WO2013137832A1 - Inhibiteurs de myostatine - Google Patents

Inhibiteurs de myostatine Download PDF

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Publication number
WO2013137832A1
WO2013137832A1 PCT/SG2013/000109 SG2013000109W WO2013137832A1 WO 2013137832 A1 WO2013137832 A1 WO 2013137832A1 SG 2013000109 W SG2013000109 W SG 2013000109W WO 2013137832 A1 WO2013137832 A1 WO 2013137832A1
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Prior art keywords
myostatin
compound
membered
substituted
alkyl
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PCT/SG2013/000109
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English (en)
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Ravi Kambadur
Ho Sup Yoon
Susana GEIFMAN SHOCHAT
Sudarsanareddy LOKIREDDY
Venkata Krishna Harikishore AMARAVADHI
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Nanyang Technological University
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Publication of WO2013137832A1 publication Critical patent/WO2013137832A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
    • C07D231/56—Benzopyrazoles; Hydrogenated benzopyrazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D233/84—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/06—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having one or two double bonds between ring members or between ring members and non-ring members
    • C07D241/08—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having one or two double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms

Definitions

  • 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.
  • Myostatin 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.
  • TGF-/3 transforming growth factor-beta
  • 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.
  • myostatin antagonists have significant therapeutic value in alleviating muscle wasting conditions seen in human diseases such as muscular dystrophy, cachexia and sarcopenia.
  • myostatin 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.
  • WT wild-type mice.
  • 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.
  • mean total body fat is significantly reduced in Mstn-/- mice, and serum leptin levels are also significantly lower.
  • the gonadal fat pads in Mstn-/- mice have approximately 25% fewer cells and reduced fat cell size.
  • loss of Mstn in genetic models of obesity leads to suppression of body fat accumulation.
  • Mstn prodomain over-expressing transgenic mice demonstrate a significant decrease in epididymal fat pad weight.
  • Mstn-/- mice nor transgenic mice that overexpress Mstn prodomain become obese when fed a high- fat diet.
  • myostatin also appears to regulate insulin sensitivity, as there is increased insulin sensitivity in myostatin null mice.
  • myostatin antagonists may not only have utility in increasing muscle growth, they may also ameliorate obesity and type II diabetes.
  • 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.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 8 , R 9 , R 10 and R 11 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,
  • R 6 , R 7 , R 12 and R 12' are independently selected from H, -OR, -NRR' and C1-C4 alkyl;
  • X is -C(O)-, -S0 2 -, -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
  • n is 1 or 2;
  • the substituent may be 1 to 9 groups independently selected from of oxo, halo, -C(0)-R 13 , -NR 13 R 14 , -OR 13 , -SR 13 , -COOR 13 , -CN, -N0 2 , -C(0)-NR ,3 R 14 , -NR 14 -C(0)-R 13 , -S0 2 -R 13 , -(S0 2 )-OR 13 , -(CH 2 ) P -R 15 , 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 13 and R 14 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, -N0 2 , -C(0)-NRR', -NR'- C(0)-R, -S0 2 -R and -(S0 2 )-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.
  • the compound is selected from:
  • a pharmaceutical composition comprising a compound of the first aspect and a pharmaceutically acceptable excipient is disclosed herein.
  • a method for the treatment or prevention of a myostatin-related disease or disorder in a subject comprises administering a therapeutically or prophylactically effective amount of a compound of the first aspect to said subject.
  • the myostatin-related disease or disorder is selected from the group consisting of cachexia, sarcopenia, obesity, insulin resistance and dystrophic muscle loss.
  • a method for increasing muscle growth or muscle regeneration in a subject comprises administering a therapeutically or prophylactically effective amount of a compound of the first aspect to said subject.
  • a method for antagonizing myostatin activity in a cell comprising contacting said cell with a compound of the first aspect is provided.
  • 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 comprising the steps:
  • 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.
  • Fig. 2 shows various embodiments of present small molecules obtained from myoblast proliferation assays.
  • 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.
  • Fig. 4 shows the effect on proliferation rate of myoblasts by increasing the
  • 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.
  • 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 ⁇ 24.7 ⁇ .
  • 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.
  • 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.
  • myostatin also herein termed antagonists or inhibitors
  • 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.
  • 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.
  • the present compounds can be injected to enhance performance in dogs, horses and all other animals that participate in competitive environment.
  • the present compounds have utility in increasing muscle mass in muscle wasting conditions like sarcopenia, cachexia, atrophy and dystrophy.
  • 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.
  • a first aspect of the present disclosure provides a compound of formula I or formula II R 6 R 7
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 8 , R 9 , R 10 and R 11 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,
  • R°, R 7 , R 12 and R 12 are independently selected from H, -OR, -NRR' and C1-C4 alkyl;
  • 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
  • n 0 or 1 ;
  • n 1 or 2;
  • the substituent may be 1 to 9 groups independently selected from of oxo, halo, -C(0)-R 13 , -NR 13 R 14 , -OR 13 , -SR 13 , -COOR 13 , -CN, -N0 2 , -C(0)-NR 13 R 14 , -NR 14 -C(0)-R 13 , -S0 2 -R 13 , -(S0 2 )-OR 13 , -(CH 2 ) P -R 15 , 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 13 and R 14 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;
  • R 15 is selected from H, -C(0)-R, -NRR', -OR, -SR, -COOR, -CN, -N0 2 , -C(0)-NRR', -NR'-
  • 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, trihalomethane
  • aliphatic 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.
  • alkyl 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.
  • 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.
  • alkenyl refers to an aliphatic hydrocarbon having one or more carbon-carbon double-bonds, such as two or three carbon-carbon double-bonds.
  • alkenyls are optionally substituted, i.e. substituted or unsubstituted.
  • 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.
  • 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.
  • alkynyl refers to an aliphatic hydrocarbon having one or more carbon-carbon triple-bonds, such as two or three carbon- carbon triple-bonds.
  • alkynyls are optionally substituted, i.e. substituted or unsubstituted.
  • 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.
  • halogen refers to fluorine (F), chlorine (CI), bromine (Br) or iodine (I).
  • heteroatom refers to an atom other than carbon or hydrogen. Heteroatoms are typically independently selected from oxygen (O), sulfur (S), nitrogen
  • N nitrogen
  • P phosphorus
  • 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.
  • 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.
  • ring system refers to two or more rings, wherein two or more of the rings are fused.
  • fused refers to structures in which two or more rings share one or more bonds.
  • 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.
  • 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.
  • an aromatic group is substituted at one or more of the para, meta, and/or ortho positions.
  • 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,
  • 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.
  • 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.
  • non-aromatic ring refers to a group comprising a covalently closed ring that is not aromatic.
  • 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).
  • a heterocyclic ring may comprise one, two, three, four, five, six, seven, eight, nine, or more than nine heteroatoms.
  • heterocycle e.g., 5- 10-membered heterocycle
  • at least one other atom i.e. the heteroatom
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • heteroaryl groups are optionally substituted with one or more substituents.
  • 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, phthal
  • 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
  • alicyclics are optionally substituted, i.e. substituted or unsubstituted.
  • 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.
  • 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.
  • 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.
  • oxo refers to a carbonyl functional group
  • cycloalkyl refers to a completely saturated hydrocarbon ring.
  • 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.
  • cycloalkyl groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • the substituent group(s) can be defined as mentioned above.
  • heterocycloalkyl refers to a cycloalkyl group wherein one or more but not all of the atoms forming the ring is a heteroatom.
  • 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.
  • 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, o
  • 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)-R 13 , -S0 2 -R 13 , -(CH 2 ) p -R 15 , 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S.
  • 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)-R 13 , - S0 2 -R 13 , -(CH 2 )p-R 15 , 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from , O and S.
  • 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)-R 13 ,
  • the piperazine may substituted with oxo.
  • the piperazine may additionally be substituted with -S0 2 -R 13 , wherein R 13 may be H.
  • 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)-R 13 , - S0 2 -R 13 , -(CH 2 ) p -R 15 , 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S.
  • the indoline may substituted with -C(0)-R 13 , wherein R 13 may be H.
  • 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)-R 13 , -SO2-R 13 , -(CH 2 ) p -R 15 , 5-14-membered heteroaryl comprising 1 to 4 heteroatoms selected from N, O and S.
  • the imidazole may substituted with -S0 2 -R 13 , wherein R 13 may be H or pyrrolidine.
  • X is -C(O)- and Y is NH or CH 2 .
  • X is -S0 2 - and Y is CHR'.
  • Z is CH 2 or missing.
  • R 1 , R 2 , R 3 , R 4 and R 5 may be halo.
  • only R 1 or R 2 or R 3 or R 4 or R 5 may be halo, or all of R 1 , R 2 , R 3 , R 4 and R 5 may be halo, or only R 1 and R 2 may be halo, or only R 1 , R 2 and R 3 may be halo, or only R 1 , R 2 , R 3 and R 4 may be halo, just to illustrate a few examples.
  • R 1 , R 2 , R 3 , R 4 and R 5 may be C1-C4 alkyl.
  • R 1 or R 2 or R 3 or R 4 or R 5 may be C1-C4 alkyl, or all of R 1 , R 2 , R 3 , R 4 and R 5 may be C1-C4 alkyl, or only R 1 and R 2 may be C1-C4 alkyl, or only R 1 , R 2 and R 3 may be C1-C4 alkyl, or only R 1 , R 2 , R 3 and R 4 may be C1-C4 alkyl, just to illustrate a few examples.
  • R 1 may be methyl and R 2 may be ethyl, or both R 1 may and R 2 may be methyl, or R 1 may be methyl and R 2 may be propyl, or R 1 may be methyl, R 2 may be ethyl, and R 3 may be propyl, just to illustrate a few examples.
  • At least one R 2 , R 3 , R 4 and R 5 may be -OR.
  • R 1 or R 2 or R 3 or R 4 or R 5 may be -OR, or all of R 1 , R 2 , R 3 , R 4 and R 5 may be -OR, or only R 1 and R 2 may be -OR, or only R 1 , R 2 and R 3 may be -OR, or only R 1 , R 2 , R 3 and R 4 may be -OR, just to illustrate a few examples.
  • R may be H or C1-C4 alkyl.
  • 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.
  • R 1 may be -OH and R 2 may be -OCH 3 , or both R 1 may and R 2 may be -OCH 3 , or R 1 may be -OCH 3 and R 2 may be -OC 2 H 5 , or R 1 may be -OH, R 2 may be -OCH3, and R 3 may be -OC 2 H 5 , just to illustrate a few examples.
  • the compound is selected from:
  • 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.
  • 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.
  • 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.
  • 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.
  • Fig. 2 shows various embodiments of present small molecules obtained from myoblast proliferation assays.
  • present small molecules may be, but not limited to:
  • Figs. 3A, 3B, and 3C show various predicted mode of binding of active hits:
  • C Overlay of four actives (D20, D21, D25, and D33) at the binding active pocket.
  • 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.
  • 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).
  • 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.
  • 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..
  • Antagonists inhibit myostatin signaling
  • 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.
  • SPR Surface Plasmon Resonance
  • compositions may comprise any one of the compounds mentioned herein and a pharmaceutically acceptable excipient.
  • a "pharmaceutically acceptable excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatine, vegetable oils and polyethylene glycols.
  • compositions including pharmaceutical compositions comprising a therapeutically effective amount of a compound of any one of the compounds mentioned herein.
  • a compound will be therapeutically effective if it is able to affect the myostatin concentration within a cell.
  • 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.
  • 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.
  • the compounds and pharmaceutical compositions are adapted to be administered in forms suitable for injectable use.
  • 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.
  • 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.
  • 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.
  • antibacterial and/or antifungal agents for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ and 20 g of active compound.
  • the tablets, troches, pills, capsules and the like may also contain other ingredients
  • 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.
  • 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,
  • the active compound(s) may be incorporated into sustained-release preparations and formulations.
  • the active ingredient may be held within a matrix which controls the release of the active agent.
  • 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.
  • SAIB sucrose acetate isobutyrate
  • the matrix sustainedly releases the drug.
  • 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,
  • 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.
  • 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.
  • 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 ⁇ , preferably 2- 5 ⁇ . Particles having a size above 20 ⁇ are generally too large when inhaled to reach the small airways.
  • 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.
  • the particles will be crystalline.
  • an excipient such as lactose
  • typically the particle size of the excipient will be much greater than the particle size of the active ingredient.
  • 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.
  • 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
  • Dosage unit form 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.
  • 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.
  • the compound or the composition may be in the form of a treatment kit comprising the dosage unit forms and instructions for use.
  • a method for the treatment or prevention of a myostatin-related disease or disorder in a subject comprises administering a therapeutically or prophylactically effective amount of a compound of any one of the compounds or compositions mentioned herein to said subject.
  • 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.
  • the subject is a mammal, and in a preferred embodiment the subject is human.
  • “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.
  • the myo statin-related disease or disorder is selected from the group consisting of cachexia, sarcopenia, obesity, insulin resistance and dystrophic muscle loss.
  • 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.
  • 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.
  • a 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 the compounds or compositions mentioned herein to said subject.
  • the subject is a mammal.
  • the mammal is a human.
  • 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.
  • a method for antagonizing myostatin activity in a cell comprising contacting said cell with a compound of any one of the compounds or compositions mentioned herein.
  • the cell is a muscle stem cell i.e. myoblast cells.
  • the cell may be a myocyte.
  • 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.
  • 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 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
  • the method further identifies compounds that can bind to myostatin and modulate its function as described herein.
  • 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).
  • HBA hydrogen bond acceptor
  • HBD hydrogen bond donor
  • Hy hydrophobic
  • 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.
  • vHTS Virtual High Throughput Screening
  • 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 ⁇ 10 5 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 % C0 2 .
  • maintenance medium [Dulbecco's modified Eagle medium (Invitrogen) containing 10 % fetal bovine serum (FBS; Sigma, St. Louis, MO), 1 ⁇ 10 5 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 % C0 2 .
  • 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).
  • CM5 carboxymethylated dextran
  • 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)
  • 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

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Abstract

L'invention concerne des inhibiteurs de myostatine, et en particulier, des inhibiteurs de myostatine comprenant des petites molécules et des compositions pharmaceutiques comprenant les inhibiteurs de myostatine. L'invention concerne également des procédés de traitement ou de prévention d'une maladie ou d'un trouble associé à la myostatine ou des procédés pour augmenter la croissance musculaire ou la régénération musculaire chez un sujet, ou des procédés permettant d'exercer une action antagoniste sur l'activité de la myostatine dans une cellule. L'invention concerne en outre des procédés d'identification de composés qui peuvent se lier à la myostatine.
PCT/SG2013/000109 2012-03-16 2013-03-18 Inhibiteurs de myostatine WO2013137832A1 (fr)

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WO2022092294A1 (fr) 2020-10-30 2022-05-05 学校法人慶應義塾 Nouveau traitement et nouvelle prévention de maladies liées à la sarcopénie

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WO2020028482A1 (fr) * 2018-07-31 2020-02-06 The Trustees Of The University Of Pennsylvania Petites molécules qui sensibilisent des cellules infectées par le vih-1 à une cytotoxicité cellulaire dépendante des anticorps
WO2022092294A1 (fr) 2020-10-30 2022-05-05 学校法人慶應義塾 Nouveau traitement et nouvelle prévention de maladies liées à la sarcopénie

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