Small molecule stimulators of steroid receptor coactivator-3 and methods of their use as cardioprotective and / or vascular regenerative agents

Abstract

To provide small molecule stimulators of steroid receptor coactivator-3 and methods of their use as cardioprotective and / or vascular regenerative agents.SOLUTION: Small molecule stimulators of steroid receptor coactivator-3 (SRC-3) and methods of their use as cardioprotective agents are provided. The small molecule stimulators are useful for promoting cardiac protection and repair and vascular regeneration after myocardial infarction. The compounds are also useful in preventing cardiac hypertrophy and collagen deposition and improving the cardiac post-infarction function.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Nos. 62 / 724,281, filed August 29, 2018, and 62 / 825,358, filed March 28, 2019, which are incorporated by reference herein in their entireties. [Background technology]

[0002] A determinant of myocardial infarction (MI)-induced heart failure is progressive remodeling of cardiac tissue associated with myocyte loss, inflammation, fibrosis, and major depression of cardiac ejection fraction. One promising therapeutic approach to improving cardiac function is to prevent adverse cardiac tissue remodeling in situ by directly preserving functional myocardium. Major hurdles to maintaining cardiac function after infarction include tissue destruction and the limited regenerative capacity of the adult heart, which poses a barrier to therapies designed to promote tissue reprogramming and repair. Summary of the Invention [Means for solving the problem]

[0003] Described herein are small molecule stimulators of steroid receptor coactivator-3 (SRC-3) and methods for their use as cardioprotective and / or revascularizing agents. The compounds described herein are useful for promoting cardiac protection and repair and revascularization after myocardial infarction. The method includes administering a compound described herein to a subject.

[0004] Small molecule SRC-3 stimulators include compounds of the formula: [ka] and pharmaceutically acceptable salts or prodrugs thereof. In these compounds, A 1 , A 2 , A 3 , A4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently 1 and N, each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 Alkyl and R 2 is a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocycloalkyl. Optionally, the compound has the formula: [ka] wherein m and n are each independently 1, 2, 3, 4, or 5.

[0005] Optionally, the compound has the formula: [ka] wherein m and n are each independently 1, 2, 3, or 4.

[0006] In the compounds described herein, R 2 is optionally selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Optionally, the compound is [ka] is selected from the group consisting of:

[0007] Optionally, the compound is [ka] or selected from the group consisting of or a pharmaceutically acceptable salt or prodrug thereof.

[0008] an effective amount of a compound of the formula: [ka] Also described herein are methods for treating ischemic injury (e.g., myocardial infarction or stroke) in a subject, comprising administering to the subject a compound selected from the group consisting of: A, B, C, D, E, E, F ... 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently 1 and N Each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 alkyl, and X is NR 2 , C.R. 3 R 4 , or O and R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6 Optionally, the compound is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. [ka] is selected from the group consisting of:

[0009] Optionally, the method may further comprise selecting a subject who has suffered a myocardial infarction, or who has suffered a stroke or other vascular injury to the central nervous system.

[0010] Further described herein is a method for reducing myocardial infarction size in a subject who has suffered a myocardial infarction. The method comprises administering an effective amount of a compound of the following formula: [ka] or a pharmaceutically acceptable salt or prodrug thereof to a subject. 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently 1 and N, each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 alkyl, and X is NR 2 , C.R. 3 R 4 , or O and R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6 Optionally, the compound is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. [ka] is selected from the group consisting of:

[0011] Optionally, the myocardial infarct size is reduced by at least 5% (eg, at least 15%) compared to the myocardial infarct size in an untreated subject who has suffered a myocardial infarction.

[0012] Also described herein are methods of preventing or reducing cardiomyocyte loss, improving cardiovascular perfusion, and / or improving central nervous system vascular perfusion in a subject who has suffered a myocardial infarction or stroke, comprising administering to a subject an effective amount of a compound of the following formula: [ka] or a pharmaceutically acceptable salt or prodrug thereof, to a subject.1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently 1 and N, each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 alkyl, and X is NR 2 , C.R. 3 R 4 , or O and R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6 Optionally, the compound is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. [ka] is selected from the group consisting of:

[0013] Also described herein are methods for improving cardiovascular function and / or central nervous system vascular function in a subject, comprising administering to a subject an effective amount of a compound of the following formula: [ka] or a pharmaceutically acceptable salt or prodrug thereof, to a subject. 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently 1 and N, each R 1is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 alkyl, and X is NR 2 , C.R. 3 R 4 , or O and R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6 Optionally, the compound is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. [ka] is selected from the group consisting of:

[0014] Optionally, the subject has suffered from an ischemic injury (e.g., a myocardial infarction or a stroke). Optionally, the subject is an elderly subject.

[0015] Also described herein is a method of promoting wound healing in a subject, comprising administering to a subject an effective amount of a compound of the following formula: [ka] or a pharmaceutically acceptable salt or prodrug thereof, to a subject. 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently 1 and N, each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 alkyl, and X is NR 2 , C.R. 3 R 4 , or O and R 2 , R 3, and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6 Optionally, the compound is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. [ka] is selected from the group consisting of:

[0016] Optionally, the subject has suffered from an ischemic injury (e.g., a myocardial infarction or a stroke). Optionally, the subject is an elderly subject.

[0017] an effective amount of a compound of the formula: [ka] Further described herein is a method for treating or preventing hypertrophic cardiomyopathy in a subject, comprising administering to the subject A or a pharmaceutically acceptable salt or prodrug thereof. 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently 1 and N, each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 alkyl, and X is NR 2 , C.R. 3 R 4 , or O and R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6 Optionally, the compound is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. [ka] is selected from the group consisting of:

[0018] Optionally, the subject has suffered an ischemic injury (eg, a myocardial infarction or stroke).

[0019] The details of one or more embodiments are set forth in the drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0020] [Figure 1] 1 includes graphs showing the expression of NCOA3 in normal human heart (left panel) and muscle tissue (right panel). [Figure 2] 10 shows an image of a mouse heart injected with adeno-SRC3 prior to harvest. [Figure 3] A shows the experimental timeline of drug treatment and echocardiographic measurements after myocardial infarction (MI). B is a graph showing the heart weight and tibia length ratio (HW / TL) of mice after MI. C is a graph showing the effect of MCB-613 treatment in mice after MI. D contains images of mouse hearts taken after MI and stained to visualize collagen fibers. [Figure 4] 1 is a graph showing the effect of compound 10-1 treatment in mice after myocardial infarction. [Figure 5A] 1 is a plot showing global single-cell transcriptional profiling of non-myocytes in the adult mouse heart. [Figure 5B] 1 is a graph showing different cell types present in MCB-613 treated hearts following myocardial infarction. [Figure 5C] Venn analysis of three cell clusters with endothelial characteristics. [Figure 6A] 1 is a heatmap showing the metabolomics of long-chain fatty acids in mouse hearts after myocardial infarction. [Figure 6B]1 is a heatmap showing the metabolomics of methylglutarylcarnitine in mouse hearts after myocardial infarction. [Figure 7] The top panel shows that MCB-613 selectively stimulates the intrinsic transcriptional activity of SRC. The center panel shows that compound 10-1 selectively stimulates the intrinsic transcriptional activity of SRC. The bottom panel shows that compound 10-2 selectively stimulates the intrinsic transcriptional activity of SRC. [Figure 8] Photographs of cross sections of the heart at the level of the papillary muscles after myocardial infarction and after treatment with compound 10-1 are included. [Figure 9] 1 includes graphs showing the results of an incremental maximal exercise test in saline-treated mice ("Saline"), MCB-613-treated mice ("MCB-613"), and non-infarcted wild-type mice ("WT"). The top panel shows carbon dioxide exhalation, and the bottom panel shows oxygen consumption. [Figure 10]MCB-613 stimulates angiogenesis in chicken eggs and mouse hearts 3 days after MI. (A) Cardiac fibroblasts were treated with DMSO or MCB-613 for 24 hours. Total protein was then isolated and immunoblotted for SRC-1, -2, and -3. Hsp90 was used as a loading control. (B) Cardiac fibroblasts were transfected with a GAL4 DNA-binding site-luciferase reporter (pG5-luc) and a GAL4-DNA-binding domain-full-length SRC-1, -2, or -3 fusion (pBIND-SRC) or a control pBIND expression vector. After transfection, cells were treated with DMSO or MCB-613 for 24 hours. Total protein was isolated and measured for luciferase activity. Relative light units (RLU) were calculated by normalizing luciferase activity to total protein concentration (n = 3) (*P < 0.05). In C, cardiac fibroblasts were treated with DMSO or MCB-613 for 24 hours and then conditioned with drug-free endothelial growth medium for an additional 24 hours. The conditioned cells were then seeded on Matrigel and allowed to form tubes overnight. The tubes were then stained with calcein AM dye and imaged. In D, chicken eggs were treated with DMSO or MCB-613, and vessel area was measured on days 1 and 3. Mouse embryonic fibroblasts (MEFs) were treated with dimethyl sulfoxide (DMSO) or MCB-613 for 24 hours and then placed in chicken egg membranes. Vessel area was measured on days 1 and 3. Data are expressed as the percent increase over the control for each condition. Six eggs were used per condition (*P<0.05). In E, mice were treated with MCB-613 or control 2 hours after MI. Hearts were fixed and immunostained for endothelial cell-specific CD31. Representative images of the infarct border zone from three control mice and three mice treated with MCB-613 are shown, along with bar graphs showing quantified CD31 immunostaining density by region of tissue for two fields per border zone (*P<0.05). [Figure 11A]

[0023] Figure 1 shows that MCB-613 improves cardiac function after myocardial infarction. Schematic diagram of the experimental procedure. Mice were treated with MCB-613 or control 2 hours after permanent ligation of the left anterior descending coronary artery, and for an additional 6 days, as well as at 8 and 16 weeks, as indicated. [Figure 11B] Figure 1 shows that MCB-613 improves cardiac function after myocardial infarction. Ejection fraction was measured by echocardiogram at the indicated times, and hearts were harvested at 24 hours and 12 weeks (*P<0.05). [Figure 11C] MCB-613 improves cardiac function after myocardial infarction. Tibia length and heart weight were compared 12 weeks after MI. [Figure 11D] This figure shows that MCB-613 improves cardiac function after myocardial infarction. Representative images of central slices of mouse hearts in axial (short axis), coronal (long axis), and sagittal planes are shown, showing the differences in morphology and 18F-FDG uptake between control (no MI), MI, and MI and MCB-613 at 2 weeks post-MI. Arrows indicate the infarct zone. n=6 control no MI, n=6 MI and vehicle control, n=4 MI and MCB-613. [Figure 11E] Figure 1 shows that MCB-613 improves cardiac function after myocardial infarction. Hearts treated with MCB-613 (n=2; infarct size 44% and 31%) and hearts treated with MCB-613 at 12 weeks (n=4; infarct size 22%, 3%, 20%, and 14%) were fixed and stained with picrosirius red. Also included is a bar graph showing quantification of percent fibrosis in the border zone of each heart. Scale bar: 2000 μm and 20 μm. [Figure 11F] Figure 1 shows that MCB-613 improves cardiac function after myocardial infarction. Representative electron micrographs of the border region 72 hours after MI are shown. My = myofibrils. Mi = mitochondria. Scale bar: 1 μm. [Figure 11G] Figure 1 shows that MCB-613 improves cardiac function after myocardial infarction. Representative TUNEL staining from control and MCB-613-treated hearts 24 hours post-MI is shown. n=4 hearts per group. Scale bars: 2000 μm and 20 μm. [Figure 12] 1 includes graphs showing the results of incremental maximal exercise tests in non-infarcted wild-type mice treated with saline ("WT Saline"), non-infarcted wild-type mice treated with MCB-613 ("WT MCB-613"), mice treated with saline after MI ("MI Saline"), and mice treated with MCB-613 after MI ("MI MCB-613"). The left panel shows oxygen consumption, and the right panel shows carbon dioxide exhalation. [Figure 13A] Figure 1 shows RNA transcriptional profiling of cardiomyocytes and single-cell analysis of interstitial cells 12 weeks post-MI revealing that the MCB-613 protective response is associated with improved oxidative phosphorylation, reduced inflammation, and reduced immune cells. Figure 2 shows a schematic of the isolation procedure to obtain cardiomyocytes for total RNA sequencing and non-cardiomyocytes for single-cell RNA-seq analysis from control-treated and MCB-613-treated mice 12 weeks post-MI. n = 2 hearts / group. [Figure 13B] Figure 1 shows RNA transcriptional profiling of cardiomyocytes and single-cell analysis of interstitial cells 12 weeks post-MI revealing that the MCB-613 protective response is associated with improved oxidative phosphorylation, reduced inflammation, and reduced immune cells. Figure 2 shows heatmap analysis of genes identified by RNA-seq and differentially expressed in cardiomyocytes from two mice treated with MCB-613 versus two mice treated with saline 10 weeks post-MI. [Figure 13C] Figure 1 shows RNA transcriptional profiling of cardiomyocytes and single-cell analysis of interstitial cells 12 weeks post-MI revealing that the MCB-613 protective response is associated with improved oxidative phosphorylation, reduced inflammation, and reduced immune cells. Gene set enrichment analysis of up- and down-regulated genes in cardiomyocytes from MCB-613 versus control-treated hearts. [Figure 13D] Figure 1 shows RNA transcriptional profiling of cardiomyocytes and single-cell analysis of interstitial cells 12 weeks post-MI revealing that the MCB-613 protective response is associated with improved oxidative phosphorylation, reduced inflammation, and reduced immune cells. Cell populations identified by unsupervised clustering are shown. Each dot represents a single cell. [Figure 13E]Figure 1 shows RNA transcriptional profiling of cardiomyocytes and single-cell analysis of interstitial cells 12 weeks post-MI revealing that the MCB-613 protective response is associated with improved oxidative phosphorylation, reduced inflammation, and reduced immune cells. Figure 2 shows a heat map depicting established cell type markers used to specifically identify each cluster. [Figure 13F] RNA transcriptional profiling of cardiomyocytes and single-cell analysis of interstitial cells 12 weeks post-MI reveal that the MCB-613 protective response is associated with improved oxidative phosphorylation, reduced inflammation, and reduced immune cells. Representative TUNEL staining from control and MCB-613-treated hearts 24 hours post-MI is shown. n=4 hearts per group. Scale bars: 2000 μm and 20 μm. [Figure 14] A. Venn analysis of fibroblast cluster gene expression. B. Venn analysis of endothelial cluster gene expression. C. Venn analysis of macrophage gene expression. D. Gene set enrichment analysis of up- and down-regulated genes in granulocytes of MCB-613 over control treated hearts. [Figure 15A] Figure 1 shows that MCB-613 modulates sustained immune and endothelial cell responses 12 weeks post-MI. Figure 2 shows the number of up- and down-regulated genes in non-myocytes from control mice compared to mice treated with MCB-613. [Figure 15B] This figure shows that MCB-613 modulates sustained immune and endothelial cell responses 12 weeks after MI. This figure includes receptor-ligand analysis of intercellular communication between cardiac cell types, excluding cardiomyocytes. Lines indicate communication between two cell types. As indicated in the figure legend, the directionality of ligand-receptor pairings begins at the node and ends at the cognate receptor. The thickness of the line reflects the number of ligand-receptor pairings. Loops represent autocrine signaling circuits. [Figure 15C] Figure 10 shows that MCB-613 modulates sustained immune and endothelial cell responses 12 weeks post-MI. Heatmap of ligand-receptor pairing between granulocytes, fibroblast clusters, and macrophage C4. [Figure 15D] Figure 1 shows that MCB-613 modulates sustained immune and endothelial cell responses 12 weeks post-MI. Figure 2 shows heat maps showing the top 50 up- and down-regulated drug-responsive genes for 277 and 310 granulocytes from control and MCB-613-treated hearts, respectively. [Figure 16] These results show that MCB-613 reduces B lymphocytes and monocytes and upregulates granulocyte genes and lysozyme as early as 24 hours after MI. (A) Quantification of cardiac immune cells by fluorescence-activated cell sorting (FACS) immunophenotyping from control and MCB-613-treated mice 24 hours after MI. (B) mRNA expression in granulocytes and neutrophils isolated from bone marrow 24 hours after MI and MCB-613 treatment. Total RNA was isolated from neutrophil-enriched and neutrophil-depleted fractions of bone marrow and converted to cDNA. Gene expression changes of S100a9, Tlr7, and Lcn2 were measured by qPCR; 18s RNA expression was used as a control. N = 6 per group. *P < 0.05. (C) Representative LYZ staining from control and MCB-613-treated hearts 24 hours after MI. The upper panel in C shows a low magnification of the endocardium to epicardium, and the lower panel shows a high magnification of the subendocardial region. Arrows indicate LYZ+ cells. The bar graph in C shows quantification of LV density of LYZ+ cells. n = 3 hearts / group, >10 mm2 image / heart, 24 hours after MI surgery. *P < 0.039. [Figure 17] 1 includes a graphical representation of the pharmacokinetic data obtained for MCB-613, Compound 1, and Compound 2 in CD-1 mice. DETAILED DESCRIPTION OF THE INVENTION

[0021] Described herein are stimulators of steroid receptor coactivator (SRC) proteins and methods for their use. Steroid receptor coactivators are members of the p160 family of nuclear receptor coactivators, including SRC-1, SRC-2 (TIF2 / GRIP1), and SRC-3 (AIB1 / RAC3 / ACTR / pCIP). The small molecules described herein are stimulators of SRC-3 and are useful as cardioprotective and / or regenerative agents. In particular, the compounds are useful for promoting cardiac protection and repair and vascular regeneration after myocardial infarction or stroke. The compounds are also useful for preventing cardiac hypertrophy and collagen deposition and improving post-infarction cardiac function. The compounds have been shown to increase angiogenesis, increase vascular perfusion in the heart and central nervous system, and promote cardiac beta-oxidation. Administration of the compounds described herein also significantly reduces the presence of methylglutarylcarnitine, a metabolite associated with dilated cardiomyopathy.

[0022] I. Compound The class of SRC stimulators described herein is represented by Formula I: [ka] and pharmaceutically acceptable salts or prodrugs thereof.

[0023] In Formula I, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently 1 and N. Each R present in formula I is selected from 1 The groups are independently selected from hydrogen, halogen, alkoxy, cyano, trifluoromethyl, and substituted or unsubstituted C 1- 6 alkyl.

[0024] Also, in formula I, X is NR 2 , C.R. 3 R 4 , or O and R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6 It is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

[0025] As used herein, the terms alkyl, alkenyl, and alkynyl include straight-chain and branched-chain monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. The range of these groups useful in the compounds and methods described herein includes C1-C 20 Alkyl, C2-C 20 Alkenyl, and C2-C 20 An additional range of these groups useful in the compounds and methods described herein includes C-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Includes alkynyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl, C2-C4 alkenyl, and C2-C4 alkynyl.

[0026] Heteroalkyl, heteroalkenyl, and heteroalkynyl are defined similarly to alkyl, alkenyl, and alkynyl, but may contain O, S, or N heteroatoms or combinations thereof in the backbone. The scope of these groups useful in the compounds and methods described herein includes C1-C 20 Heteroalkyl, C2-C 20 Heteroalkenyl, and C-C 20 An additional range of these groups useful in the compounds and methods described herein includes C-C heteroalkynyl. 12 Heteroalkyl, C2-C 12 Heteroalkenyl, C2-C 12Includes heteroalkynyl, C1-C6 heteroalkyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, C1-C4 heteroalkyl, C2-C4 heteroalkenyl, and C2-C4 heteroalkynyl.

[0027] The terms cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclic alkyl groups having a single cyclic ring or multiple condensed rings. Examples include cyclohexyl, cyclopentylethyl, and adamantanyl. The range of these groups useful in the compounds and methods described herein includes C3-C6 20 Cycloalkyl, C3-C 20 Cycloalkenyl, and C3-C 20 An additional range of these groups useful in the compounds and methods described herein includes C5-C 12 Cycloalkyl, C5-C 12 Cycloalkenyl, C5-C 12 Includes cycloalkynyl, C5-C6 cycloalkyl, C5-C6 cycloalkenyl, and C5-C6 cycloalkynyl.

[0028] The terms heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl are defined similarly to cycloalkyl, cycloalkenyl, and cycloalkynyl, but may contain O, S, or N heteroatoms or combinations thereof within the cyclic backbone. The scope of these groups useful in the compounds and methods described herein includes C3-C 20 Heterocycloalkyl, C3-C 20 Heterocycloalkenyl, and C-C 20 An additional scope of these groups useful in the compounds and methods described herein includes C5-C 12 Heterocycloalkyl, C5-C 12 Heterocycloalkenyl, C5-C 12 Includes heterocycloalkynyl, C5-C6 heterocycloalkyl, C5-C6 heterocycloalkenyl, and C5-C6 heterocycloalkynyl.

[0029] Aryl molecules include, for example, cyclic hydrocarbons incorporating one or more planar sets of typically six carbon atoms connected by alternating single and double covalent bonds and the same number of delocalized electrons. An example of an aryl molecule is benzene. Heteroaryl molecules include substitutions of atoms such as O, N, or S along their main cyclic chain. When heteroatoms are introduced, sets of five atoms, e.g., four carbons and a heteroatom, can create aromatic systems. Examples of heteroaryl molecules include furan, pi, and phenyl. Examples of aryl and heteroaryl groups include benzofuran, indole, benzothiophene, imadazole, oxazole, pyridine, and pyrazine. The aryl and heteroaryl groups may also contain additional fused rings, such as benzofuran, indole, benzothiophene, naphthalene, anthracene, and quinoline. The aryl and heteroaryl groups may be bonded at any position on the ring, unless otherwise specified.

[0030] The term alkoxy, as used herein, is an alkyl group attached through a single, terminal ether linkage. Similarly, the term aryloxy, as used herein, is an aryl group attached through a single, terminal ether linkage.

[0031] The term hydroxyl as used herein is represented by the formula —OH.

[0032] As used herein, the term amine or amino refers to a group of the formula -NZ 1 Z 2 and Z 1 and Z 2 may each be a substituent described herein, such as hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

[0033] As used herein, alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl moieties can be substituted or unsubstituted. As used herein, the term substituted includes the addition of an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl group to a position attached to the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl backbone, e.g., replacement of a hydrogen atom with one of these moieties. Examples of substituents include, but are not limited to, hydroxyl, halogen (e.g., F, Br, Cl, or I), and carboxyl groups. Conversely, as used herein, the term unsubstituted indicates that the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl, or heterocycloalkyl has a full complement of hydrogens, i.e., does not contain substitution, commensurate with its level of saturation, e.g., straight-chain decane (—(CH)—CH).

[0034] In some examples, formula I is represented by structure IA. [ka] In structure IA, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , A 10 , and R 2 is as defined above for Formula I. In some examples of structure IA, A1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 Each of the 1 and each R 1 are independently selected from the groups as defined above for formula I. For example, a compound of structure IA can be represented by structure I-A1. [ka] In Structure I-A1, m and n are each independently 1, 2, 3, 4, or 5. In other words, the phenyl ring of the molecule is bound to 1 to 5 R 1 R 1 Each of the groups may be independently selected from the groups as defined above for Formula I.

[0035] In some examples of structure IA, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 can be N. For example, a compound of structure IA can be represented by structure I-A2, structure I-A3, or structure I-A4. [ka] In Structure I-A2, Structure I-A3, and Structure I-A4, m and n are each independently 1, 2, 3, or 4. In other words, the phenyl ring of the molecule is bound to one to four R 1 R 1 Each of the groups may be independently selected from the groups as defined above for Formula I.

[0036] Optionally, in Structure I-A1, Structure I-A2, Structure I-A3, and / or Structure I-A4, R 2 is a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocycloalkyl. In some examples, R 2 is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0037] In some examples, formula I is represented by structure IB. [ka] In structure IB, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , A 10 , R 3 , and R 4 is as defined above for formula I. In some examples of structure IB, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 Each We are CR 1 and each R 1 are independently selected from the groups as defined above for formula I. For example, a compound of structure IB can be represented by structure I-B1.

[0038] [ka] In structure I-B1, m and n are each independently 1, 2, 3, 4, or 5. In other words, each phenyl ring of the molecule is independently substituted with 1 to 5 R 1 R1 Each of the groups may be independently selected from the groups as defined above for Formula I.

[0039] In some examples of structure IB, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 can be N. For example, a compound of structure IB can be represented by structure I-B2, structure I-B3, or structure I-B4. [ka] In Structure I-B2, Structure I-B3, and Structure I-B4, m and n are each independently 1, 2, 3, or 4. In other words, each phenyl ring of the molecule is independently substituted with 1 to 4 R 1 R 1 Each of the groups may be independently selected from the groups as defined above for Formula I.

[0040] In some instances, formula I is represented by structure IC. [ka] In the structure IC, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 is as defined above for Formula I. In some examples of structure IC, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9, and A 10 Each of the 1 and each R 1 are independently selected from the groups as defined above for formula I. For example, a compound of structure IC can be represented by structure I-C1.

[0041] [ka] In Structure I-C1, m and n are each independently 1, 2, 3, or 4. In other words, each phenyl ring of the molecule is independently substituted with 1 to 4 R 1 R 1 Each of the groups may be independently selected from the groups as defined above for Formula I.

[0042] In some examples of structural IC, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 can be N. For example, a compound of structure IC can be represented by structure I-C2, structure I-C3, or structure I-C4. [ka] In Structure I-C2, Structure I-C3, and Structure I-C4, m and n are each independently 1, 2, 3, or 4. In other words, each phenyl ring of the molecule is independently substituted with 1 to 4 R 1 R 1 Each of the groups may be independently selected from the groups as defined above for Formula I.

[0043] Examples of Formula I include the following compounds: [ka] [ka] [ka] [ka] [ka] [ka]

[0044] In some embodiments, the compound is SYC-944 (Compound 2-8) (also referred to herein as MCB-613). In some embodiments, the compound is not SYC-944 (Compound 2-8) (also referred to herein as MCB-613). In some embodiments, the compound is Compound 9-2, Compound 9-8, Compound 10-1, or Compound 10-2.

[0045] II. How to Make Compounds The compounds described herein can be prepared in a variety of ways. The compounds can be synthesized using a variety of synthetic methods. At least some of these methods are known in the art of synthetic organic chemistry. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art by routine optimization procedures. can be determined.

[0046] Variations of Formula I include the addition, subtraction, or movement of various components described for each compound. Similarly, when one or more chiral centers are present in a molecule, all possible chiral variants are included. Furthermore, compound synthesis may involve the protection and deprotection of various chemical groups. The use of protection and deprotection, as well as the selection of appropriate protecting groups, can be determined by those skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts, Greene's Protective Groups in Organic Synthesis, 5th Ed., Wiley & Sons, 2014, which is incorporated herein by reference in its entirety.

[0047] The reactions producing the compounds described herein can be carried out in a solvent that can be selected by one skilled in the art of organic synthesis. The solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions, i.e., temperature and pressure, at which the reaction is carried out. The reaction can be carried out in one solvent or a mixture of more than one solvent. The formation of the product or intermediate can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by nuclear magnetic resonance spectroscopy (e.g., 1 H-NMR or 13 The activity may be monitored by spectroscopic means, such as C-NMR, infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography, such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0048] Exemplary methods for synthesizing the compounds described herein are provided in Example 1 below and in International Patent Application Publication No. WO2016 / 109470, which is incorporated herein by reference.

[0049] III. Pharmaceutical Formulations The compounds described herein or their derivatives can be provided in pharmaceutical compositions.Depending on the intended mode of administration, the pharmaceutical composition can be in the form of a solid, semi-solid, or liquid dosage form, such as tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in a unit dosage form suitable for single administration of a precise dose.The composition comprises a therapeutically effective amount of the compounds described herein or their derivatives in combination with a pharmaceutically acceptable carrier, and may further comprise other medicinal agents, pharmaceutical agents, carriers, or diluents.Pharmaceutically acceptable means a non-biological or otherwise undesirable substance that can be administered to an individual together with the selected compound without causing unacceptable biological effects or interacting in a harmful manner with other components contained in the pharmaceutical composition.

[0050] As used herein, the term carrier includes any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other substance known in the art for use in pharmaceutical formulations.The choice of carrier for use in a composition will depend on the intended route of administration of the composition.The preparation of pharmaceutically acceptable carriers and formulations containing these substances is described, for example, in Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005. Examples of physiologically acceptable carriers include buffers such as phosphate buffer, citrate buffer, and buffers containing other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; mannitol, ... sugar alcohols such as tetritol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS® (BASF; Florham Park, NJ).

[0051] Compositions containing the compounds described herein or their derivatives suitable for parenteral injection can include physiologically acceptable aqueous or non-aqueous sterile solutions, dispersions, suspensions or emulsions, and sterile powders that can be reconstituted into sterile injectable solutions or dispersions.Suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (olive oil, etc.), and injectable organic esters such as ethyl oleate.Appropriate fluidity can be maintained, for example, by using a coating agent such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0052] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be enhanced by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. Isotonic agents, for example, sugars, sodium chloride, etc., may also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0053] Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof are dispersed in at least one inert conventional excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, such as glycerol; and (d) disintegrants, such as agar, calcium carbonate, and the like. The formulation may be mixed with (e) solution retarders such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as cetyl alcohol and glycerol monostearate, (h) adsorbents such as kaolin and bentonite, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0054] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

[0055] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. These may contain pacifying agents and may be of a composition that releases the active compound(s) in a delayed manner in a certain part of the intestinal tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compound can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0056] The liquid dosage form for oral administration of the compound described herein or its derivatives includes pharmaceutically acceptable emulsion, solution, suspension, syrup and elixir.In addition to active compound, liquid dosage form can contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters, or the mixture of these substances.

[0057] Besides inert diluents, compositions can also include additional agents, such as wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, and perfuming agents.

[0058] In addition to the active compounds, suspensions may contain additional agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.

[0059] Compositions of the compounds described herein or derivatives thereof for rectal administration are optionally suppositories which can be prepared by mixing the compound with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol, or a suppository wax, which is solid at ordinary temperatures but liquid at body temperature and therefore will melt in the rectum and vaginal cavity and release the active ingredient.

[0060] The dosage form for topical administration of the compound described herein or its derivatives includes ointments, powders, sprays and inhalants.The compound described herein or its derivatives are mixed under sterile conditions with a physiologically acceptable carrier and, if necessary, any preservative, buffer or propellant.Ophthalmic preparations, ointments, powders and solutions are also contemplated within the scope of the composition.

[0061] Compositions may include one or more of the compounds described herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds described herein or derivatives thereof, and, where possible, zwitterionic forms of the compounds described herein, that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, etc., commensurate with a reasonable benefit / risk ratio, and are effective for their intended use. The term salt refers to the relatively non-toxic inorganic and organic acid addition salts of the compounds described herein. These salts can be prepared in situ during the isolation and purification of the compounds, or by separately reacting the purified compound in its free form with a suitable organic or inorganic acid and isolating the salt so formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthyl mesylate, glucoheptonate, lactobionate, methanesulfonate, and laurylsulfonate salts, etc. These are based on alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. (See S. M. Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated herein by reference in its entirety, at least for the compositions taught therein.)

[0062] Administration of the compounds and compositions described herein, or pharmaceutically acceptable salts thereof, can be carried out using a therapeutically effective amount of the compounds and compositions described herein, or pharmaceutically acceptable salts thereof described herein, for a period of time effective to treat the disorder. Effective amounts of the compounds and compositions described herein, or pharmaceutically acceptable salts thereof described herein, can be determined by one of skill in the art and include exemplary dosages of the active compound for a mammal of about 0.5 to about 200 mg / kg body weight per day, which can be administered in the form of a single dose or individual divided doses, such as 1 to 4 times per day. Alternatively, the dosage may be from about 0.5 to about 150 mg / kg of body weight per day of the active compound, from about 0.5 to about 100 mg / kg of body weight per day of the active compound, from about 0.5 to about 75 mg / kg of body weight per day of the active compound, from about 0.5 to about 50 mg / kg of body weight per day of the active compound, from about 0.01 to about 50 mg / kg of body weight per day of the active compound, from about 0.05 to about 25 mg / kg of body weight per day of the active compound, from about 0.1 to about 25 mg / kg of body weight per day of the active compound, or from about 0.5 to about 25 mg / kg of body weight per day of the active compound. Body weight of active compound, about 1 to about 20 mg / kg body weight of active compound per day, about 1 to about 10 mg / kg body weight of active compound per day, about 20 mg / kg body weight of active compound per day, about 10 mg / kg body weight of active compound per day, about 5 mg / kg body weight of active compound per day, about 2.5 mg / kg body weight of active compound per day, about 1.0 mg / kg body weight of active compound per day, or about 0.5 mg / kg body weight of active compound per day, or any range derivable therein. Optionally, the dosage is about 0.01 mg / kg to about 10 mg / kg body weight of active compound per day. Optionally, the dosage is about 0.01 mg / kg to about 5 mg / kg. Optionally, the dosage is about 0.01 mg / kg to about 2.5 mg / kg.

[0063] Those skilled in the art will understand that the specific dose level and frequency of administration for any particular subject may vary and will depend on a variety of factors, including the activity of the particular compound used, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex, and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and the severity of the particular condition.

[0064] The exact dosage used in the formulation also depends on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the expert and each subject's circumstances.Effective dosages can be extrapolated from dose-response curves derived from in vitro or animal model test systems.In addition, depending on the route of administration, those skilled in the art will know how to determine the dosage that will produce a plasma concentration for the desired level of response in the cells, tissues, and / or organs of the subject.

[0065] IV.How to use Provided herein is a method for treating myocardial infarction or other ischemic damage (e.g., stroke) in a subject.The method comprises administering to the subject an effective amount of one or more compounds or compositions described herein, or their pharmaceutically acceptable salts or prodrugs.When used to describe the amount of a compound in a method, an effective amount refers to the amount of the compound that achieves the desired pharmacological effect or other biological effect.

[0066] Also contemplated are methods that include administering to a subject an amount of one or more compounds described herein such that an in vivo concentration in target cells in the subject is achieved that corresponds to the concentration administered in vitro.

[0067] Further described herein is a method for reducing myocardial infarction size in a subject suffering from myocardial infarction.The method comprises administering to the subject an effective amount of one or more compounds or compositions as described herein.Compared with the myocardial infarction size of an untreated subject suffering from myocardial infarction (for example, a subject suffering from myocardial infarction and not receiving any treatment for myocardial infarction, or a subject suffering from myocardial infarction and receiving a therapeutic agent other than the compound or composition as described herein), the myocardial infarction size can be reduced by at least 5%.Optionally, compared with the myocardial infarction size of an untreated subject suffering from myocardial infarction, the myocardial infarction size can be reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. The compounds and compositions described herein are also useful for preventing or reducing cardiomyocyte loss in a subject who has suffered a myocardial infarction. A method for preventing or reducing cardiomyocyte loss in a subject who has suffered a myocardial infarction comprises administering to the subject an effective amount of one or more compounds or compositions described herein.

[0068] Further described herein are methods for improving cardiovascular function, improving cardiovascular perfusion, improving central nervous system vascular function, improving central nervous system vascular perfusion, promoting wound healing, and / or preventing or treating hypertrophic cardiomyopathy in a subject.The method comprises administering an effective amount of one or more compounds or compositions described herein to the subject.Optionally, the subject suffers from ischemic damage, such as myocardial infarction or stroke.Optionally, the subject is an elderly individual, an obese individual, an individual with diabetes, an individual with metabolic syndrome, an individual exposed to smoke (e.g., smokers and individuals exposed to secondhand smoke for long periods), an individual with elevated blood pressure, blood cholesterol, or triglyceride levels, or an individual with an autoimmune condition.

[0069] Methods for treating myocardial infarction, reducing myocardial infarct size, preventing or reducing cardiomyocyte loss, improving cardiovascular function, improving cardiovascular perfusion, improving central nervous system vascular function, improving central nervous system vascular perfusion, promoting wound healing, and / or preventing or treating hypertrophic cardiomyopathy in a subject may further include administering one or more additional agents to the subject. One or more additional agents and compounds described herein, or pharmaceutically acceptable salts or prodrugs thereof, can be administered in any order, including combined, simultaneous, or sequential administration. Sequential administration can be administration in a time sequence separated by up to several days. The method may also include two or more administrations of one or more additional agents and / or compounds described herein, or pharmaceutically acceptable salts or prodrugs thereof. Administration of one or more additional agents and compounds described herein, or pharmaceutically acceptable salts or prodrugs thereof, can be by the same or different routes, simultaneously, or sequentially.

[0070] Additional therapeutic agents include, but are not limited to, antiplatelet agents, statins, beta-blockers, and renin-angiotensin-aldosterone system (RAAS) blockers (e.g., angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs)). Illustrative, non-limiting examples of antiplatelet agents useful as additional therapeutic agents described herein include glycoprotein (GP) IIb / IIIa receptor antagonists (e.g., abciximab, eptifibatide, and tirofiban), dipyridamole, cyclooxygenase inhibitors (e.g., acetylsalicylic acid, ibuprofen, indomethacin, and sulfinpyrazone), adenosine diphosphate (ADP) receptor antagonists (e.g., clopidogrel and ticlopidine), and phosphodiesterase inhibitors (e.g., cilostazol).

[0071] Illustrative, non-limiting examples of statins useful as additional therapeutic agents described herein include: , atorvastatin, cerivastatin, pravastatin, lovastatin, mevastatin, simvastatin, rosuvastatin, fluvastatin, and pitavastatin.

[0072] Illustrative examples of beta-blockers useful as additional therapeutic agents described herein include, but are not limited to, acebutolol, atenolol, betaxolol, bisoprolol fumarate, carteolol, carvedilol, esmolol, labetalol, metoprolol, nadolol, nebivolol, penbutolol, pindolol, propranolol, sotalol, and timolol.

[0073] Illustrative examples of renin-angiotensin-aldosterone system (RAAS) blockers (e.g., angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs)) useful as additional therapeutic agents described herein include, but are not limited to, aliskiren, enalkiren, remikirem, benazepril, benazeprilat, captopril, enalapril, lisinopril, perindopril, quinapril, ramipril, trandolapril, fosinopril, moexipril, perindopril, losartan, valsartan, irbesartan, candesartan, telmisarsan, tasosartan, eprosartan, spironolactone, and eplerenone.

[0074] Any of the above-mentioned therapeutic agents can be used in any combination with the compositions described herein.Combination can be administered either in combination (for example, as a mixture), separately but simultaneously (for example, through separate intravenous lines to the same subject), or sequentially (for example, one of the compounds or drugs is administered first, and then the second is administered).Therefore, the term combination is used to refer to the combined, simultaneous, or sequential administration of two or more drugs.

[0075] Optionally, the compounds or therapeutic agents described herein may be administered in conjunction with surgery (eg, coronary artery bypass surgery), angioplasty, stenting, or another transplant procedure.

[0076] The methods and compounds described herein are useful for both prophylactic and therapeutic treatments. For prophylactic use, a therapeutically effective amount of the compounds and compositions described herein, or a pharmaceutically acceptable salt thereof, is administered to a subject pre-onset (e.g., before overt signs of myocardial infarction), during early onset (e.g., at the time of early signs and symptoms of myocardial infarction), or after the occurrence of myocardial infarction. Prophylactic administration can occur from several days to several years before the appearance of symptoms of myocardial infarction. Therapeutic treatment involves administering a therapeutically effective amount of the compounds and compositions described herein, or a pharmaceutically acceptable salt thereof, to a subject after the occurrence of myocardial infarction.

[0077] The methods for preventive and therapeutic treatment herein optionally include selecting a subject who is suffering from or at high risk of suffering from ischemic injury such as myocardial infarction or stroke (e.g., an obese individual, an elderly individual, or an individual who has previously suffered from ischemic injury such as myocardial infarction or stroke). Those skilled in the art can make such a determination using various prognostic and diagnostic methods, including, for example, personal or family history of the disease or condition, clinical testing (e.g., genetic testing), etc. Optionally, the methods herein can be used to prevent a subject who has suffered from a myocardial infarction from suffering from a subsequent myocardial infarction.

[0078] The compounds and compositions described herein, or pharmaceutically acceptable salts thereof, are useful for treating myocardial infarction, reducing myocardial infarct size, preventing or reducing cardiomyocyte loss, improving cardiovascular function, promoting wound healing, and / or preventing or treating hypertrophic cardiomyopathy in humans, including, but not limited to, pediatric and geriatric populations, as well as animals, e.g., veterinary applications.

[0079] V. Kit Also provided herein are kits for treating myocardial infarction, reducing myocardial infarct size, preventing or reducing cardiomyocyte loss, improving cardiovascular function, promoting wound healing, and / or preventing or treating hypertrophic cardiomyopathy in a subject. The kits may include any of the compounds or compositions described herein. For example, the kits may include one or more compounds of Formula I. The kits may further include one or more additional agents, such as antiplatelet agents, statins, beta-blockers, renin-angiotensin-aldosterone system (RAAS) blockers (e.g., angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs)), and combinations thereof.

[0080] The kit may include an oral formulation of any of the compounds or compositions described herein. The kit may include an intravenous or intraperitoneal formulation of any of the compounds or compositions described herein. The kit may further include instructions for using the kit (e.g., instructions for treating a subject), a container, a means for administering the compound or composition (e.g., a syringe), and / or a carrier.

[0081] As used herein, the terms "treatment," "treating," or "treating" refer to a method of reducing one or more symptoms of a disease or condition. Thus, in the disclosed methods, treatment can refer to a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of one or more symptoms of a disease or condition. For example, a method for treating a disease is considered therapeutic if there is a 5% reduction in one or more symptoms or signs of the disease (e.g., myocardial infarction size) in a subject compared to a control. As used herein, a control refers to an untreated condition (e.g., myocardial infarction size in an untreated subject who has suffered a myocardial infarction). Thus, this reduction can be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 5% and 100% compared to the natural or control level. It is understood that treatment does not necessarily refer to a cure or complete elimination of the disease, condition, or symptoms of the disease or condition.

[0082] As used herein, the terms prevent, preventing, and prevention of a disease or disorder refer to the action of, e.g., administering a composition or therapeutic agent before or at about the same time as a subject begins to exhibit one or more symptoms of a disease or disorder, which inhibits or delays the onset or severity of one or more symptoms of the disease or disorder.

[0083] As used herein, reference to decrease, reduction, or inhibition includes a change of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control level. Such terms can include, but do not necessarily include, complete elimination.

[0084] As used herein, subject refers to both mammals and non-mammals. Mammals include, for example, humans, non-human primates, such as apes and monkeys, cows, horses, sheep, rats, mice, pigs, and goats. Non-mammals include, for example, fish and birds.

[0085] Throughout this application, various publications are referenced, the disclosures of which in their entireties are hereby incorporated by reference into this application.

[0086] The following examples are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims. [Example]

[0087] Example 1: Synthesis of compounds All chemicals for synthesis were purchased from Alfa Aesar (Ward Hill, MA) or Aldrich (Milwaukee, WI). Compound identity was confirmed by NMR spectroscopy on a Varian (Palo Alto, CA) 400-MR spectrometer. 1 The purity of the synthesized compounds was monitored by UV at 254 nm and analyzed by HNMR using a Shimadzu HPLC system equipped with a Zorbax C18 (or C8) column (4.6 x 250 mm). The purity of the reported compound was found to be >95% as determined by Prominence HPLC.

[0088] Synthesis of 1-cyclopropylpiperidin-4-one: [ka] Cyclopropylamine (6.9 mL, 100 mmol) and ethyl acrylate (22.3 mL, 210 mmol, 2.1 equiv.) were dissolved in absolute ethanol (50 mL). The mixture was stirred at room temperature (rt) for 4 days. The volatiles were removed in vacuo to give a crude oil, which was purified by column chromatography (silica gel, 10:1 to 1:1 n-hexane:ethyl acetate) to give diethyl 3,3'-(cyclopropylazanediyl)dipropanoate as a colorless liquid (15.68 g, 61%).

[0089] Sodium hydride (60% dispersion in oil, 3.0 g, 75 mmol, 1.5 equiv.) and tetrahydrofuran (THF, 30 mL) were placed in an oven-dried flask, to which a solution of diethyl 3,3'-(cyclopropylazaniyl)dipropanoate (12.8 g, 50 mmol) in THF (20 mL) was added dropwise. Absolute ethanol (2.9 mL, 50 mmol, 1.0 equiv.) was then added, and the resulting mixture was stirred at reflux for 24 hours. The reaction was quenched with saturated ammonium chloride (50 mL). The mixture was extracted with diethyl ether (3 × 100 mL), and the combined organic layers were washed with water and brine and dried over Na2SO4. The volatiles were removed in vacuo to give a crude oil, which was purified by column chromatography (silica gel, 10:1 to 2:1 n-hexane:ethyl acetate) to give 1-cyclopropylpiperidin-4-one as a colorless liquid (4.52 g, 65% yield). 1 H NMR (400MHz, CDCl3): δ2.92 (t, J=6.2Hz, 4H), 2.42 (t, J=6.2Hz, 4H), 1.80~1.70 (m, 1H), and 0.55~0.47 (m, 4H).

[0090] Synthesis of 1-isopentylpiperidin-4-one: [ka] Piperidin-4-one hydrochloride (13.56 g, 100 mmol), 1-bromo-3-methylbutane (14.4 mL, 120 mmol, 1.2 equiv.), and potassium carbonate (27.6 g, 200 mmol, 2.0 equiv.) were dissolved in a mixed solvent of acetonitrile / water (50 / 50 mL). The mixture was stirred at reflux for 12 hours. The mixture was cooled to room temperature and extracted with diethyl ether (3 × 100 mL). The combined organic layers were washed with water and brine and dried over NaSO. The volatiles were removed in vacuo to give a crude oil, which was purified by column chromatography (silica gel, 10:1 to 2:1 n-hexane:ethyl acetate) to give 1-isopentylpiperidin-4-one as a colorless liquid (10.32 g, 61% yield). 1H NMR (400 MHz, CDCl): δ 2.71 (t, J = 5.9 Hz, 4H), 2.43 (t, J = 5.9 Hz, 6H), 1.61 (septet, J = 6.6 Hz, 1H), 1.40 (td, J = 7.6, 6.6 Hz, 2H), and 0.90 (d, J = 6.6 Hz, 6H).

[0091] General method for synthesizing α,β-unsaturated ketones: [ka] N-Protected piperidin-4-one (5 mmol) and aldehyde (11 mmol, 2.2 equiv.) were dissolved in acetic acid (10 mL) and concentrated hydrochloric acid (3 mL) was added. The mixture was stirred at room temperature for 12 h. The reaction was carefully quenched with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate (3 × 30 mL), and the combined organic layers were washed with water and brine and dried over NaSO. The volatiles were removed in vacuo to give a crude oil, which was purified by column chromatography (silica gel, 5:1 to 1:1 n-hexane:ethyl acetate) to afford the α,β-unsaturated ketone (60–76% yield). The compounds described herein, including compounds 9-2, 9-8, 10-1, and 10-2, were prepared according to the general method. The properties of each of these compounds are provided below.

[0092] (3E,5E)-1-Cyclopropyl-3,5-bis(3-methoxybenzylidene)piperidin-4-one (9-2). [ka] Yellow powder. 1 H NMR (400MHz, CDCl3): δ7.75(s, 2H), 7.36(t, J=8.0Hz, 2H), 7.03(d, J=8.0Hz, 2H), 6.96(s, 2H), 6.93(d , J=8.0Hz, 2H), 3.99(s, 4H), 3.85(s, 6H), 1.96~1.91(m, 1H), 0.51~0.47(m, 2H), and 0.41~0.38(m, 2H); 13C NMR (100 MHz, CDCl): 187.6, 159.7, 136.7, 136.4, 133.8, 129.7, 123.0, 116.1, 114.7, 54.0, 51.3, 38.1, and 6.9; MS (ESI) [M+H] + 376.5.

[0093] (3E,5E)-1-Cyclopropyl-3,5-bis(pyridin-3-ylmethylene)piperidin-4-one (9-8). [ka] Orange powder. 1 H NMR (400MHz, CDCl3): δ8.67(s, 2H), 8.59(d, J=4.8Hz, 2H), 7.73(s, 2H), 7.70(d, J=8.0Hz, 2H), 7.3 7(dd, J=8.0, 4.8Hz, 2H), 3.99(s, 4H), 1.99~1.94(m, 1H), 0.58~0.51(m, 2H), and 0.47~0.42(m, 2H); 13 C NMR (100 MHz, CDCl): 187.2, 151.2, 149.9, 137.3, 135.8, 132.4, 131.1, 123.6, 55.8, 40.1, and 6.9; MS (ESI) [M+H] + 318.5.

[0094] (3E,5E)-1-Isopentyl-3,5-bis(2-methoxybenzylidene)piperidin-4-one (10-1). [ka] Compound 10-1 was prepared as the hydrochloride salt (yellow powder). 1H NMR(400 MHz, CDCl3): δ13.19(br, 1H), 8.36(s, 2H), 7.45(t, J=7.3Hz, 2H), 7.10(d, J=6.6Hz, 2H), 7.03(t, J=7.3Hz, 2H), 6.97(d, J=8.3Hz, 2H), 4.49(d, J=15 .6Hz, 2H), 4.28(d, J=15.6Hz, 2H), 3.88(s, 6H), 2.87~2.82(m, 2H), 1.43(s) putet, J=6.6Hz, 1H), 1.30(td, J=7.6, 6.6Hz, 2H), and 0.70(d, J=6.6Hz, 6H); 13 C NMR (100 MHz, CDCl): 182.2, 158.2, 141.4, 132.5, 130.5, 124.1, 122.2, 120.9, 111.3, 55.7, 50.8, 49.6, 32.8, 26.0, and 22.1; MS (ESI) [M+H] + 406.5.

[0095] (3E,5E)-1-Isopentyl-3,5-bis(3-methoxybenzylidene)piperidin-4-one (10-2). [ka] Compound 10-2 was prepared as the hydrochloride salt (yellow powder). 1 H NMR (400 MHz, CDCl3): δ13.46 (br, 1H), 8.17 (s, 2H), 7.40 (t, J=8.0Hz, 2H), 7.01 (dd, J=8.0, 2.3Hz, 2H), 6.8 9(d, J=8.0Hz, 2H), 6.86(d, J=2.3Hz, 2H), 4.59(d, J=15.6Hz, 2H), 4.48(d, J=15.6Hz, 2H), 3.84(s , 6H), 2.89~2.84(m, 2H), 1.46(septet, J=6.6Hz, 1H), 1.37(td, J=7.6, 6.6Hz, 2H), and 0.72(d, J=6.6 Hz, 6H); 13C NMR (100 MHz, CDCl): 181.8, 160.0, 144.8, 134.3, 130.4, 123.9, 122.2, 116.3, 116.1, 55.5, 50.5, 50.2, 32.7, 26.0, and 22.1; MS (ESI) [M+H] + 406.5.

[0096] Example 2: Promotion of cardioprotection and repair after myocardial infarction The data herein demonstrate that administration of the compounds described herein after myocardial infarction promotes cardiac protection and repair. Among other functions, representative compounds prevented an increase in infarct size, cardiac hypertrophy, and collagen deposition. The compounds significantly improved cardiac post-infarction function. The compounds also increased angiogenesis, promoted cardiac beta-oxidation, and significantly reduced levels of methylglutarylcarnitine, a metabolite associated with dilated cardiomyopathy.

[0097] method All animal studies and protocols were approved by the Baylor College of Medicine's Institutional Animal Care and Use Committee and were performed in strict compliance with the National Institutes of Health guidelines for the care and use of laboratory animals. Adult (8-10 weeks old) ICR (CD1) mice were used for all studies.

[0098] Model of heart failure in adult mice. To induce myocardial infarction (MI) in 8-10 week-old mice, the left anterior descending artery (LAD) was permanently ligated. Briefly, mice were anesthetized with 2% isoflurane and then intubated. The heart was exposed by performing a thoracotomy through the fourth or fifth intercostal space, and an 8-0 nylon suture was tied around the LAD artery. Titer 9 × 10 9Ten microliters of adenovirus SRC-3 (Ad-SRC-3) or adenovirus GFP (Ad-GFP) at pfu / ml was injected into the anterior free wall of the left ventricle near the descending coronary artery. To assess cell cycle entry, the analog 5-ethynyl-2'-deoxyuridine (EdU; 0.2 g / L, Santa Cruz, SC-284628A) was added to the drinking water for the 9-day experimental period. Cell proliferation was measured using a Click-iT EdU kit (Invitrogen, C10339). The first dose of MCB-613 was administered intraperitoneally at 20 mg / kg 2 hours after surgery. The same dose was then injected for six more days, followed by three repeat doses at weeks 9 and 16. Mice were harvested at the indicated time points for analysis.

[0099] Echocardiogram. Cardiac function was determined by echocardiography (VisualSonics, Vevo 2100, 40 Mhz-550S probe). After alignment with the papillary muscles and transverse B-mode, cardiac function was measured on M-mode images.

[0100] Histological analysis. Whole hearts were fixed in 10% formalin, embedded in paraffin, and sectioned at 7 μm intervals. Each slide contained 3 or 10 sections, starting from the apex and ending at the suture ligation site (approximately 30–50 slides). Sections at the papillary level (slides 20–30) were stained with picrosirius red to identify areas of fibrosis. Infarct size was determined using a length-based approach.

[0101] Immunostaining analysis. Immunohistochemistry and immunofluorescence staining experiments were performed on FFPE (formalin-fixed and paraffin-embedded) sections. TUNEL staining to detect apoptotic cells was performed using the DeadEnd™ Fluorometric TUNEL System manufactured by the manufacturer. The assay was performed using the manufacturer's protocol (Promega, GS3250).

[0102] Isolation of cardiac cells for single-cell transcriptional profiling. Before cervical dislocation, mice were placed under a surgical surface of anesthesia. The heart was removed, and cells were isolated by Langendorff retrograde perfusion with calcium-free Tyrode's solution, pH 7.4 (130 mM NaCl, 74.55 mM KCl, 0.5 mM MgCl, 0.33 mM NaH2PO4, 0.25 mM HEPES, 22 mM glucose) containing collagenase at 1 mg / mL for 15 minutes. The heart was then removed from the apparatus and minced in the same Tyrode's buffer containing 15 mg / mL bovine serum albumin (BSA) before triturating with a glass pipette. Cardiomyocytes were then pelleted by differential centrifugation at 300 RPM for 3 minutes. The supernatant, containing the non-cardiomyocyte population of cells, was then filtered through a 70-micron filter, pelleted at 750G, and resuspended in 1.1 mL of 2% fetal bovine serum (FBS) in phosphate-buffered saline (PBS). 0.1 mL was removed for a "no stain" control for fluorescence-activated cell sorting (FACS). The remaining 1 mL was incubated with 4 μg / mL calcein blue and 10 μM DyeCycleRuby and incubated at 37°C for 10 minutes. The cells were then spun down at 600G and resuspended in 0.5 mL of 2% FBS / PBS containing Sytox Green (30 nM). The cells were then sorted into Sytox Green-, Calcein+, and DyeCycleRuby+ in 0.4% FBS in PBS using a FACS Aria II cell sorter. Cells were then pelleted, resuspended in 100uL of 0.4% FBS in PBS, counted, and then run on a 10x Genomics Chromium system for single-cell transcriptome profiling.

[0103] Metabolic profiling. Hearts from four control and four MCB-613-treated mice were isolated 24 hours post-MI, perfused with 10 mM KCl, snap-frozen in liquid nitrogen, and stored at -80°C. 10 mg of tissue was analyzed for fatty acids and carnitine and normalized to three normal liver control samples. Fatty acids were normalized to the internal standard L-tryptophan, and carnitine was normalized to the internal standard L-zeatin.

[0104] Measurement of RER, VO2, and VCO2. Respiratory exchange ratio (RER), oxygen consumption (VO2), and carbon dioxide exhalation (VCO2) were measured by indirect calorimetry using an incremental maximal exercise test until mice reached exhaustion.

[0105] result Activation of SRC-3 has low toxicity. Nuclear receptor coactivator 3 (NCOA3) is expressed at low levels in the adult human heart, indicating that SRC-3 activation has few side effects. In addition, MCB-613 has a low toxicity profile in vitro and in vivo. NCOA3 expression was measured in normal human heart and compared with muscle tissue analyzed from the GTEx database of approximately 1,000 autopsy donors. Figure 1 includes graphs showing NCOA3 expression in normal human heart (left panel) and muscle tissue (right panel). Fragments per kb gene length per million mapped reads (FPKM) are shown on the y-axis, with data points representing samples. Data are categorized by NCOA3 expression level.

[0106] Expression of SRC-3 induces proliferation of non-cardiomyocytes in the heart. Adenovirus-derived SRC-3 (adeno-SRC3) was injected into the left ventricular free wall of wild-type adult mouse hearts. Mice were given 5-ethynyl-2'-deoxyuridine-treated water (EdU water) as a marker for cell proliferation. Hearts were harvested 9 days later and stained with 4',6-diamidino-2-phenylindole (Dapi) to identify nuclei, PCM1 cardiomyocyte markers, the cell surface marker wheat germ agglutinin (WGA), and the cell proliferation marker EdU. Figure 2 shows images of the hearts. We show that SRC-3 expression induces proliferation of non-cardiomyocytes in the hearts of wild-type adult mice.

[0107] The compounds described herein protect against the early and progressive loss of cardiac function after myocardial infarction. Following myocardial infarction, mice were treated with representative compounds described herein. Figure 3A shows the experimental timeline of drug treatment and echocardiographic measurements after myocardial infarction. MCB-613 or saline was administered 2 hours after ligation and every 24 hours for an additional 6 days. Repeat injections were administered three times weekly at weeks 8 and 16. Heart weight and tibia length were measured at week 10, as shown in Figure 3B. Six mice received saline and seven mice received MCB-613. *P<0.03. Left ventricular ejection fraction was determined by echocardiography (n=14, up to 12 weeks; n=3, 19 weeks) (Figure 3C). Data were analyzed by ANOVA using repeated measures and expressed as mean + / - SEM. *P<0.001. Figure 3D includes images of mouse hearts harvested after myocardial infarction and stained to visualize collagen fibers. Specifically, Figure 3D shows picrosirius red staining of cross sections at the level of the papillary muscles at 4x magnification of the infarct border zone and corresponding 20x magnification 10 weeks after myocardial infarction. Infarct size is expressed as % length. The data show a reduction in infarct size in MCB-613-treated mice compared to control mice administered saline. Left ventricular ejection fraction was determined by echocardiography in mice treated with compound 10-1 after myocardial infarction (see Figure 4). Figure 8 shows picrosirius red staining of cross sections at the level of the papillary muscles 6 weeks after myocardial infarction. Infarct size is expressed as % length. The data show a reduction in infarct size in mice treated with compound 10-1 compared to control mice administered saline.

[0108] MCB-613 induces changes in primary and secondary non-cardiomyocyte cell types in the heart after myocardial infarction. Comprehensive single-cell transcriptional profiling of non-cardiomyocytes in adult mouse hearts was performed. Single-cell sequencing was performed on non-cardiomyocytes in the heart 10 weeks after myocardial infarction. Cell clusters were generated by tSNE analysis and identified by gene expression signatures. See Figure 5A for a plot showing comprehensive single-cell transcriptional profiling of non-cardiomyocytes in the adult mouse heart. Figure 5B shows the different cell types present in hearts treated with MCB-613 after myocardial infarction. Figure 5C shows Venn analysis of three cell clusters with an endothelial signature, demonstrating that MCB-613 stimulates endothelial cell growth of two distinct endothelial cell populations in the heart 10 weeks after myocardial infarction.

[0109] MCB-613 increases cardioprotective carnitine metabolites. Figure 6A is a heat map showing the metabolomics of long-chain fatty acids in the hearts of mice 24 hours after myocardial infarction. Figure 6B is a heat map showing the metabolomics of methylglutarylcarnitine in the hearts of mice after myocardial infarction (full set FDR=1). The heat map shows that MCB-613 increases cardioprotective carnitine metabolites, increases beta-oxidation of long-chain fatty acids, and decreases methylglutarylcarnitine.

[0110] The compounds described herein stimulate the intrinsic transcriptional activity of SRC. HeLa cells transfected with a Gal4-responsive luciferase reporter (pG5-luc) and constructs encoding SRC-1, SRC-2, or SRC-3 fused to the Gal4 DNA-binding domain (pBIND-SRC-1, pBIND-SRC-2, or pBIND-SRC-3) were exposed to treatment with the compounds described herein, including MCB-613, compound 10-1, and compound 10-2. The top panel of Figure 7 shows that MCB-613 selectively stimulates the intrinsic transcriptional activity of SRC. The center panel of Figure 7 shows that compound 10-1 selectively stimulates the intrinsic transcriptional activity of SRC. The bottom panel of Figure 7 shows that compound 10-2 selectively stimulates the intrinsic transcriptional activity of SRC.

[0111] Compound 10-1 improves cardiovascular endurance after myocardial infarction. 9 includes graphs showing the results of incremental maximal exercise tests in treated mice ("Saline"; n=3), MCB-613-treated mice ("MCB-613"; n=3), and non-infarcted wild-type mice ("WT"; n=2). The top panel shows exhaled carbon dioxide (VCO2), and the bottom panel shows oxygen consumption (VO2). As shown for representative compound 10-1 from FIG. 9, compounds described herein improve cardiovascular and peripheral vascular endurance after myocardial infarction.

[0112] overview As shown by the data presented herein, the compounds described herein stimulate angiogenesis in vivo and improve the function of damaged myocardium.Therefore, the compounds described herein are exceptional therapeutic agents useful for repairing and preventing chronic wounds, promoting angiogenesis, restoring blood flow in vascular disease, and preventing adverse structural remodeling of vulnerable myocardium by preventing metabolic remodeling.These compounds are useful for cardiac repair after coronary artery failure.

[0113] Example 3: Promoting Cardioprotection and Repair After Myocardial Infarction Using Steroid Receptor Coactivator Stimulators Progressive remodeling of cardiac tissue, accompanied by myocyte loss, inflammation, fibrosis, and reduced cardiac ejection fraction, is a hallmark of myocardial infarction (MI)-induced heart failure. Important therapeutic goals after MI are to protect the myocardium, minimize infarct size, prevent progression to heart failure, and support functional recovery. Data herein demonstrate that the small molecule stimulators of steroid receptor coactivators described herein promote new blood vessel growth and improve cardiac function after MI. As demonstrated through representative compounds, administration of small molecule receptor coactivator stimulators reduces infarct size, apoptosis, cardiac hypertrophy, collagen deposition, and activates cardiomyocyte energy pathways. Single-cell transcriptional profiling identified distinct interstitial cell types and transcriptional responses associated with improved cardiac function. The compounds described herein represent novel therapeutic options for preventing the early and progressive loss of cardiac function after MI.

[0114] method Animals. All animal studies and protocols were approved by the Baylor College of Medicine's Institutional Animal Care and Use Committee and performed in strict compliance with the National Institutes of Health guidelines for the care and use of laboratory animals. Adult (8-10 weeks old) ICR (CD1) mice were used in all studies.

[0115] Angiogenesis assay for small molecule stimulator treatment. Five- to seven-day-old, specific pathogen-free (SPF) certified fertilized chicken eggs (White Leghorn) were used to access the chorioallantoic membrane (CAM). The total vascular area of ​​the CAM was measured using images obtained before drug application. 100 μL of MCB-613 at a concentration of 0.6 μM was topically applied to the surface of the CAM. The vascular area of ​​the CAM was monitored daily after each drug application. A vehicle control was maintained throughout the experiment. Treatment continued for four days, at the end of which the total vascular area of ​​the CAM was quantified using image thresholding (ImageJ). The percent growth in vascular area was compared between control and treated eggs. n = 6 eggs per condition. This experiment was repeated three times.

[0116] Angiogenesis assay using drug-treated mouse embryonic fibroblasts (MEFs). Five to seven-day-old SPF-certified fertilized chicken eggs (White Leghorn) were used to access the chorioallantoic membrane (CAM). Mouse embryonic fibroblasts (MEFs) were treated with 0.6 μM MCB-613 for 24 hours. After treatment, 2 million MEFs were suspended in 60 μL of PBS containing magnesium and calcium and 40 μL of Matrigel (2 M / egg) (Corning Inc.; Corning, NY). The MEFs were then thoroughly mixed by pipetting and the CAM was then incubated for 24 hours. MEFs were transferred to the surface of the CAM. The vascular area of ​​the CAM was monitored daily. A vehicle control was maintained throughout the experiment. MEFs were grown on the CAM surface for 4 days, at the end of which the total vascular area of ​​the CAM was quantified using image thresholding (ImageJ). The percent growth in vascular area was then compared between control and treated eggs (MEF-treated vs. untreated). n = 6 eggs per condition.

[0117] Reporter assay. Cardiac fibroblasts were seeded in 6-well plates and transfected with expression vectors for a GAL4-responsive luciferase reporter (pG5-luc) and GAL4 DNA-binding domain (GAL4-DBD) full-length SRC-1, -2, or -3 fusion constructs (pBIND-SRC-1, pBIND-SRC-2, or pBIND-SRC-3) or pBIND control using Lipofectamine 3000 (Invitrogen; Carlsbad, CA). 24 h after transfection, cells were treated with 6 μM MCB-613 or dimethyl sulfoxide (DMSO) and incubated overnight. Treated cells were lysed, and total protein was isolated using the Promega Luciferase Assay System (Promega Life Sciences; Madison, WI). Protein concentration was measured using a Bradford assay (Bio-Rad Laboratories; Hercules, CA). Relative light units were measured and normalized to total protein concentration.

[0118] A model of heart failure in adult mice. To induce MI in 8-10-week-old mice, the left anterior descending artery (LAD) was permanently ligated. Briefly, mice were anesthetized with 2% isoflurane and then intubated. The heart was exposed by performing a thoracotomy through the fourth or fifth intercostal space, and an 8-0 nylon suture was tied around the LAD. The first dose of MCB-613 was administered intraperitoneally at 20 mg / kg 2 hours after surgery. Subsequently, injections of the same dose were administered at the same time of day for six additional days, followed by three-day repeat doses at weeks 9 and 16. Mice were harvested at the indicated time points for analysis.

[0119] Echocardiography. Cardiac function was determined by echocardiography (VisualSonics, Vevo 2100, 40 MHz-550S probe). After alignment with the papillary muscles and transverse B-mode imaging, cardiac function was measured with M-mode imaging. The animal numbers for cardiac function in Figure 11A are: Day 0 control (17); MCB-613 (15); Day 1 control (10); MCB-613 (12); Day 14 vehicle (19); control (19); Day 56 control (12); MCB-613 (15); Day 70 control (8); MCB-613 (10); Day 80 control (8); MCB-613 (11); Day 133 control (3); and MCB-613 (3).

[0120] Histological analysis. Whole hearts were fixed in 10% formalin, embedded in paraffin, and sectioned at 7 μm intervals. Each slide contained 3–10 sections, starting from the apex and ending at the suture ligation site (approximately 30–50 slides). Papillary-level sections (slides 20–30) were stained with picrosirius red to identify areas of fibrosis. Infarct size was determined using a length-based approach. TUNEL staining to detect apoptotic cells was performed using the DeadEnd™ Fluorometric TUNEL System (Promega, GS3250).

[0121] Electron microscopy. Animals were sacrificed, and hearts were quickly removed and placed directly into cold primary fixative (2% paraformaldehyde + 2.5% glutaraldehyde + 2 mM CaCl2 in 0.1 M cacodylate buffer, pH 7.4). They were cross-sectioned and then kept in cold primary fixative for 4 days. After fixation, tissues were stained with 0.1% tannic acid in 0.1 M cacodylate buffer, rinsed, and infiltrated for 1 hour. Afterwards, tissues were rinsed in dH2O and counterstained with aqueous uranyl acetate. Tissues were rinsed again in dH2O and then stained with a gradient series of ethanol. The tissue was dehydrated in a series of columns (50, 70, 80, 90, 95, and 100%). Over a 4-day period, the tissue was slowly infiltrated with increasing dilutions of plastic resin in ethanol until 100% plastic was reached. After a full day of infiltration with three changes of 100% plastic, the tissue was embedded in freshly made Spurr's low-viscosity resin and polymerized overnight at 60°C. Ultrathin sections of 55-65 nm were cut with a Diatome Ultra 45 diamond knife using a Leica UC7 ultramicrotome. Sections were collected on 150-cell hexagonal mesh copper grids and viewed with a Hitachi H7500 transmission electron microscope. Images were captured using an AMTXR-16 digital camera and AMT Image Capture, v602.600.51 software.

[0122] Cardiac Cell Isolation. Before cervical dislocation, mice were placed under a surgical surface of anesthesia. The hearts were removed, and cells were isolated by Langendorff retrograde perfusion with calcium-free Tyrode's solution, pH 7.4 (130 mM NaCl, 74.55 mM KCl, 0.5 mM MgCl, 0.33 mM NaH2PO4, 0.25 mM HEPES, 22 mM glucose) containing collagenase at 1 mg / mL for 15 minutes. The hearts were then removed from the apparatus and minced in the same Tyrode's buffer containing 15 mg / mL BSA before triturating with a glass pipette. Cardiomyocytes were then pelleted by differential centrifugation at 300 RPM for 3 minutes. The supernatant, containing the non-cardiomyocyte population of cells, was then filtered through a 70-micron filter, pelleted at 750 g, and resuspended in 1.1 mL of 2% fetal bovine serum (FBS) in phosphate-buffered saline (PBS). 0.1 mL of the mixture was then removed for a "no stain control" for fluorescence-activated cell sorting (FACS). The remaining 1 mL of the mixture was incubated with 4 μg / mL calcein blue and 10 μM DyeCycleRuby and incubated at 37°C for 10 minutes. The cells were then spun down at 600g and resuspended in 0.5 mL of 2% FBS / PBS containing Sytox Green (30 nM). The cells were then sorted using a FACS Aria ii cell sorter. Green-, Calcein+, DyeCycle Ruby+ cells were sorted in 0.4% FBS in PBS, then pelleted, resuspended in 100 μL of 0.4% FBS in PBS, counted, and run through a 10x Genomics Chromium system.

[0123] Single-cell RNA sequencing. Raw fastq files were imported into Cell Ranger 2.1.1 (10X Genomics) for alignment with STAR, filtering, barcode counting, and UMI counting. Cell Ranger results were analyzed using the Seurat suite version 3.0.0 implemented in R (version 3.4.3) to identify cell clusters and differentially expressed genes. Cells expressing <200 or >5,000 unique genes, or >25% of reads mapping to mitochondria, were removed as quality control measures. Filtered data were normalized and scaled within each sample, and both wild-type and treated samples were aligned using Seurat's alignment procedure for integrated analysis. Differentially expressed genes across cell types or treatments were identified using the Wilcoxon rank-sum test built into Seurat. Gene ontology analysis was performed using custom code developed in Python, which utilizes the hypergeometric distribution to identify enriched pathways (P value <0.05).

[0124] To study the effect of treatment on intercellular communication in the infarcted heart, human curated putative ligand-receptor pairs were obtained. For each cell type, drug treatment signatures were obtained by applying filters of P value < 0.05 and log2FC > 0.25 or < -0.25. Intercellular communication was constructed by connecting cell types A and B, where the ligand was differentially expressed in cell type A, while the receptor was differentially expressed in cell type B. The network was modeled using the igraph R package. It was lotted.

[0125] Total RNA-Seq analysis. Sequencing reads were trimmed using trimGalore software. Reads were then mapped to the UCSC mm10 human genome build using HISAT and quantified against the Gencode gene model using StringTie. Gene expression (FPKM) was quantile-normalized using the R statistical system. Differentially expressed genes between tumor and normal samples were determined using a parametric t-test with a p-value <0.05 and a fold change of 1.25. Pathway enrichment analysis was performed using the GSEA software package, and significance was achieved with an adjusted q-value (q <0.25). Heatmaps were generated in Python using the Matplotlib, NumPy, and SciPy libraries.

[0126] RNA isolation and qPCR. Total RNA was isolated from cells using a Qiagen RNA isolation kit. cDNA was prepared with VILO Master Mix reagent. qPCR analysis was performed using a Taqman kit containing primers for Tlr7, Lcn2, and 18s.

[0127] Granulocyte isolation. Bone marrow cells were isolated from the hind limbs of control or MCB-613-treated mice for 24 hours. The hind limbs were removed and placed in ice-cold Hank's balanced salt solution (HBSS) (Ca / Mg-free) and 2% FBS. Both ends of the bone were cut, and the bone marrow was flushed with ice-cold HBSS containing 2% FBS using a 26G needle. Clumps were broken up with an 18G needle, filtered through a 70-μm filter, and centrifuged at 400 g for 10 minutes at 4°C. The pellet was suspended in RBC lysis buffer (BD Biosciences Pharmigen; San Diego, CA) and incubated at room temperature for 2 minutes. HBSS buffer (8 mL) was added, and the cells were spun at 400 g for 10 minutes at 4°C. Viable cells were counted by trypan blue exclusion, and bone marrow granulocytes were isolated using a mouse neutrophil isolation kit from Miltenyi Biotec (Bergisch Gladbach, Germany).

[0128] Flow cytometry and cardiac immunophenotyping. Hearts and spleens isolated from control and MCB-613-treated mice 24 h after MI or sham surgery were digested in 4 ml (sufficient for approximately 12 spleens at 1x) of RPMI 1640 with digestion buffer: 500 μL of DNAse I (10 mg / ml), 500 μL of collagenase II (50 mg / ml). Cells were placed in a GentleMacs dissociator and run twice through "IMPC_step2" and incubated at 25°C for 15 min. The "IMPC_step2" program was repeated, and the samples were incubated at 25°C for an additional 15 min, followed by another run of the "IMPC_step2" program. Then, 400 μL of 4°C stop buffer (1x PBS, 0.1 M EDTA) was added to each sample and centrifuged at approximately 100 g for 1 second to collect the liquid at the bottom of the tube. The sample was filtered through a mesh filter cap into a 50 mL conical tube. The tube was then washed with 1 mL of FACS buffer, which was also passed through the filter. The heart preparation was more viscous and was washed with 20 mL of cold filtered saline. The sample was centrifuged at 500 g for 6 minutes. The supernatant was discarded, and the pellet was suspended in 1 mL of 4°C FACS buffer. Following red blood cell (RBC) lysis and blocking, the single-cell suspension was stained with an immunocytochemistry panel and quantified using an LSR II flow cytometer. 500,000 viable events were then counted for the spleen control, and the entire tube of heart cells was recorded and analyzed.

[0129] Western Blot. Frozen whole hearts were pulverized using a mortar and pestle. Approximately 20 mg of powdered tissue was added to 300 μL of radioimmunoprecipitation assay (RIPA) buffer and homogenized using a tissue homogenizer. Samples were then incubated on a rotating platform at 4°C for 1 hour, followed by 10 minutes at 12,000 g. The cells were centrifuged to remove debris. The supernatant was collected and stored at -80°C for future use. Protein concentration was determined using the bicinchoninic acid assay (BCA) reagent system. For cell lysates, cells were lysed using NETN buffer containing 10% glycerol, and total protein was isolated. All lysis buffers were supplemented with protease and phosphatase inhibitors. Tissue lysate proteins (30–50 μg) or cell lysate proteins (50–70 μg) were loaded onto a 4–15% gradient gel (Bio-Rad) and transferred to a polyvinylidene fluoride (PVDF) membrane. Immunoblotting was performed using antibodies against SRC-1, SRC-2, SRC-3, actin, and Hsp90. HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were used at a dilution of 1:2,500. Chemiluminescent detection was performed using Pierce ECL.

[0130] Tube formation assay. Cardiac fibroblasts were treated with either DMSO or 6 μM MCB-613. 24 hours after treatment, the drug was washed off by rinsing the cells twice with PBS. The cells were then conditioned for 24 hours with endothelial cell growth medium. After conditioning, the cells were seeded in growth factor-reduced Matrigel (10 mg / ml) and allowed to form tubes overnight. The following day, the tubes were stained with Calcein AM and imaged using a Cytation imaging system.

[0131] Immunostaining. Hearts were perfused with cardioplegic 20 mM KCL-PBS, followed by 10% neutral buffered formalin, followed by drop-fixation and processing into paraffin wax. Sections (7 microns) were then cut and placed on slides. Immunofluorescence was performed by first removing the paraffin and then rehydrating the sections. Antigen retrieval was then performed (antigen unmasking solution, Tris-based, Vector Labs catalog number H-3301; Vector Labs, Burlingame, CA). Sections were permeabilized with 0.1% Tween 20-PBS, blocked with 10% donkey serum in 1% Tween 20-PBS, and then incubated with a primary antibody (1:200 rabbit anti-lysozyme, Abcam catalog no. AB108508; Abcam, Cambridge, United Kingdom) in blocking solution, followed by a secondary antibody (1:200 donkey anti-rabbit, Alexa 647, Thermo Fisher Scientific catalog no. A-31573; Thermo Fisher Scientific, Waltham, MA), followed by rhodamine-conjugated WGA (1:250 Vector Labs catalog no. RL-1022) and DAPI (1:500 Thermo Fisher Scientific catalog no. 62248). Images were taken with a Zeiss LSM780 confocal microscope. LYZ+ cells were manually counted from random images spanning the entire left ventricular myocardium after left anterior descending coronary artery occlusion surgery. n = 3 hearts / group, >10 mm 2 Image / heart, 24 hours after MI surgery.

[0132] RER, VO2, and VCO2 measurements. RER, VO2, and VCO2 were measured by indirect calorimetry using an incremental maximal exercise test until mice reached fatigue.

[0133] result MCB-613 stimulates angiogenesis. Specifically, the effects of MCB-613 on angiogenesis and stromal responses in adult cardiac fibroblasts were investigated. SRC-1, 2, and 3 proteins were expressed in adult cardiac fibroblasts isolated from 10-week-old mice (Fig. 10A). Cardiac fibroblasts were transfected with expression vectors for GAL4 DNA-binding domain-SRC-1, 2, and 3 fusion proteins and a GAL4-responsive luciferase reporter to measure SRC activation after MCB-613 treatment (Fig. 10B). SRC-3 activity was induced to a greater extent than SRC-1 and SRC-2 in response to MCB-613, indicating that MCB-613 preferentially stimulates SRC-3 activity in cardiac fibroblasts. Functionally, MCB-613 stimulated the formation of tubes in adult cardiac fibroblasts in vitro. MCB-613 stimulated angiogenesis in vitro (Figure 10C). To investigate the angiogenesis stimulation of MCB-613 in vivo, a chick egg angiogenesis assay was performed (Figure 10D). Direct administration of MCB-613 into chick eggs stimulated angiogenesis in vivo. Furthermore, introduction of mouse embryonic fibroblasts pre-stimulated with MCB-613 promoted robust angiogenesis, likely in a cell-nonautonomous manner. Without being bound by theory, these findings indicate that MCB-613's stimulation of angiogenesis may occur through multiple mechanisms.

[0134] To determine whether MCB-613 improves recovery after ischemia-induced myocardial injury, MCB-613 or vehicle control was administered to mice 2 hours after myocardial injury induced by permanent surgical ligation of the left anterior descending coronary artery. Increased angiogenesis was observed in the infarct border zone 3 days post-MI, indicating that MCB-613 promotes angiogenesis in injured tissue and restores blood flow in the setting of vascular disease (Figure 10E).

[0135] MCB-613 prevents the loss of cardiac function after MI. Surgical ligation of the left anterior carotid artery in mice is a commonly used preclinical MI model for testing cardiovascular therapeutic interventions. To investigate the role of SRC stimulation on early and late cardiac function and remodeling after infarction, mice undergoing MI were treated with MCB-613 or vehicle control. Mice received 20 mg / kg MCB-613 or vehicle control by intraperitoneal injection 2 hours after MI surgery and every 24 hours for an additional 6 days (Figure 11A). The early and progressive loss of cardiac function after MI was measured by echocardiography before surgery, 24 hours after surgery, and at 2, 8, 12, and 19 weeks. 24 hours after MI, ejection fraction decreased to an average of 30% in control-treated animals. In contrast, mice treated with MCB-613 2 hours post-MI had a mean ejection fraction of 43%, indicating that MCB-613 prevented the early decline in ejection fraction and provided early protection to vulnerable myocardium (Figure 11B). The ejection fraction of control-treated mice further decreased after 24 hours and was lowest at 19 weeks post-MI, indicating a progressive loss of cardiac function over time. In contrast, the ejection fraction remained above 40% from 24 hours post-MI to 19 weeks post-MI after MCB-613 administration, indicating that the early myocardial protective effect of MCB-613 prevents the progressive loss of cardiac function. Repeat injections for 3 days at weeks 8 and 16 did not alter the ejection fraction, indicating that MCB-613 had no further effect on cardiac function at later time points. The maintenance of cardiac function up to 19 weeks post-MI indicates that short-term early intervention can be effective in preventing congestive heart failure after MI. Analysis of heart weight revealed that MCB-613 attenuated the MI-induced cardiac hypertrophy compensatory response 12 weeks after MI (Figure 11C), indicating that preservation of cardiac function correlates with prevention of another important hallmark of heart failure. Cardiac positron emission tomography (PET) imaging was then used to spatially assess myocardial viability. Improved cardiac function in the infarct zone was also observed. 18F-FDG uptake demonstrates that MCB-613 maintains healthy myocardium 2 weeks post-MI (Figure 11D). Heart tissue sections were stained with picrosirius red to assess infarct size and the degree of fibrosis (Figure 11E). Infarct size measured 12 weeks post-MI was larger in control-treated hearts (31% and 44%) compared with hearts from MCB-613-treated mice (3%, 14%, 20%, and 22%). Furthermore, cardiomyocytes were smaller, associated with reduced fibrosis in the infarct border zone, indicating that MCB-613 prevents two additional key molecular hallmarks of progressive heart failure (Figure 11E). Cardiac metabolic dysfunction is a common feature of heart failure. SRC coordinates the diverse metabolic requirements of tissues, including skeletal and cardiac muscle. Indirect calorimetry with exercise was performed to determine the effect of MCB-613 on energy expenditure 3 weeks post-MI compared with age-matched mice without MI (Figure 12). VCO2 and VO2 are elevated in MCB-613-treated animals 3 weeks after MI, indicating that MCB-613 can improve energy utilization during exercise in mice after MI. Therefore, improved cardiac function is associated with improved energy expenditure. Electron micrographs of hearts 72 hours after MI showed that MCB-613 can prevent the collapse of myofibrillar structure and abnormal mitochondrial crystalline structure, suggesting that MCB-613 can protect myocardium and mitochondria from MI-induced damage. These results demonstrate that MCB-613 protects against inflammatory injury (Figure 11F). Supporting early myocardial protection, MCB-613 prevents apoptosis 24 hours post-MI (Figure 11G). These findings demonstrate that MCB-613 directly maintains functional myocardium and acts to prevent adverse remodeling of cardiac tissue.

[0136] MCB-613 prevents the cardiomyocyte injury response. To gain insight into the cardiomyocyte- and non-cardiomyocyte-specific transcriptional functions of MCB-613 associated with attenuation of myocardial remodeling and improved cardiac function, transcriptome profiling was performed on purified cardiac cells from control- and MCB-613-treated mice 12 weeks post-MI (Figure 13A). Differential gene expression analysis of cardiomyocytes revealed that 122 up-regulated and 107 down-regulated genes were associated with improved cardiac function 12 weeks post-MI (Figure 13B). Gene set enrichment analysis of differentially expressed genes showed strong enrichment for gene ontology classifications representing oxidative phosphorylation and adipogenesis, as well as suppression of apoptosis and inflammatory responses (Figure 13C), providing further support that MCB-613 improves cardiac energy utilization in addition to preventing signaling associated with cardiomyocyte injury.

[0137] MCB-613 reduces inflammatory macrophages. Single-cell transcriptome profiling was performed to identify cell types and cell-type-specific signaling responses associated with improved cardiac function in MCB-613-treated mice 12 weeks post-MI. Metabolically active live single-cell suspensions of non-cardiomyocytes were prepared from whole hearts after Langendorff perfusion (Figure 13A). Minimal procedural manipulations were performed to prevent cell type loss and minimize impact on transcriptional activity. Transcriptional profiles of 21,894 cells from two saline-treated mice and 21,474 cells from two MCB-613-treated mice that passed RNA quality control were analyzed using the 10x Chromium platform with Seurat analysis. Based on cell expression patterns, unsupervised clustering, and dimensionality reduction analysis using Seurat software analysis, 15 distinct cell clusters were identified (Figure 13D). Cluster sizes ranged from 101 to 6,085 cells. Cell populations were identified based on known mouse cardiac cell type markers (Figure 13E). Macrophages, accounting for 42% of all cells analyzed, were the predominant non-cardiomyocyte population 12 weeks post-MI, distinct from normal adult mouse hearts, where non-cardiomyocytes comprise only 10% hematopoietic-derived cells. Evaluation of the largest changes in cell number indicated that epicardial cells, NK / T lymphocytes, fibroblasts, and endothelial cells, including lymphatic cells, were increased in cell number in the hearts of MCB-613-treated mice, while the numbers of macrophage cluster 1 and B lymphocytes were decreased (Figure 13F). Cardiac macrophage, fibroblast, and endothelial cell populations exhibited transcriptional heterogeneity, consisting of four, three, and two subclusters, respectively. Evaluation of unique gene signatures in cardiac fibroblasts revealed the presence of an injury-reactive fibroblast population expressing Postn (fibroblast cluster 2), while cluster 3 fibroblasts uniquely expressed Comp, supporting the recently reported "homeostatic fibroblast" in the remodeled heart (Figure 14A). Genes uniquely enriched in fibroblast cluster 1 indicate the presence of a fibroblast subpopulation involved in secretory functions that promote angiogenesis (Bmp4, Ecm1, Ccl11, Pgf) and extracellular matrix organization (Ecm2 and Pdgfra).The transcriptional signature of endothelial cells indicates the presence of three subpopulations. Endothelial cluster 2 and lymphatic endothelial cells exhibit increased transcriptional activation of 218 and 308 unique genes, respectively, compared with endothelial cluster 1, indicating distinct roles in cardiac maintenance 12 weeks after MI (Figure 14B). The lymphatic endothelial cluster is defined by unique expression of the lymphatic endothelial genes Prox1 and Lyve1. In addition to increased cell numbers, co-expression of the pro-angiogenic regulators HIF1A and Lrg1 and the lymphangiogenic regulator Ccl121a indicates that MCB-613 stimulates lymphangiogenesis 12 weeks after MI. The gene expression signature of endothelial cluster 2, defined by unique expression of the early cardiac genes Mkl2, Tek, and Hand2, indicates a transcriptional reversion to a more primitive cellular state, likely due to injury stress response. Without being bound by theory, the largest endothelial cluster, Endothelial Cluster 1, represents endogenous homeostatic endothelial cells characterized by a small number of unique genes and the absence of any associated GO terms or signaling pathways. The transcriptional signatures of the four macrophage subclusters were clearly separable (Figure 14C). Macrophage Cluster 1, the largest subpopulation of macrophages, is defined by the expression of an inflammatory gene signature including Ccl8, Ccl24, and Ly96, compatible with the role of these genes in the resolution of myocardial inflammation. Cluster 2 represents a population of Ccr2+ monocyte-derived macrophages expressing the inflammatory genes Cxcl1, Ccr2, Ccr5, and Tlr2, which are known to be short-lived infiltrating macrophages derived from the bone marrow in response to injury. In contrast, macrophages in Cluster 3 express Ccr2+, which are known to be maintained by local proliferation that plays a role in tissue repair and myogenesis. -Cluster 4 macrophages uniquely express 110 cell cycle proliferation genes, indicating the presence of a small subpopulation of proliferating macrophages. Cluster 4 macrophages were identified by expression of genes involved in phagocytic activation, including Cd209 and Coro1a, indicating the presence of a small subpopulation of phagocytic macrophages at 12 weeks post-MI. Surprisingly, the rather small changes in cell subpopulation numbers associated with improved cardiac function preservation indicate that MCB-613 cardioprotection is instead likely the result of altered cellular function.

[0138] MCB-613 promotes beneficial paracrine signaling. To determine the functional responses of stromal cell types associated with MCB-613-mediated cardiac function improvement 12 weeks after MI, transcriptome profiles were compared between non-cardiomyocytes from control-treated and MCB-613-treated mice (Figure 15A). The large variation in transcriptional responses of cell populations indicates that MCB-613-selective cellular responses contribute to improved cardiac function. Smaller populations of cells, consisting of lymphoid and immune cell populations, experienced the greatest drug-induced transcriptome changes. To identify potential stromal cell signaling interactions contributing to improved cardiac function, we calculated the number of interactions between ligands and receptors for each cell type in control hearts compared to MCB-613-treated hearts (Figure 15B). The highest frequency of interactions occurred between endothelial / SMC populations distributing ligands for each fibroblast population, one macrophage subtype, and granulocyte receptors. This pattern of stromal cell signaling to granulocytes implies extensive paracrine regulation of granulocyte function in the MCB-613 cardioprotective response 12 weeks after MI. Ligand-receptor pairing suggests coordinated regulation of tissue architecture and anti-inflammatory signaling pathways, including MMP9-LRP1, HSP90B1-TLR7, and SERPINE1-TAUR (Figure 15C). In support of this, gene set enrichment analysis of the up- and down-regulated gene signature in granulocytes indicates that MCB-613 suppresses inflammatory granulocyte function (Figure 14D). The top up- and down-regulated genes in cardiac granulocytes from MCB-613-treated mice compared with controls reveal increased expression of granules involved in innate defense and decreased cytokines, enzymes, and chemokines involved in inflammatory signaling (Figure 15D). These findings indicate that the myocardial response to MCB-613 is characterized by a sustained paracrine anti-inflammatory signaling landscape that underlies improved cardiac function.

[0139] Because administration of MCB-613 at the time of injury resulted in an immediate response at 24 hours (Figures 11B and 11E), we measured the effect of MCB-613 on immune cells 24 hours after MI. We quantified immune cell composition using immunophenotyping by FACS analysis of single cells isolated from whole hearts 24 hours after MI. Similar to what was seen at 12 weeks after MI, there was a significant decrease in B cells, a trend toward a decrease in monocytes, and no change in the proportion of granulocytes in hearts from MCB-613-treated mice compared to controls (Figure 16A). We then measured the acute granulocyte transcriptional response to MCB-613 and the robust transcriptional response in granulocytes 12 weeks after MI due to the presence of paracrine signaling. Granulocytes are the first innate immune cells to reach the myocardium after acute ischemic injury and are key mediators of the extent of the inflammatory response caused by an acute heart attack and the resulting extent of myocardial damage. Mouse hearts Because it is difficult to isolate sufficient amounts of intact granulocytes from the liver, we isolated bone marrow granulocytes, which reflect myocardial granulocyte responses 24 h after MI, to investigate the granulocyte response. Increased mRNA expression of the granulocyte marker S100A9 in granulocytes compared with granulocyte-depleted bone marrow indicates successful granulocyte isolation (Figure 16B). Increased expression of Tlr7 and Lcn2 in granulocytes from mice treated with MCB-613 supports single-cell transcriptome analysis, revealing that regulation of granulocyte function may contribute to the acute myocardial response to MCB-613. To control for the possibility that MCB-613 regulates Tlr7 or Lcn2 in bone marrow granulocytes as a result of tissue trauma due to surgical procedures in the absence of MI, we isolated granulocytes from mice 24 h after sham surgery administered with either vehicle or MCB-613. No changes in cell number or gene expression were observed, indicating that changes in granulocyte gene expression are the result of an MCB-613-mediated myocardial injury response. MCB-613 induction of a robust transcriptome response in granulocytes indicates that neutrophil granules can modulate the compound's post-MI inflammatory effects. In support of this, LYZ1 granule expression was significantly increased in the myocardium of MCB-613-treated mice compared with control animals 24 hours post-MI (Figure 16C).

[0140] Example 4: Pharmacokinetic (PK) studies of MCB-613, Compound 10-1, and Compound 10-2 The pharmacokinetics of MCB-613, compound 10-1, and compound 10-2 were studied in CD-1 mice. Each of the three compounds was dissolved in DMSO (20 mg / mL) and mixed with 30% hydroxypropyl-β-cyclodextrin at a 1:9 ratio. The compounds were administered intraperitoneally (ip) or orally (po) to CD-1 mice by gavage. After compound administration, blood samples (three mice per compound) were collected from the tail vein at nine time points: 5 min, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h. Plasma was isolated from these blood samples, and the plasma concentrations of the compounds were determined by HPLC-MS / MS. Pharmacokinetic parameters were calculated using the program PKSolver, an add-in program used for the analysis of pharmacokinetic and pharmacodynamic data analysis, as described in Zhang et al., Computer Methods and Programs in Biomedicine, 99:306-314 (2010). The results are summarized in Tables 1 and 2, and the half-life (t 1 / 2 ), terminal half-life (terminal t 1 / 2 ), the time after administration of the compound at which the maximum concentration is reached (t 最大 ), the maximum observed concentration of the compound (C 最大 ), area under the curve to the last measurable concentration (AUC 0-t ), area under the curve to infinity (AUC 0-inf ), and the clearance rate of the compound (Cl).

[0141] Table 1 contains the pharmacokinetic data from the above study in which MCB-613, compound 10-1, and compound 10-2 were administered intraperitoneally to CD-1 mice. [Table 1]

[0142] Table 2 contains the pharmacokinetic data from the above study in which MCB-613, Compound 10-1, and Compound 10-2 were administered orally to CD-1 mice. [Table 2]

[0143] Pharmacokinetic data are also shown in Figure 17. The graphs in Figure 17 show the mean plasma concentrations measured over time. The top row of graphs shows pharmacokinetic data from the study described above in which MCB-613 (top left graph), compound 10-1 (top center graph), and compound 10-2 (top right graph) were administered intraperitoneally to CD-1 mice. The bottom row of graphs shows pharmacokinetic data from the study described above in which MCB-613 (bottom left graph), compound 10-1 (bottom center graph), and compound 10-2 (bottom right graph) were administered orally to CD-1 mice.

[0144] The compounds and methods of the following claims are not limited in scope by the specific compounds and methods described therein, which may be used in conjunction with any of the embodiments of the claims. Any compounds and methods that are intended as examples and are functionally equivalent are within the scope of this disclosure. In addition to the modifications shown and described herein, various modifications of the compounds and methods are intended to be included within the scope of the appended claims. Furthermore, while only certain representative compounds, methods, and aspects of these compounds and methods are specifically described, other compounds and methods are intended to be included within the scope of the appended claims. Thus, although a combination of steps, elements, components, or constituents may be explicitly recited herein, all other combinations of steps, elements, components, and constituents are included, even if not explicitly recited. The present invention provides, for example, the following items: (Item 1) A compound of the formula: [ka] or a pharmaceutically acceptable salt or prodrug thereof, wherein: A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently, CR 1 and N, each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 is alkyl, R 2 is substituted or unsubstituted cycloalkyl or substituted or unsubstituted heterocycloalkyl, or a pharmaceutically acceptable salt or prodrug thereof. (Item 2) The compound has the formula: [ka] During the ceremony, Item 1. The compound according to item 1, wherein m and n are each independently 1, 2, 3, 4, or 5. (Item 3) The compound has the formula: [ka] During the ceremony, Item 1. The compound according to item 1, wherein m and n are each independently 1, 2, 3, or 4. (Item 4) The compound has the formula: [ka] During the ceremony, Item 1. The compound according to item 1, wherein m and n are each independently 1, 2, 3, or 4. (Item 5) The compound has the formula: [ka] During the ceremony, Item 1. The compound according to item 1, wherein m and n are each independently 1, 2, 3, or 4. (Item 6) R 2 is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. (Item 7) The compound is [ka] Item 1. The compound according to item 1, (Item 8) The compound is [ka] Item 1. The compound according to item 1, (Item 9) [ka] a compound selected from the group consisting of: or a pharmaceutically acceptable salt or prodrug thereof. (Item 10) 1. A method for treating ischemic injury in a subject, comprising: an effective amount of a compound of the formula: [ka] or a pharmaceutically acceptable salt or prodrug thereof to said subject, wherein: A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently, CR 1 and N, each R 1is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 is alkyl, X is NR 2 , C.R. 3 R 4 , or O and R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6 The method wherein the alkyl is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. (Item 11) The compound is [ka] 11. The method of claim 10, selected from the group consisting of: (Item 12) Item 13. The method according to item 10 or 11, wherein the ischemic injury comprises myocardial infarction or stroke. 13. The method of any one of items 10 to 12, further comprising selecting a subject suffering from ischemic damage, wherein the ischemic damage comprises myocardial infarction or stroke. (Item 14) 1. A method of reducing myocardial infarction size in a subject who has suffered a myocardial infarction, comprising: an effective amount of a compound of the formula: [ka] or a pharmaceutically acceptable salt or prodrug thereof to said subject, wherein: A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently, CR 1 and N, each R 1is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 is alkyl, X is NR 2 , C.R. 3 R 4 , or O and R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6 The method wherein the alkyl is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. (Item 15) The compound is [ka] Item 15. The method of item 14, selected from the group consisting of: (Item 16) 16. The method of item 14 or 15, wherein the myocardial infarction size is reduced by at least 5% compared to the myocardial infarction size in an untreated subject who has suffered a myocardial infarction. (Item 17) 17. The method according to any one of items 14 to 16, wherein the myocardial infarction size is reduced by at least 15% compared to the myocardial infarction size in an untreated subject who has suffered a myocardial infarction. (Item 18) 1. A method of preventing or reducing cardiomyocyte loss, improving cardiovascular perfusion, and / or improving central nervous system vascular perfusion in a subject who has suffered a myocardial infarction or stroke, comprising: an effective amount of a compound of the formula: [ka] or a pharmaceutically acceptable salt or prodrug thereof to said subject, wherein: A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8, A 9 , and A 10 are each independently, CR 1 and N, each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 is alkyl, X is NR 2 , C.R. 3 R 4 , or O and R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6 The method wherein the alkyl is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. (Item 19) The compound is [ka] Item 19. The method of item 18, selected from the group consisting of: (Item 20) 1. A method for improving cardiovascular function and / or central nervous system vascular function in a subject, comprising: an effective amount of a compound of the formula: [ka] or a pharmaceutically acceptable salt or prodrug thereof to said subject, wherein: A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently, CR 1 and N, each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 is alkyl, X is NR2 , C.R. 3 R 4 , or O and R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6 The method wherein the alkyl is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. (Item 21) The compound is [ka] 21. The method of claim 20, selected from the group consisting of: (Item 22) 22. The method of item 20 or 21, wherein the subject is suffering from an ischemic injury. (Item 23) 23. The method of claim 22, wherein the ischemic injury is myocardial infarction or stroke. (Item 24) 22. The method of item 20 or 21, wherein the subject is an elderly subject. (Item 25) 1. A method of promoting wound healing in a subject, comprising administering to a subject an effective amount of a compound of the following formula: [ka] or a pharmaceutically acceptable salt or prodrug thereof to said subject, wherein: A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently, CR 1 and N, each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 is alkyl, X is NR2 , C.R. 3 R 4 , or O and R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6 The method wherein the alkyl is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. (Item 26) The compound is [ka] 26. The method of claim 25, selected from the group consisting of: (Item 27) 1. A method for treating or preventing hypertrophic cardiomyopathy in a subject, comprising: an effective amount of a compound of the formula: [ka] or a pharmaceutically acceptable salt or prodrug thereof to said subject, wherein: A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently, CR 1 and N, each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 is alkyl, X is NR 2 , C.R. 3 R 4 , or O and R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted C 1-6The method wherein the alkyl is selected from the group consisting of alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. (Item 28) The compound is [ka] 28. The method of claim 27, selected from the group consisting of: (Item 29) 29. The method of item 27 or 28, wherein the subject is suffering from an ischemic injury. (Item 30) 30. The method of claim 29, wherein the ischemic injury is a myocardial infarction or a stroke.

Claims

1. Formula (I): 【Chemistry 61】 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient or diluent, wherein A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently selected from the group consisting of —CR 1 — and N—, and each R 1 is independently selected from the group consisting of hydrogen, halogen, alkoxy, cyano, trifluoromethyl, substituted C 1-6 alkyl, and unsubstituted C 1-6 alkyl; X is —NR 2 —, where R 2 is substituted C 1-6 alkyl, unsubstituted C 1-6 alkyl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, or unsubstituted heterocycloalkyl; Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1, wherein the compound has formula (I-A1): 【Transformation 62】 having the structure m is 1, 2, 3, 4, or 5, and n is 1, 2, 3, 4, or 5; Pharmaceutical compositions.

3. The pharmaceutical composition of claim 2, wherein m is 1 and n is 1.

4. The pharmaceutical composition of claim 2, wherein each R 1 is alkoxy, and optionally each R 1 is methoxy.

5. The pharmaceutical composition of claim 2, wherein R 2 is unsubstituted C 1-6 alkyl, and optionally R 2 is —(CH 2 ) 2 CH(CH 3 ) 2 .

6. The pharmaceutical composition of claim 2, wherein R 2 is unsubstituted cycloalkyl, and optionally, R 2 is cyclopropyl.

7. The pharmaceutical composition of claim 2, wherein the compound is the hydrochloride salt of formula (I-A1).

8. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is a solid dosage form for oral administration.

9. A pharmaceutical composition as described in claim 1, wherein the pharmaceutically acceptable excipient or diluent is sodium citrate or dicalcium phosphate.

10. A pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition is for use in treating myocardial infarction, reducing myocardial infarction size, preventing or reducing cardiomyocyte cell loss, improving cardiovascular function, promoting wound healing, or preventing or treating hypertrophic cardiomyopathy in a human subject in need thereof.

11. A kit for use in treating myocardial infarction, reducing myocardial infarction size, preventing or reducing cardiomyocyte loss, improving cardiovascular function, promoting wound healing, or preventing or treating hypertrophic cardiomyopathy in a human subject in need thereof, said kit comprising a compound of formula (I): 【Transformation 63】 or a pharmaceutically acceptable salt thereof, and instructions for use, wherein A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 are each independently selected from the group consisting of —CR 1 — and —N—, and each R 1 is independently selected from the group consisting of hydrogen, halogen, alkoxy, cyano, trifluoromethyl, substituted C 1-6 alkyl, and unsubstituted C 1-6 alkyl; X is —NR 2 —, where R 2 is substituted C 1-6 alkyl, unsubstituted C 1-6 alkyl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, or unsubstituted heterocycloalkyl; kit.

12. The kit of claim 11, wherein the compound has formula (I-A1): 【Chemistry 64】 having the structure m is 1, 2, 3, 4, or 5, and n is 1, 2, 3, 4, or 5; kit.

13. The kit of claim 12, wherein m is 1 and n is 1.

14. The kit of claim 12, wherein each R 1 is alkoxy, and optionally each R 1 is methoxy.

15. The kit of claim 12, wherein R 2 is unsubstituted C 1-6 alkyl, and optionally R 2 is —(CH 2 ) 2 CH(CH 3 ) 2 .

16. The kit of claim 12, wherein R 2 is unsubstituted cycloalkyl, and optionally, R 2 is cyclopropyl.

17. The kit of claim 12, wherein the compound is the hydrochloride salt of formula (I-A1).

18. The kit of claim 11, further comprising one or more additional drugs selected from the group consisting of antiplatelet agents, statins, beta-blockers, and renin-angiotensin-aldosterone system (RAAS) blockers.

19. The kit described in claim 11, wherein the RAAS blocker is an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin receptor blocker (ARB).

20. A kit according to any one of claims 11 to 19, wherein the oral formulation is in tablet form, pill form, or capsule form.

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