are not limited to those atoms. In embodiments in which two or more heteroatoms are present, the two or more heteroatoms may all be the same as one another, or some or all of the two or more heteroatoms may each be different from the others.

[0039] In the present context, the term "ring" refers to any covalently closed structure. Rings include, for example, carbocycles (e.g., aryls and alicyclics), heterocycles (e.g., heteroaryls and non-aromatic heterocycles), aromatics (e.g., aryls and heteroaryls), and non-aromatics (e.g., alicyclics and non-aromatic heterocycles). Rings may be optionally substituted. The term "ring system" refers to two or more rings, wherein two or more of the rings are fused. The term "fused" refers to structures in which two or more rings share one or more bonds. [0040] In the present context, the term "aromatic" refers to a group comprising a covalently closed planar ring having a delocalized [pi]-electron system comprising 4n+2 [pi] electrons, where n is an integer. Aromatic rings may be formed by five, six, seven, eight, nine, or more than nine atoms. Aromatics may be optionally substituted. Examples of aromatic groups include, but are not limited to phenyl, naphthalenyl, phenanthrenyl, anthracenyl, tetralinyl, fluorenyl, indenyl, and indanyl. The term aromatic includes, for example, benzenoid groups, connected via one of the ring- forming carbon atoms, arid optionally carrying one or more substituents selected from an aryl, a heteroaryl, a cycloalkyl, a non-aromatic heterocycle, a halo, a hydroxy, an amino, a cyano, a nitro, an alkylamido, an acyl, a C1-C6 alkoxy, a C1-C6 alkyl, a C1-C6 hydroxyalkyl, a C1-C6 aminoalkyl, an alkylsulfenyl, an alkylsulfinyl, an alkylsulfonyl, an sulfamoyl, or a trifluoromethyl. In certain embodiments, an aromatic group is substituted at one or more of the para, meta, and/or ortho positions. Examples of aromatic groups comprising substitutions include, but are not limited to, phenyl, 3-halophenyl, 4-halophenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 3-aminophenyl, 4- aminophenyl, 3-methylphenyl, 4-methylphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 4- trifluoromethoxyphenyl, 3-cyanophenyl, 4-cyanophenyl, dimethylphenyl, naphthyl,

hydroxynaphthyl, hydroxymethylphenyl, (trifluoromethyl)phenyl, alkoxyphenyl, 4-morpholin-4- ylphenyl, 4-pyrrolidin-l-ylphenyl, 4-pyrazolylphenyl, 4-triazolylphenyl, and 4-(2-oxopyrrolidin-l- yl)phenyl. [0041] In the present context, the term "aryl" refers to an aromatic ring wherein each of the atoms forming the ring is a carbon atom. Aryl rings may be formed by five, six, seven, eight, nine, or more than nine carbon atoms. In certain embodiments, the aryl may be a 5-14-membered aryl, such as 5-membered aryl, 6-membered aryl, 7-membered aryl, 8-membered aryl, 9-membered aryl, 10-membered aryl, 1 1-membered aryl, 12-membered aryl, 13-membered aryl, or 14-membered aryl. Designations such as "5-14-membered aryl" refer to the total number of atoms in the ring. Aryl groups may be optionally substituted.

[0042] In the present context, the term "non-aromatic ring" refers to a group comprising a covalently closed ring that is not aromatic.

[0043] In the present context, the term "heterocycle" refers to a group comprising a covalently closed ring wherein at least one atom forming the ring is a carbon atom and at least one atom forming the ring is a heteroatom. Heterocyclic rings may be formed by three, four, five, six, seven, eight, nine, or more than nine atoms. Any number of those atoms may be heteroatoms (i.e., a heterocyclic ring may comprise one, two, three, four, five, six, seven, eight, nine, or more than nine heteroatoms). Herein, whenever the number of atoms or members in a heterocycle is indicated

(e.g., 5- 10-membered heterocycle), at least one other atom (i.e. the heteroatom) must be present in the ring. Designations such as "5- 10-membered heterocycle" refer to the total number of atoms in the ring. It is understood that the heterocylic ring will have additional heteroatoms in the ring. In heterocycles comprising two or more heteroatoms, those two or more heteroatoms may be the same or different from one another. Heterocycles may be optionally substituted. Binding to a heterocycle can be at a heteroatom or via a carbon atom. Examples of heterocycles include, but are not limited to the following:

wherein D, E, F, and G independently represent a heteroatom. Each of D, E, F, and G may be the same or different from one another.

[0044] In the present context, the term "heteroaryl" refers to an aromatic heterocycle.

Heteroaryl rings may be formed by three, four, five, six, seven, eight, nine, or more than nine atoms or members wherein at least one of the atoms or members in the ring is a heteroatom. In certain embodiments, the heteroaryl may be a 5-10-membered aromatic heterocycle, 5-1 1- membered aromatic heterocycle, 5-12-membered aromatic heterocycle, 5-13-membered aromatic heterocycle, or 5-14-membered aromatic heterocycle. Heteroaryls may be optionally substituted. Examples of heteroaryl groups include, but are not limited to, aromatic C3-C8 heterocyclic groups comprising one oxygen or sulfur atom or up to four nitrogen atoms, or a combination of one oxygen or sulfur atom and up to two nitrogen atoms, and their substituted as well as benzo- and pyrido-fused derivatives, for example, connected via one of the ring-forming carbon atoms. In certain embodiments, heteroaryl groups are optionally substituted with one or more substituents. Examples of heteroaryl groups include, but are not limited to, unsubstituted and mono- or di- substituted derivatives of furan, be zofuran, thiophene, benzothiophene, pyrrole, pyridine, indole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole, quinoline, isoquinoline, pyridazine, pyrimidine, purine and pyrazine, furazan, 1,2,3- oxadiazole, 1,2,3-thiadiazole, 1 ,2,4-thiadiazole, triazole, benzotriazole, pteridine, phenoxazole, oxadiazole, benzopyrazole, quinolizine, cinnoline, phthalazine, quinazoline, and quinoxaline.

[0045] In the present context, the term "alicyclic" refers to a group comprising a non-aromatic ring wherein each of the atoms forming the ring is a carbon atom. Alicyclic groups may be formed by three, four, five, six, seven, eight, nine, or more than nine carbon atoms. In certain

embodiments, alicyclics are optionally substituted, i.e. substituted or unsubstituted. In certain embodiments, an alicyclic comprises one or more unsaturated bonds, such as one or more carbon- carbon double-bonds. Alicyclics include cycloalkyls and cycloalkenyls. Examples of alicyclics include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclopentadiene, cyclohexane, cyclohexene, 1,3-cyclohexadiene, 1 ,4-cyclohexadiene, cycloheptane, and cycloheptene. Accordingly, the term "heteroalicyclic ring" refers to a group comprising a non-aromatic ring wherein one or more but not all of the atoms forming the ring is a heteroatom. In certain embodiments, the heteroalicyclic ring may be a 5-10-membered non- aromatic heterocycle, 5-1 1-membered non-aromatic heterocycle, 5-12-membered non-aromatic heterocycle, 5- 13 -membered non-aromatic heterocycle, or 5- 14-membered non-aromatic heterocycle. Heteroalicyclic rings may be optionally substituted.

[0046] In the present context, the term "oxo" refers to a carbonyl functional group.

[0047] In the present context, the term "cycloalkyl" refers to a completely saturated hydrocarbon ring. In certain embodiments, the cycloalkyl may be a 5-14-membered cycloalkyl, such as, 5-membered cycloalkyl, 6-membered cycloalkyl, 7-membered cycloalkyl, 8-membered cycloalkyl, 9-membered cycloalkyl, 10-membered cycloalkyl, 1 1-membered cycloalkyl, 12- membered cycloalkyl, 13-membered cycloalkyl, or 14-membered cycloalkyl. The cycloalkyl group can for example be optionally substituted. Examples of cycloalkyl groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. When substituted, the substituent group(s) can be defined as mentioned above. Accordingly, the term "heterocycloalkyl" refers to a cycloalkyl group wherein one or more but not all of the atoms forming the ring is a heteroatom. In certain embodiments, the heterocycloalkyl may be a 5-14-membered

heterocycloalkyl, such as 5-membered heterocycloalkyl, 6-membered heterocycloalkyl, 7- membered heterocycloalkyl, 8-membered heterocycloalkyl, 9-membered heterocycloalkyl, 10- membered heterocycloalkyl, 1 1-membered heterocycloalkyl, 12-membered heterocycloalkyl, 13- membered heterocycloalkyl, or 14-membered heterocycloalkyl. [0048] In various embodiments, HAr may be selected from the group consisting of pyrrol, pyrrolidine, imidazol, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, triazole, tetrazole, piperidine, pyridine, piperazine, pyrazine, pyrimidine, pyridazine, triazines, tetrazines, indazole, indole, isoindole benzimidazole, indoline, quinoline, isoquinoline, purine, oxazolidine, oxazole, oxazoline, isoxazolidine, isoxazole, thiazolidine, thiazole, thiazoline, isothiazolidine, isothiazole, furazane, oxadiazole, oxadiazine, thiadiazole, thiadiazine, morpholine, oxazine, thiomorpholine, and thiazine, all of which may be substituted or unsubstituted.

[0049] For example, HAr may be selected from the group consisting of indazole, piperazine, thiadiazine, indole, indoline, imidazole and benzimidazole, all of which may be substituted or unsubstituted and when substituted, the substituent may be selected from oxo, -C(0)-R¹³, -S0₂-R¹³, -(CH₂)_p-R¹⁵, 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S.

[0050] In some embodiments, HAr may be indazole. The indazole may be substituted or unsubstituted. If the indazole is substituted, the substituent may be selected from oxo, -C(0)-R¹³, - S0₂-R¹³, -(CH₂)p-R¹⁵, 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from , O and S.

[0051] In some embodiments, HAr may be piperazine. The piperazine may be substituted or unsubstituted. If the piperazine is substituted, the substituent may be selected from oxo, -C(0)-R¹³,

-S0₂-R¹³, -(CH₂)p-R¹⁵, 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S. For example, the piperazine may substituted with oxo. The piperazine may additionally be substituted with -S0₂-R¹³, wherein R¹³ may be H.

[0052] In some embodiments, HAr may be indoline. The indoline may be substituted or unsubstituted. If the indoline is substituted, the substituent may be selected from oxo, -C(0)-R¹³, - S0₂-R¹³, -(CH₂)_p-R¹⁵, 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S. For example, the indoline may substituted with -C(0)-R¹³, wherein R¹³ may be H.

[0053] In some embodiments, HAr may be imidazole. The imidazole may be substituted or unsubstituted. If the imidazole is substituted, the substituent may be selected from oxo, -C(0)-R¹³, -SO2-R¹³, -(CH₂)_p-R¹⁵, 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S. For example, the imidazole may substituted with -S0₂-R¹³, wherein R¹³ may be H or pyrrolidine.

[0054] In various embodiments, in formula (I) X is -C(O)- and Y is NH or CH₂.

[0055] In further embodiments, in formula (I) X and Y are missing.

[0056] In yet other embodiments, in formula (I) X is -S0₂- and Y is CHR'.

[0057] In one or more embodiments, in formula (I) Z is CH₂ or missing.

[0058] In some embodiments, at least one of R¹, R², R³, R⁴ and R⁵ may be halo. For example, only R¹ or R² or R³ or R⁴ or R⁵ may be halo, or all of R¹, R², R³, R⁴ and R⁵ may be halo, or only R¹ and R² may be halo, or only R¹, R² and R³ may be halo, or only R¹, R², R³ and R⁴ may be halo, just to illustrate a few examples.

[0059] In alternative or additional embodiments, at least one of R¹, R², R³, R⁴ and R⁵ may be C1-C4 alkyl. For example, only R¹ or R² or R³ or R⁴ or R⁵ may be C1-C4 alkyl, or all of R¹, R², R³, R⁴ and R⁵ may be C1-C4 alkyl, or only R¹ and R² may be C1-C4 alkyl, or only R¹, R² and R³ may be C1-C4 alkyl, or only R¹, R², R³ and R⁴ may be C1-C4 alkyl, just to illustrate a few examples. Further, in each case where two or more of R', R², R³, R and R⁵ are C1-C4 alkyl, the alkyl group may be the same or different. For example, R 1 may be methyl and R 2 may be ethyl, or both R 1 may and R² may be methyl, or R¹ may be methyl and R² may be propyl, or R¹ may be methyl, R² may be ethyl, and R³ may be propyl, just to illustrate a few examples.

[0060] In yet alternative or additional embodiments, at least one

R², R³, R⁴ and R⁵ may be -OR. For example, only R¹ or R² or R³ or R⁴ or R⁵ may be -OR, or all of R¹, R², R³, R⁴ and R⁵ may be -OR, or only R¹ and R² may be -OR, or only R¹, R² and R³ may be -OR, or only R¹, R², R³ and R⁴ may be -OR, just to illustrate a few examples. R may be H or C1-C4 alkyl. Further, in each case where two or more

R², R³, R⁴ and R⁵ are -OR, R may be the same or different, and in each case where R is C1-C4 alkyl, the alkyl group may be the same or different. For example, R¹ may be -OH and R² may be -OCH₃, or both R¹ may and R² may be -OCH₃, or R¹ may be -OCH₃ and R² may be -OC₂H₅, or R¹ may be -OH, R² may be -OCH3, and R³ may be -OC₂H₅, just to illustrate a few examples.

[0061] In certain embodiments, the compound is selected from:

I

D20, E002-1236 D21 , E135-1183 D25, E646-7423

D29, E746-0699 D33, G271-0164

[0062] Identification of antagonists

[0063] The present inventors have built a homology model of myostatin using its closest homologs BMP-7, BMP-6 and GDF-5. A comparison of the homology model to the crystal structure of myostatin revealed the presence of extra cysteine knot between Cys6 and Cysl6, except for these changes at first 12 residues of myostatin for which there is no homology, most of the coordinates are well modelled.

[0064] Mutation in cysteine knot such as the natural mutation C47Y (Cys313) alters the ability of myostatin to inhibit proliferation of myoblasts. Therefore, cysteine knot and consensus TGF super family motif surrounding region was considered as active site for model development and docking studies.

[0065] Fig. 1A shows a structure-based pharmacophore model comprising a minimum of three features: an acceptor (HBA), a donor (HBD), and a hydrophobic (Hy) feature mainly targeting the R17, C16 residues; Fig. IB shows a structure-based pharmacophore model along with exclusion volume spheres added around lOA region of active site to improve the binding by preventing clashes with proteins atoms. As shown in the Figs. 1A and IB, Argl7 and Cysl6, which lie close to the natural variant Cys74 (Cys313) and are varied residues in comparison to the BMP family members ( 1 ) as a donor and an acceptor anchoring the main chain and side chain nitrogen atoms of R17 and an hydrophobic group in the vicinity of Cysl6, are chosen in the model (Fig. lA) and inclusion of exclusion volume spheres (Fig. IB) filters the ligand that could have clashes with protein atoms at the active site.

[0066] Structure-based pharmacophore and virtual screening, as well as consensus scoring enabled the identification of potential hits that could bind to myostatin. Most of the active hits selected for myoblast proliferation assays are computationally predicted to sit at the groove formed by the residues Y55, C16, R17, N41 residues. [0067] Fig. 2 shows various embodiments of present small molecules obtained from myoblast proliferation assays. Thus, in various embodiments, present small molecules may be, but not limited to:

D20, E002-1236 D21 , E135-1183 D25, E646-7423

D29, E746-0699 D33, G271 -0164

[0068] Figs. 3A, 3B, and 3C show various predicted mode of binding of active hits: (A) Indazole moiety positions into the active pocket lined by Y55, CI 6, R17 residues, Nl, N2 atoms of indazole form hydrogen bonding interactions with R17, while the o-methyl-phenyl makes stacking interactions with Y 18; (B) D25, piperazine moiety fits into active pockets making bonding contacts with CI 6 and R17 while the phenyl group makes stacking interactions with Y18; (C) Overlay of four actives (D20, D21, D25, and D33) at the binding active pocket. As an illustration, the top five hits identified from myoblast proliferation assays are computationally predicted to bind at the groove between the Y55, R17, C16 and N41. Compound D20, (Fig. 3 A) the most active ligand in myoblast proliferation assays, fits into the pocket and interacts with R17 via its indazole nitrogen (Nl, N2) atoms, while the phenyl group forms van der Waals interactions with Y18, R17 and N41. Similarly, piperazine moiety in compound D25 fits into pocket fonning close van der Waal contacts with Y55, CI 6, while linker with its carbonyl groups interacts with R17and the phenyl group from stacking interactions with Y18 residues (Fig. 3B). Overlay of all the top hits indicates that the ligands bind into the pocket formed by Y55, C16, and R17 residues (Fig. 3C).

[0069] Myostatin antagonism by small molecules

[0070] Myostatin antagonistic activity of presently disclosed small molecules were tested in vitro assays. Myostatin is an inhibitor of myoblast proliferation. Therefore, neutralization of myostatin by the antagonist would increase the proliferation rate of myoblasts. Hence, myoblasts were plated and grown in the presence of increasing concentrations of different small molecules and the growth of myoblasts was monitored. Fig. 4 shows the effect on proliferation rate of myoblasts by increasing the concentrations of present small molecules to growth media of C2C12 myoblasts. The results demonstrated that addition of increasing concentrations of small molecules to the growth media of C2C12 myoblasts significantly increase the proliferation rate of myoblasts..

[0071] Antagonists inhibit myostatin signaling

[0072] Myostatin has been shown to inhibit signal by activating the Smad3 protein. Therefore, the ability of antagonist (compound D20) to inhibit myostatin signaling using a Smad3 binding reporter construct, the SBE4-Luc in the promoter-reporter assay was demonstrated and confirmed. Fig. 5 shows the effect on luciferase activity (as compared to control untreated cells) when C2C12 cells harboring the SBE4-Luc vector were treated with myostatin. The results showed that when C2C12 cells harboring the SBE4-Luc vector were treated with myostatin, significantly higher luciferase activity was noted as compared to control untreated cells. As expected, antagonists treated cells showed lower luciferase activity as compared to the untreated control cells indicating interference with myostatin signaling. Consistent with inhibition of myostatin signaling, cells are treated with combinations. Collectively, these results confirmed that the presently disclosed myostatin antagonists are able to interfere with myostatin signaling in vitro.

[0073] Binding of small molecules to myostatin

[0074] Surface Plasmon Resonance (SPR) was used to measure the binding of compound D20 to myostatin and the ability of myostatin to its receptor Act IIB. Fig. 6 shows the results of using surface plasmon resonance to measure the binding of small molecule D20 to myostatin and the binding ability of myostatin to its receptor Act IIB. The results show that compound D20 binds to myostatin at Kd of 662.5 ± 24.7μΜ.

[0075] Consistent with the binding of small molecule to the myostatin, an inhibition study performed on surface resonance plasma suggested that in the presence of increased concentration of small molecule D20, there is a reduced level of myostatin binding to Act IIB receptor (Fig. 7) [0076] Pharmaceutical composition

[0077] Pharmaceutical compositions may comprise any one of the compounds mentioned herein and a pharmaceutically acceptable excipient.

[0078] In the present context, a "pharmaceutically acceptable excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatine, vegetable oils and polyethylene glycols.

[0079] Thus, the present invention also relates to compositions including pharmaceutical compositions comprising a therapeutically effective amount of a compound of any one of the compounds mentioned herein. As used herein a compound will be therapeutically effective if it is able to affect the myostatin concentration within a cell. Preferably, a compound will be therapeutically effective if it is able to affect the myostatin concentration within a cell where it is able to treat or prevent a myostatin-retated disease or disorder in a subject after the compound has been administered to a subject.

[0080] Methods of measuring myostatin concentration can be conducted by any of the known methods for measuring protein expression such as mR A issolation, antibody detection, microarrays, magnetic separation or any other suitable method known in the art to determine the cell concentration of myostatin. A qualitative method of measuring the therapeutic effect of a compound on the concentration of myostatin is to use the myoblast assay as described herein.

[0081] In a preferred embodiment the compounds and pharmaceutical compositions are adapted to be administered in forms suitable for injectable use. In such embodiments the pharmaceutically acceptable excipient may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions and or one or more carrier. Alternatively, injectable solutions may be delivered encapsulated in liposomes to assist their transport across cell membrane. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating/destructive action of microorganisms such as, for example, bacteria and fungi.

[0082] The pharmaceutically acceptable excipient may include carriers. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as, for example, lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Preventing the action of microorganisms in the compositions of the invention is achieved by adding antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0083] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, to yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

[0084] When the active ingredients, in particular small molecules contemplated within the scope of the invention, are suitably protected they may be orally administered, for example, with an inert diluent or with an edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that a dosage unit form contains between about 0.1 μ and 20 g of active compound.

[0085] The tablets, troches, pills, capsules and the like may also contain other

pharmaceutically acceptable excipients. For example the composition may include binding agents, such as, for example, gum, acacia, corn starch or gelatin. They may also contain an excipient, such as, for example, dicalcium phosphate. They may also contain a disintegrating agent such as, for example, corn starch, potato starch, alginic acid and the like. They may also contain a lubricant such as, for example, magnesium stearate. They may also contain a sweetening agent such a sucrose, lactose or saccharin. They may also contain a flavouring agent such as, for example, peppermint, oil of wintergreen, or cherry flavouring.When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier.

[0086] Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparaben as preservatives, a dye and flavouring such as, for example, cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be

pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

[0087] To this extent the active ingredient may be held within a matrix which controls the release of the active agent. Preferably, the matrix comprises a substance selected from the group consisting of lipid, polyvinyl alcohol, polyvinyl acetate, polycaprolactone, poly(glycolic)acid, poly(lactic)acid, polycaprolactone, polylactic acid, polyanhydrides, polylactide-co-glycolides, polyamino acids, polyethylene oxide, acrylic terminated polyethylene oxide, polyamides, polyethylenes, polyacrylonitriles, polyphosphazenes, poly(ortho esters), sucrose acetate isobutyrate (SAIB), and combinations thereof and other polymers known in the art. Preferably, the matrix sustainedly releases the drug.

[0088] In another embodiment the the compounds and pharmaceutical compositions are adapted to be administered in forms suitable for inhalation into the lungs. Aerosol compositions suitable for inhalation can be presented either as suspensions or as solutions and typically contain the active compound and a suitable propellant such as a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixtures thereof, particularly hydro fluoroalkanes such as

dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and especially 1,1, 1,

2-tetrafluoroethane, 1,1, 1,2, 3,3, 3-heptafluoro-n-propane and mixtures thereof. [0089] The aerosol composition may optionally contain additional excipients typically associated with such compositions, for example surfactants such as oleic acid or lecithin and cosolvents such as ethanol. Pressurised formulations will generally be contained within a canister (for example an aluminium canister) closed with a metering valve and fitted into an actuator provided with a mouthpiece.

[0090] Medicaments for administration by inhalation desirably have a controlled particle size. The optimum particle size for inhalation into the bronchial system is usually 1-10 μηι, preferably 2- 5 μτη. Particles having a size above 20 μια are generally too large when inhaled to reach the small airways. To achieve these particle sizes the particles of the active ingredient may be subjected to a size reducing process such as micronisation. The desired size fraction may be separated out by air classification or sieving. Preferably, the particles will be crystalline. When an excipient such as lactose is employed, typically the particle size of the excipient will be much greater than the particle size of the active ingredient.

[0091] In another embodiment subligual absorbable formulations or Intranasal sprays may be formulated with aqueous or non-aqueous vehicles with the addition of agents such as thickening agents, buffer salts or acid or alkali to adjust the pH, isotonic adjusting agents or anti-oxidants.

[0092] A pharmaceutically acceptable excipient may include carriers and/or diluents may also include any and all solvents, dispersion media, coatings, antibactenals and/or antifungals, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is

contemplated.

[0093] It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable excipient. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

[0094] The principal active ingredient is compounded for convenient and effective

administration in effective amounts with a suitable pharmaceutically acceptable excipient in dosage unit form. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 pg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of-the said ingredients.

[0095] The compound or the composition may be in the form of a treatment kit comprising the dosage unit forms and instructions for use.

[0096] Method for treatment

[0097] A method for the treatment or prevention of a myostatin-related disease or disorder in a subject is contempated, comprises administering a therapeutically or prophylactically effective amount of a compound of any one of the compounds or compositions mentioned herein to said subject.

[0098] Subject, for the purposes of the present invention includes humans and other animals, particularly mammals. Thus the methods are applicable to both human therapy and veterinary applications. In certain embodiments the subject is a mammal, and in a preferred embodiment the subject is human.

[00991 "Treatment" and "treat" and synonyms thereof refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a myostatin-related disease or disorder. [00100] Preferably, the myo statin-related disease or disorder is selected from the group consisting of cachexia, sarcopenia, obesity, insulin resistance and dystrophic muscle loss.

[00101] As used herein, in the context of a treatment or prevention of a myostatin-related disease or disorder, a "therapeutically effective amount" or "prophylactically effective amount" of a compound will be an amount of active agent that is capable of treating, preventing or at least slowing down (lessening) myostatin-related disease or disorder. Dosages and administration of an antagonist of the invention in a pharmaceutical composition may be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics. An effective amount of the compound or composition to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the mammal. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from about 10 ng/kg to up to 100 mg/kg of the mammal's body weight or more per day, preferably about 1 g/kg/day to 10 mg/kg/day.

[00102] A method for increasing muscle growth or muscle regeneration in a subject is contempated, comprising administering a therapeutically or prophylactically effective amount of a compound of any one of the compounds or compositions mentioned herein to said subject.

[00103] Preferably, the subject is a mammal. Preferably, the mammal is a human. [00104] As used herein, in the context of increasing muscle growth or muscle regeneration in a subject, a "therapeutically or prophylactically effective amount" of a compound will be an amount of active agent that is capable of increasing muscle growth or muscle regeneration in a subject. Dosages and administration of a compound of the invention in a pharmaceutical composition may be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics.

[00105] A method for antagonizing myostatin activity in a cell is also contemplated, comprising contacting said cell with a compound of any one of the compounds or compositions mentioned herein.

[00106] Preferably the cell is a muscle stem cell i.e. myoblast cells. Similarly the cell may be a myocyte.

[00107] Preferably, measuring the antagonising effect on the myostatin activity can be determined using the myoblast assay as described herein whereby a decrease in absorbance of the cells at 655 nm treated with an antagonist in comparison to cells not treated with the antagonist.

[00108] Method of identifying compounds that can bind to myostatin

[00109] A method of identifying compounds that can bind to myostatin by comparing the 3-D structure of candidate compounds with the 3-D molecular model of myostatin shown in Fig. 1 is contemplated. The method comprises the steps of (a) calculating the distances between hydrogen bonding moieties of different candidate compounds and the amino acid residues that form the binding site of myostatin in the 3-D molecular model, wherein the binding site is defined by amino acid residues CI 6, R17, Y18, N41 and Y55 of myostatin and wherein the main chain and one side chain guanidinium nitrogen of R17 act as a hydrogen bond donor and hydrogen bond acceptor, respectively,' and the side chain of C16 provides for a hydrophobic interaction site, to identify compounds that can bind to the binding site of myostatin; and

(b) including size exclusion volume spheres around 10 A of the binding site region to discriminate against candidate compounds that spatially clash with protein atoms.

[00110] The method further identifies compounds that can bind to myostatin and modulate its function as described herein.

[00111] In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following non-limiting examples.

Examples

Modeling of myostatin structure and design of small molecules

[00112] De novo structure based pharmacophore screening. The 3D structure of myostatin

(apo-form) was built using Insight II homology modeling program. The 3D-coordinates of closest homology members (BMP7, BMP6, and GDF5) were used as reference templates to build a homology model. The modeled protein structure was prepared by adjusting hydrogen atoms at pH 7.0, partial charges, potentials and energy minimized using a cascade of energy minimization protocols in Accelrys suite of programs. Thus, energy minimized molecule was saved and further used for a structure-based pharmacophore generation and virtual high throughput screening.

Binding site analysis of myostatin shows a site adjoining the cysteine knot and the consensus motif of TGF beta super family. This site point was used to develop Ludi interactive map and structure based pharmacophore model. Ludi interaction map identifies the possible interactive site vectors which can make favorable interaction with target residues. These interaction site vectors are hierarchically clustered using a rms distance of 0.7 A and a three feature model: hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), hydrophobic (Hy) features anchoring CI 6, R17, Y18, N44, Y55 residues are used during development of structure based pharmacophore model (Fig. 1A). Further, to filter the ligands that could bump with protein atoms, a total of 160 size exclusion volume spheres around 10 A of active site region was included in the pharmacophore model (Fig. IB). Database screening with this model on in-house built chemdiv virtual library yielded a focused library of about 50,000 molecules.

[00113] Virtual High Throughput Screening (vHTS). Virtual high throughput screening was carried out on refined protein by defining a region around 10 A region from the cysteine knot residues. Initial vHTS docking was performed by default docking parameters to screen the 60,000 molecules. Among these, molecules that were having better interactions at the key residues of TGF-_/6 motif and in conjunction with ADMET filters, the dataset were reduced to 500 molecules. These 500 molecules are further docked by Gold docking software and the resulting solutions are rescored with Ligscore, PLP, PMF, Jain and Ludi scoring functions with Accelrys suite and consensus scoring theme was applied to pick the hits. Ligands which were having interactions at cysteine knot and in conjunction with consensus scoring are prioritized and 38 molecules were selected for further experimental studies.

[00114] Myoblasts assay. For the standard myoblast proliferation assay, C2C12 myoblasts (American Tissue Culture Collection, Manassas, VA) were seeded at 1000 cells per well in 96- well Nuncmicrotiter plates in maintenance medium [Dulbecco's modified Eagle medium (Invitrogen) containing 10 % fetal bovine serum (FBS; Sigma, St. Louis, MO), 1 ^χ 10⁵ IU/L penicillin (Sigma) 100 mg/L streptomycin (Sigma), and 17.02 mM NaHC03] and incubated at 37 °C in a humidified atmosphere of 5 % C0₂. Following a 16 h attachment period, maintenance medium was discarded and replaced with myostatin antagonist test media (maintenance medium with 5 % FBS and 0-5 μg/ml Mstn-antl or Mstn-ant2). C2C12 myoblasts were grown in test media for 72 h after which proliferation was assessed using a methylene blue photometric end point assay. In this assay, absorbance at 655 nm is directly proportional to the final cell number (Luciferase assay). [00115] Binding studies. All SPR experiments were carried out on carboxymethylated dextran (CM5) sensor chips and all buffers were degassed and filter-sterilized through 0.2 yum filters prior to use. Myostatin were immobilized onto CM5 chip using standard amine coupling chemistry, at a flow rate of 5 ^L/min until approximately 10000 Resonance Unit (RU) was reached. The dextran matrix was first activated by 1 : 1 mixture of 0.4 M l -ethyl-3-(3-dimethylaminopropyl)

carbodiimide (EDC) and O. lM N-hydroxysuccinimide (NHS) for 10 min to create reactive succinimide esters, followed by covalent binding of ligand (ActRIIB or its mutants) dissolved in 10 mM sodium acetate pH 4.5. Unreacted esters were deactivated by injecting 1 M ethanolamine-HCl pH 8.5 for 10 min. The reference surface was treated as the ligand surface except the protein injection was omitted. D20 was dissolved in PBS-P containing 5 % methanol/DMSO, which was also used as the running buffer. For kinetic analysis, a concentration series of D20 was injected over myostatin and reference surfaces at a flow rate of 30 jiiL/min. For each concentration, association and dissociation were measured for 60 s. All sensograms were aligned and double- referenced. Affinity constant (KD) were determined by fitting the corrected sensorgrams with steady state model using BIAevaluation 4.1 software.

[00116] For inhibition studies, ActRIIB was immobilized using thiol coupling method. The surface was first activated by 1 : 1 mixture of 0.4 M EDC and 0.1 M NHS for 10 min, followed by

10 min injection of 80 mM 2-(2-pyridinyldithio)ethaneamine (PDEA) in 0.1 M sodium borate pH 8.5 to introduce reactive disulfide groups, and 6 min injection of 1 M EA. ActRIIB was diluted in 10 mM sodium acetate pH 4.5 and immobilized to 10000 RU. Excess reactive groups were deactivated using 50 mM cysteine in 1 M NaCl and 0.1 M sodium acetate pH 4.0 for 10 min. A similar level of IgG was immobilized on a reference flow cell, using the same protocol as described above. For inhibition studies, slow flow rate (10 μΐ/min) was used to generate complete mass transfer condition. Different concentrations of D20 were mixed with 100 nM of myostatin, and these mixtures were then injected across ActRIIB surface. PBS-P containing 5 % methanol was used as sample and running buffer. Association and dissociation were measured over 60 s and 120 s, respectively. The surface is regenerated by 30 s injection of 5 mMHCl. Inhibition profile was determined by plotting the initial slope of the association curves as a function of the inhibitor.

[00117] By "comprising" it is meant including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[00118] By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present.

[00119] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00120] By "about" in relation to a given numberical value, such as for temperature and period of time, it is meant to include numerical values within 10% of the specified value.

[00121] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00122] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

Claims

1. Compound of formula I or formula II

Formula I

Formula II

wherein

R¹, R², R³, R⁴, R⁵, R⁸, R⁹, R¹⁰ and Rⁿ are independently selected from the group consisting of H, halo, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, -C(0)-R, -NRR', -OR, -SR, -COOR,

-CN, -N0₂, -C(0)-NRR', -NR'-C(0)-R, -S0₂-R and -(S0₂)-OR;

R⁶, R⁷, R¹² and R¹² are independently selected from H, -OR, -NRR' and C1-C4 alkyl; X is -C(O)-, -S0₂-, -NH- or is missing;

Y is -NH-, -(CRR')-, -C(O)- or is missing;

Z is -NH-, -(CRR')- or is missing;

HAr is a substituted or unsubstituted 5-10-membered heteroaryl or heteroalicyclic ring comprising 1 to 4 heteroatoms selected from N, O and S, provided that at least one of the heteroatoms is nitrogen;

R and R' are independently selected from H and C1-C4 alkyl;

m is 0 or 1 ;

n is 1 or 2;

wherein when HAr is substituted, the substituent may be 1 to 9 groups independently selected from of oxo, halo, -C(0)-R¹³, -NR¹³R¹⁴, -OR¹³, -SR¹³, -COOR¹³, -CN, -N0₂, -C(0)-NR¹³R¹⁴, -NR¹⁴-C(0)-R¹³, -SC-2-R¹³, -(S0₂)-OR¹³ , -(CH₂)_P-R¹⁵, 5-14-membered aryl, 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S, 5-14-membered cycloalkyi, and 5-14-membered heterocycloalkyl comprising 1 to 4 heteroatoms selected from N, O and S;

R¹³ and R¹⁴ are independently selected from H, C1-C4 alkyl, 5-14-membered aryl, 5-14- membered heteroaryl comprising 1 to 4 heteroatoms selected from , O and S, 5-14- membered cycloalkyl, and 5- 14-membered heterocycloalkyl comprising 1 to 4 heteroatoms selected from N, O and S;

R¹⁵ is selected from H, -C(0)-R, -NRR', -OR, -SR, -COOR, -CN, -N0₂, -C(0)-NRR', -NR'- C(0)-R, -S0₂-R and -(S0₂)-OR, 5-14-membered aryl, 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from , O and S, 5-14-membered cycloalkyl and 5- 14-membered heterocycloalkyl comprising 1 to 4 heteroatoms selected from N, O and S; and p is an integer from 1 to 4.

The compound of claim 1, wherein HAr is selected from the group consisting of pyrrol, pyrrolidine, imidazol, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, triazole, tetrazole, piperidine, pyridine, piperazine, pyrazine, pyrimidine, pyridazine, triazines, tetrazines, indazole, indole, isoindole benzimidazole, indoline, quinoline, isoquinoline, purine, oxazolidine, oxazole, oxazoline, isoxazolidine, isoxazole, thiazolidine, thiazole, thiazoline, isothiazolidine, isothiazole, furazane, oxadiazole, oxadiazine, thiadiazole, thiadiazine, morpholine, oxazine, thiomorpholine, and thiazine, all of which may be substituted or unsubstituted.

The compound of claim 2, wherein HAr is selected from the group consisting of indazole, piperazine, thiadiazine, indole, indoline, imidazole and benzimidazole all of which may be substituted or unsubstituted and when substituted, the substituent is selected from oxo, - C(0)-R¹³, -S0₂-R¹³, -(CH₂)p-R¹⁵, 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S.

The compound of any one of claims 1-3, wherein

(i) X is -C(O)- and Y is NH or CH₂;

(ii) X and Y are missing; or

(iii) X is -S0₂- and Y is CHR'.

The compound of any one of claims 1-4, wherein Z is CH₂ or missing.

The compound of any one of claims 1-5, wherein

(i) at least one of R¹, R², R³, R⁴ and R⁵ is halo; and/or

(ii) at least one of R¹ , R², R³, R⁴ and R⁵ is C 1 -C4 alkyl; and/or

(iii) at least one of R¹, R², R³, R⁴ and R⁵ is -OR.

The compound of any one of claims 1-6, wherein the compound is selected from:

D29, E746-0699 D33, G271 -0164

8. Pharmaceutical composition comprising a compound according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient.

9. Method for the treatment or prevention of a myostatin-related disease or disorder in a subject, comprising administering a therapeutically or prophylactically effective amount of a compound of any one of claims 1 to 7 to said subject.

10. The method according to claim 9, wherein the myostatin-related disease or disorder is

selected from the group consisting of cachexia, sarcopenia, obesity, insulin resistance and dystrophic muscle loss.

11. Method for increasing muscle growth or muscle regeneration in a subject, comprising

administering a therapeutically or prophylactically effective amount of a compound of any one of claims 1 to 7 to said subject.

12. The method of any one of claims 9-1 1, wherein the subject is a mammal.

13. The method of claim 12, wherein the mammal is a human.

14. Method for antagonizing myostatin activity in a cell, comprising contacting said cell with a compound of any one of claims 1-7.

15. The method of claim 14, wherein the cell is a muscle stem cell.

16. Method of identifying compounds that can bind to myostatin by comparing the 3-D structure of candidate compounds with the 3-D molecular model of myostatin shown in Figure 1, the method comprising the steps:

(a) calculating the distances between hydrogen bonding moieties of different candidate compounds and the amino acid residues that form the binding site of myostatin in the 3-D molecular model, wherein the binding site is defined by amino acid residues CI 6, R17, Y18, N41 and Y55 of myostatin and wherein the main chain and one side chain guanidinium nitrogen of Rl 7 act as a hydrogen bond donor and hydrogen bond acceptor, respectively, and the side chain of C16 provides for a hydrophobic interaction site, to identify compounds that can bind to the binding site of myostatin; and

(b) including size exclusion volume spheres around 10 A of the binding site region to discriminate against candidate compounds that spatially clash with protein atoms.

17. The method of claim 16, wherein the method identifies compounds that can bind to myostatin and modulate its function.