Small molecule stimulators of steroid receptor coactivation-3 and methods of their use as cardiac protective agents and / or revascularization agents

Abstract

The present invention relates to small molecule stimulators of steroid receptor coactivation-3 and methods of their use as cardiac protective agents and / or revascularization agents. Small molecule stimulators of steroid receptor co-activator-3 (SRC-3) and methods of their use as cardiac protective agents are provided. The small molecule stimulant can be used for promoting cardiac protection and repair and vascular regeneration after myocardial infarction. The compounds may also be used to prevent cardiac hypertrophy and collagen deposition and to improve post-cardiac infarction function.

Description

[0001] This application is a divisional application of the invention application with patent application number 201980071611.6, application date August 29, 2019, and invention title "Small molecule agonists of steroid receptor coactivator-3 and their use as cardioprotective agents and / or angiogenesis agents".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 724,281, filed August 29, 2018, and U.S. Provisional Application No. 62 / 825,358, filed March 28, 2019, which are hereby incorporated by reference in their entireties. Background of the Invention

[0004] The determinants of myocardial infarction (MI)-induced heart failure are the progressive remodeling of cardiac tissue associated with the loss of cardiomyocytes, inflammation, fibrosis, and a severe reduction in cardiac ejection fraction. A promising therapeutic approach to improving cardiac function is to prevent the detrimental remodeling of in situ cardiac tissue by directly protecting functional myocardium. The major obstacles to maintaining cardiac function after infarction include tissue destruction and the limited and restricted regenerative potential of the adult heart, which pose a barrier to therapies aimed at promoting tissue reprogramming and repair. Summary of the Invention

[0005] Small molecule agonists of steroid receptor coactivator-3 (SRC-3) and their use as cardioprotective agents and / or angiogenesis agents are described herein. The compounds described herein can be used to promote cardioprotection and repair as well as angiogenesis after myocardial infarction. The methods include administering a compound as described herein to a subject.

[0006] Small molecule SRC-3 agonists include compounds of the formula:

[0007]

[0008] and their pharmaceutically acceptable salts or prodrugs. In these compounds, 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 CR 1 and N, where each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 alkyl; and R2 is a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocycloalkyl. Optionally, the compound has the following formula:

[0009]

[0010] wherein m and n are each independently 1, 2, 3, 4, or 5.

[0011] Optionally, the compound has the following formula:

[0012]

[0013] wherein m and n are each independently 1, 2, 3, or 4.

[0014] 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 selected from the group consisting of:

[0015]

[0016] Optionally, the compound is selected from the group consisting of:

[0017]

[0018] or a pharmaceutically acceptable salt or prodrug thereof.

[0019] Also described herein is a method of treating an ischemic injury (e.g., myocardial infarction or stroke) in a subject, which comprises administering to the subject an effective amount of a compound of the following formula:

[0020]

[0021] or a pharmaceutically acceptable salt or prodrug thereof. In the compounds used in such a method, 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 CR 1 and N, wherein each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or a substituted or unsubstituted C 1-6 alkyl; and X is NR 2 、CR 3 R 4 or O, wherein R 2, R 3 and R 4 each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. Optionally, the compound is selected from the group consisting of:

[0022]

[0023]

[0024] Optionally, the method may further include selecting a subject who has suffered from myocardial infarction or has suffered from stroke or other vascular damage to the central nervous system.

[0025] The present invention further describes a method for reducing the size of myocardial infarction in a subject who has suffered from myocardial infarction. The method may include administering to the subject an effective amount of a compound of the following formula:

[0026]

[0027] or a pharmaceutically acceptable salt or prodrug thereof. In the compounds used in the methods described herein, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 5 , A 9 and A 10 each independently selected from CR 1 and N, where each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 alkyl; and X is NR 2 , CR 3 R 4 or O, where R 2 , R 3 and R 4 each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. Optionally, the compound is selected from the group consisting of:

[0028]

[0029] Optionally, compared with the size of myocardial infarction in an untreated subject who has suffered from myocardial infarction, the size of myocardial infarction is reduced by at least 5% (e.g., reduced by at least 15%).

[0030] The present invention also describes methods for preventing or reducing cardiomyocyte loss, improving cardiac vascular perfusion, and / or improving central nervous system vascular perfusion in subjects who have suffered a myocardial infarction or stroke, which comprise administering to the subject an effective amount of a compound of the following formula:

[0031]

[0032] or a pharmaceutically acceptable salt or prodrug thereof. Among the compounds used in such methods, 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 CR 1 and N, where each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl, or substituted or unsubstituted C 1-6 alkyl; and X is NR 2 、CR 3 R 4 or O, where R 2 、R 3 and R 4 are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. Optionally, the compound is selected from the group consisting of:

[0033]

[0034]

[0035] The present invention also describes methods for improving the cardiovascular function and / or central nervous system vascular function of a subject, which comprise administering to the subject an effective amount of a compound of the following formula:

[0036]

[0037] or a pharmaceutically acceptable salt or prodrug thereof. Among the compounds used in such methods, A 1 、A 2 、A 3 、A 4 、A 5 、A 6 、A 7 、A 8 、A 9 and A 10each independently selected from CR 1 and N, where each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl or substituted or unsubstituted C 1-6 alkyl; and X is NR 2 , CR 3 R 4 or O, where R 2 , R 3 and R 4 each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted cycloalkyl and substituted or unsubstituted heterocycloalkyl. Optionally, the compound is selected from the group consisting of:

[0038]

[0039] Optionally, the subject has suffered an ischemic injury (e.g., myocardial infarction or stroke). Optionally, the subject is an elderly subject.

[0040] Also described herein is a method of promoting wound healing in a subject, which comprises administering to the subject an effective amount of a compound of the following formula:

[0041]

[0042] or a pharmaceutically acceptable salt or prodrug thereof. Among the compounds used in such a method, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 and A 10 each independently selected from CR 1 and N, where each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl or substituted or unsubstituted C 1-6 alkyl; and X is NR 2 , CR 3 R 4 or O, where R 2 , R 3 and R 4 each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted cycloalkyl and substituted or unsubstituted heterocycloalkyl. Optionally, the compound is selected from the group consisting of:

[0043]

[0044] Optionally, the subject has suffered an ischemic injury (e.g., myocardial infarction or stroke). Optionally, the subject is an elderly subject.

[0045] Also described herein is a method of treating or preventing hypertrophic cardiomyopathy in a subject, comprising administering to the subject an effective amount of a compound of the following formula:

[0046]

[0047] or a pharmaceutically acceptable salt or prodrug thereof. Among the compounds used in such methods, 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 CR 1 and N, where each R 1 is hydrogen, halogen, alkoxy, cyano, trifluoromethyl or substituted or unsubstituted C 1-6 alkyl; and X is NR 2 、CR 3 R 4 or O, where R 2 、R 3 and R 4 are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted cycloalkyl and substituted or unsubstituted heterocycloalkyl. Optionally, the compound is selected from the group consisting of:

[0048]

[0049] Optionally, the subject has suffered an ischemic injury (e.g., myocardial infarction or stroke).

[0050] Details of one or more embodiments are set forth in the accompanying drawings and description below. Other features, objects, and advantages will be apparent from the description and drawings and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Contains a graph showing the expression of NCOA3 in normal human heart (left panel) and muscle tissue (right panel).

[0052] Figure 2 Is an image of the heart from a mouse injected with adenovirus-SRC3 before harvest.

[0053] Figure 3A Experimental timeline depicting the drug treatment and echocardiogram measurements after myocardial infarction (MI). Figure 3B Graph showing the heart weight to tibia length ratio (HW / TL) of mice after myocardial infarction. Figure 3C Graph showing the effect of MCB-613 treatment in mice after myocardial infarction. Figure 3D Image containing a mouse heart harvested and stained to visualize collagen fibers after myocardial infarction.

[0054] Figure 4 Graph showing the effect of compound 10-1 treatment in mice after myocardial infarction.

[0055] Figure 5A Figure describing a comprehensive single-cell transcriptional profiling of non-myocytes in the adult mouse heart. Figure 5B Graph showing the different cell types present in the heart treated with MCB-613 after myocardial infarction. 5C is a Venn analysis of three cell clusters with endothelial markers.

[0056] Figure 6A Heatmap showing the metabolomics of long-chain fatty acids in the mouse heart after myocardial infarction. Figure 6B Heatmap showing the metabolomics of methylglutaryl carnitine in the mouse heart after myocardial infarction.

[0057] Figure 7 The upper panel shows that MCB-613 selectively stimulates the intrinsic transcriptional activity of SRC. Figure 7 The middle panel shows that compound 10-1 selectively stimulates the intrinsic transcriptional activity of SRC. Figure 7 The lower panel shows that compound 10-2 selectively stimulates the intrinsic transcriptional activity of SRC.

[0058] Figure 8 Picture containing a cross-sectional view of the papillary muscle of the heart after myocardial infarction and after treatment with compound 10-1.

[0059] Figure 9 Graph containing the results of progressive maximal exercise tests conducted in mice treated with saline ("Saline"), mice treated with MCB-613 ("MCB-613"), and non-infarcted wild-type mice ("WT"). The upper panel shows carbon dioxide exhalation, and the lower panel shows oxygen consumption.

[0060] Figure 10A - 10E Shows that MCB-613 stimulates angiogenesis in chicken eggs and mouse hearts three days after MI. For Figure 10A, cardiac fibroblasts were treated with DMSO or MCB-613 for 24 hours. Total protein was then isolated and immunoblot assays were performed against SRC-1, -2, and -3. Hsp90 was used as a loading control. For Figure 10B , cardiac fibroblasts were transfected with the GAL4 DNA binding site-luciferase reporter (pG5-luc) and the GAL4-DNA binding domain-full length SRC-1, -2, or -3 fusions (pBIND-SRC) or a control pBIND expression vector. After transfection, the cells were treated with DMSO or MCB-613 for 24 hours. Total protein was isolated and luciferase activity was measured. Relative light units (RLU) were calculated by normalizing luciferase activity to total protein concentration (n = 3) (*P < 0.05). For Figure 10C , cardiac fibroblasts were treated with DMSO or MCB-613 for 24 hours and then re-conditioned with endothelial growth medium for 24 hours in the absence of the drug. The conditioned cells were then plated in Matrigel to allow tube formation overnight, and the tubes were stained with calcein AM dye and imaged. For Figure 10D , chicken eggs were treated with DMSO or MCB-613, and the vascular area was measured on the first and third days. Mouse embryonic fibroblasts (MEFs) were treated with dimethyl sulfoxide (DMSO) or MCB-613 for 24 hours and then placed on the membrane in the chicken eggs. The vascular area was measured on the first and third days. Data are presented as the percentage increase relative to the control for each condition. Six eggs were used for each condition *P < 0.05. For Figure 10E , mice were treated with MCB-613 or control two hours after MI. The hearts were fixed and immunostained for endothelial cell-specific CD31. Figure 10E Representative images of the infarct border zones from three control mice and three mice treated with MCB-613 are shown. Figure 10E Also shown is a bar graph of the quantitative CD31 immunostaining density per unit area of tissue showing two fields of view of each border zone *P < 0.05.

[0061] Figure 11A - 11G Shows that MCB-613 improves cardiac function after myocardial infarction. Figure 11A Is a schematic of the experimental protocol. Mice were treated with MCB-613 or control two hours after permanent ligation of the left anterior descending coronary artery and at six additional days and at 8 and 16 weeks as indicated. For Figure 11B , ejection fraction was measured by echocardiography at the indicated times, and the hearts were harvested at 24 hours and 12 weeks. *P < 0.05. For Figure 11C , heart weight was compared to tibia length at 12 weeks after MI. Figure 11DRepresentative images of central slices of mouse hearts showing the axial (short axis), coronal (long axis), and sagittal planes, which show the morphological and 18 F-FDG uptake differences between control (no MI), MI, and MI plus MCB-613 at two weeks after MI. Arrows indicate the infarct area. For the no-MI control, n = 6; for the MI plus vehicle control, n = 6; for MI plus MCB-613, n = 4. For Figure 11E , hearts treated with MCB-613 at 12 weeks (n = 2; infarct sizes 44% and 31%) and hearts treated with MCB-613 (n = 4; infarct sizes 22%, 3%, 20%, and 14%) were fixed and stained with Sirius red. Figure 11E Also contains bar graphs showing the quantification of the percentage of fibrosis in the border zone of each heart. Scale bars: 2000 μm and 20 μm. Figure 11F Representative electron micrographs of the border zone at 72 hours after MI are shown. My = myofibril. Mi = mitochondrion. Scale bar = 1 μm. Figure 11G Representative TUNEL staining from control and MCB-613-treated hearts at 24 hours after MI is shown. n = 4 hearts per group. Scale bars: 2000 μm and 20 μm.

[0062] Figure 12 Contains graphs showing the results of progressive maximal exercise tests performed 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.

[0063] Figure 13A - 13F Shows RNA transcriptional profiling of cardiomyocytes and single-cell analysis of interstitial cells at 12 weeks after MI, which reveals that the protective response of MCB-613 is associated with improved oxidative phosphorylation, reduced inflammation, and reduced immune cells. Figure 13A Is a schematic diagram of the isolation procedure for cardiomyocytes for total RNA sequencing and non-cardiomyocytes for single-cell RNA-seq analysis obtained from control-treated and MCB-613-treated mice at 12 weeks after MI. n = 2 hearts / group. Figure 13B Is a heat map analysis of genes identified by RNA-seq and differentially expressed in cardiomyocytes of two mice treated with MCB-613 compared to two mice treated with saline at 10 weeks after MI. Figure 13CGene set enrichment analysis of genes upregulated and downregulated in cardiomyocytes of MCB-613-treated hearts compared to control-treated hearts. Figure 13D Describes cell populations identified by unsupervised clustering. Each point represents a single cell. Figure 13E Is a heatmap indicating established cell type markers used to specifically identify each cluster. Figure 13F Shows representative TUNEL staining from control-treated and MCB-613-treated hearts at 24 hours after MI. n = 4 hearts per group. Scale bars: 2000 μm and 20 μm.

[0064] Figure 14A Is a Venn analysis of fibroblast cluster gene expression. Figure 14B Is a Venn analysis of endothelial cell cluster gene expression. Figure 14C Is a Venn analysis of macrophage gene expression. Figure 14D Gene set enrichment analysis of genes upregulated and downregulated in granulocytes of MCB-613-treated hearts compared to control-treated hearts.

[0065] Figure 15A - 15D Shows that MCB-613 regulates persistent immune and endothelial cell responses at 12 weeks after MI. Figure 15A Shows the number of genes upregulated and downregulated in non-myocytes of control mice compared to MCB-613-treated mice. Figure 15B Receptor-ligand analysis of intercellular communication between cardiac cell types excluding cardiomyocytes. Lines indicate communication between two cell types. The directionality of ligand-receptor pairs starts at the node and ends at the cognate receptor, as shown in the legend. The thickness of the lines reflects the number of ligand-receptor pairs. Loops represent autocrine signaling circuits. Figure 15C Is a heatmap of ligand-receptor pairings between granulocytes, fibroblast clusters, and macrophage C4. Figure 15D Is a heatmap showing the top 50 upregulated and downregulated drug response genes in 277 and 310 granulocytes from control-treated and MCB-613-treated hearts, respectively.

[0066] Figure 16A - 16C Shows that MCB-613 reduces B lymphocytes and monocytes as early as 24 hours after MI and upregulates granulocyte genes and lysozyme. Figure 16A Shows quantification of cardiac immune cells from control-treated and MCB-613-treated mice by fluorescence-activated cell sorting (FACS) immunophenotyping at 24 hours after MI. Figure 16BShows mRNA expression in granulocytes and neutrophils isolated from bone marrow 24 hours after MI and MCB613 treatment. Total RNA was isolated from the neutrophil-enriched fraction and neutrophil-depleted fraction of bone marrow and converted to cDNA. Gene expression changes of S100a9, Tlr7, and Lcn2 were measured by qPCR, and 18s RNA expression was used as a control. N = 6 per group *P<0.05. Figure 16C Shows representative LYZ staining from control-treated hearts and MCB-613-treated hearts 24 hours after MI. Figure 16C The upper panel of shows low magnification from endocardium to epicardium, and the lower panel shows high magnification of the subendocardial region. Arrows indicate LYZ+ cells. Figure 16C The bar graph in shows quantification of the LV density of LYZ+ cells. n = 3 hearts / group, >10mm 2 Imaging / heart, 24 hours after MI surgery *P < 0.039.

[0067] Figure 17 Graph showing the pharmacokinetic data of MCB-613, Compound 1, and Compound 2 obtained in CD-1 mice. Detailed Description

[0068] Stimulants of steroid receptor coactivator (SRC) proteins and methods of using them are described herein. Steroid receptor coactivators are members of the p160 family of nuclear receptor coactivators and include SRC-1, SRC-2 (TIF2 / GRIP1), and SRC-3 (AIB1 / RAC3 / ACTR / pCIP). The small molecules described herein are stimulants of SRC-3 and can be used as cardioprotective agents and / or angiogenesis agents. In particular, the compounds can be used to promote cardioprotection and repair as well as angiogenesis after myocardial infarction or stroke. The compounds can also be used to prevent cardiac hypertrophy and collagen deposition and to improve function after cardiac infarction. The compounds have been shown to increase angiogenesis, increase vascular perfusion in the heart and central nervous system, and promote cardiac β-oxidation. Administration of the compounds described herein also significantly reduces the presence of the metabolite methylglutaryl carnitine associated with dilated cardiomyopathy.

[0069] I. Compounds

[0070] One class of SRC stimulants described herein is represented by Formula I:

[0071]

[0072] and their pharmaceutically acceptable salts or prodrugs.

[0073] 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 selected from CR 1 and N. Each R 1 group present in Formula I is independently selected from hydrogen, halogen, alkoxy, cyano, trifluoromethyl, and substituted or unsubstituted C 1-6 alkyl.

[0074] In addition, in Formula I, X is NR 2 , CR 3 R 4 or O, where R 2 , R 3 and R 4 are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.

[0075] As used herein, the terms alkyl, alkenyl, and alkynyl include straight-chain and branched-chain monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, etc. The scope of these groups useful in the compounds and methods described herein includes C 1 -C 20 alkyl, C 2 -C 20 alkenyl, and C 2 -C 20 alkynyl. Additional scopes of these groups useful in the compounds and methods described herein include C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 4 alkyl, C 2 -C 4 alkenyl, and C 2 -C 4 alkynyl.

[0076] Heteroalkyl, heteroalkenyl, and heteroalkynyl are defined similarly to alkyl, alkenyl, and alkynyl, but may contain O, S, or N heteroatoms or combinations thereof within the backbone. The scope of these groups useful in the compounds and methods described herein includes C 1 -C 20 heteroalkyl, C 2 -C 20 heteroalkenyl, and C 2 -C 20 heteroalkynyl. Additional scopes of these groups useful in the compounds and methods described herein include C 1 -C 12 heteroalkyl, C 2 -C 12 heteroalkenyl, C 2 -C 12 heteroalkynyl, C 1 -C 6 heteroalkyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 heteroalkynyl, C 1 -C 4 heteroalkyl, C 2 -C 4 heteroalkenyl, and C 2 -C 4 heteroalkynyl.

[0077] The terms cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclic alkyls having a single cyclic ring or multiple fused rings. Examples include cyclohexyl, cyclopentylethyl, and adamantyl. The scope of these groups useful in the compounds and methods described herein includes C 3 -C 20 cycloalkyl, C 3 -C 20 cycloalkenyl, and C 3 -C 20 cycloalkynyl. Additional scopes of these groups useful in the compounds and methods described herein include C 5 -C 12 cycloalkyl, C 5 -C 12 cycloalkenyl, C 5 -C 12 cycloalkynyl, C 5 -C 6 cycloalkyl, C 5 -C 6 cycloalkenyl, and C 5 -C 6 cycloalkynyl.

[0078] The terms heterocycloalkyl, heterocycloalkenyl, and heterocycloalkynyl are similarly defined as 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 C 3 -C 20 heterocycloalkyl, C 3 -C 20 heterocycloalkenyl, and C 3 -C 20 heterocycloalkynyl. Additional scope of these groups useful in the compounds and methods described herein includes C 5 -C 12 heterocycloalkyl, C 5 -C 12 heterocycloalkenyl, C 5 -C 12 heterocycloalkynyl, C 5 -C 6 heterocycloalkyl, C 5 -C 6 heterocycloalkenyl, and C 5 -C 6 heterocycloalkynyl.

[0079] Aryl molecules include, for example, cyclic hydrocarbons incorporating one or more planar groups of six carbon atoms typically having delocalized electrons numbered the same as if they were composed of alternating single and double covalent bonds. An example of an aryl molecule is benzene. Heteroaryl molecules include substitution of atoms such as O, N, or S along the main cyclic chain of the heteroaryl molecule. When a heteroatom is introduced, a set of five atoms, for example, four carbons and the heteroatom, can produce an aromatic system. Examples of heteroaryl molecules include furan, pyrrole, thiophene, imidazole, oxazole, pyridine, and pyrazine. Aryl and heteroaryl molecules can also include additional fused rings, such as benzofuran, indole, benzothiophene, naphthalene, anthracene, and quinoline. Unless otherwise specified, aryl and heteroaryl molecules can be attached at any position on the ring.

[0080] As used herein, the term alkoxy is an alkyl group bonded through a single terminal ether linkage. Similarly, as used herein, the term aryloxy is an aryl group bonded through a single terminal ether bond.

[0081] As used herein, the term hydroxy is represented by the formula -OH.

[0082] As used herein, the term amine or amino is represented by the formula -NZ 1 Z 2 wherein Z 1 and Z 2 can each be a substituent group as described herein, such as hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl as described above.

[0083] The alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl molecules used herein may be substituted or unsubstituted. As used herein, the term "substituted" includes adding an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl to a position on the backbone attached to the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl, for example, a hydrogen is replaced by one of these molecules. Examples of substituents include, but are not limited to, hydroxyl, halogen (e.g., F, Br, Cl or I), and carboxyl. In contrast, as used herein, the term "unsubstituted" means that the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl has a full complement of hydrogens (i.e., hydrogens commensurate with its saturation level) without substitution, for example, straight-chain decane (-(CH 2 ) 9 -CH 3 ).

[0084] In some instances, Formula I is represented by Structure I-A:

[0085]

[0086] In Structure I-A, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , A 10 , and R 2 are as defined above for Formula I. In some instances of Structure I-A, each of A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , and A 10 is CR 1 , where each R 1 is independently selected from the groups as defined above for Formula I. For example, a compound of Structure I-A can be represented by Structure I-A1:

[0087]

[0088] In Structure I-A1, m and n are each independently 1, 2, 3, 4, or 5. In other words, the benzene ring of the molecule may include one to five R 1 groups. Each of the R 1 groups may independently be selected from the groups as defined above for Formula I.

[0089] In some instances of Structure I-A, A 1 、A 2 、A 3 、A 4 、A 5 、A 6 、A 7 、A 8 、A 9 and A 10 One or more of may be N. For example, the compound of Structure I-A may be represented by Structure I-A2, Structure I-A3, or Structure I-A4:

[0090]

[0091] 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 benzene ring of the molecule may include one to four R 1 groups. Each of the R 1 groups may independently be selected from the groups as defined above for Formula I.

[0092] 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 instances, R 2 is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0093] In some instances, Formula I is represented by Structure I-B:

[0094]

[0095] In Structure I-B, 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 are as defined above for Formula I. In some instances of Structure I-B, A1 、A 2 、A 3 、A 4 、A 5 、A 6 、A 7 、A 8 、A 9 and A 10 each of which is CR 1 where each R 1 is independently selected from the groups defined above for formula I. For example, a compound of structure I-B can be represented by structure I-B1:

[0096]

[0097] In structure I-B1, m and n are each independently 1, 2, 3, 4, or 5. In other words, the benzene rings of the molecule can each independently include one to five R 1 groups. Each of the R 1 groups can be independently selected from the groups defined above for formula I.

[0098] In some instances of structure I-B, one or more of 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 I-B can be represented by structure I-B2, structure I-B3, or structure I-B4:

[0099]

[0100] In structures I-B2, I-B3, and I-B4, m and n are each independently 1, 2, 3, or 4. In other words, the benzene rings of the molecule can each independently include one to four R 1 groups. Each of the R 1 groups can be independently selected from the groups defined above for formula I.

[0101] In some instances, formula I is represented by structure I-C:

[0102]

[0103] In structure I-C, A 1 、A 2 、A 3 、A 4 、A5 and A 6 and A 7 and A 8 and A 9 and A 10 as defined above for formula I. In some instances of structure I-C, A 1 and A 2 and A 3 and A 4 and A 5 and A 6 and A 7 and A 8 and A 9 and A 10 each of which is CR 1 , where each R 1 is independently selected from the groups defined above for formula I. For example, a compound of structure I-C can be represented by structure I-C1:

[0104]

[0105] In structure I-C1, m and n are each independently 1, 2, 3, or 4. In other words, the benzene rings of the molecule can each independently include one to four R 1 groups. Each of the R 1 groups can be independently selected from the groups defined above for formula I.

[0106] In some instances of structure I-C, one or more of A 1 and A 2 and A 3 and A 4 and A 5 and A 6 and A 7 and A 8 and A 9 and A 10 can be N. For example, a compound of structure I-C can be represented by structure I-C2, structure I-C3, or structure I-C4:

[0107]

[0108]

[0109] In structures I-C2, I-C3, and I-C4, m and n are each independently 1, 2, 3, or 4. In other words, the benzene rings of the molecule can each independently include one to four R 1 groups. Each of the R 1 groups can be independently selected from the groups defined above for formula I.

[0110] Examples of Formula I include the following compounds:

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] 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.

[0118] II. Methods for Preparing the Compounds

[0119] The compounds described herein can be prepared in a variety of ways. The compounds can be synthesized using various synthetic methods. At least some of these methods are known in the field of synthetic organic chemistry. The compounds described herein can be prepared from readily available starting materials. The optimal reaction conditions can vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art through routine optimization procedures.

[0120] Changes to Formula I include the addition, reduction, or movement of various components as described for each compound. Similarly, when one or more chiral centers are present in the molecule, all possible chiral variants are included. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection and 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 Edition, Wiley & Sons, 2014, which is incorporated herein by reference in its entirety.

[0121] The reactions for generating the compounds described herein can be carried out in a solvent, which can be selected by those skilled in the art of organic synthesis. Under the conditions (i.e., temperature and pressure) under which the reaction proceeds, the solvent can substantially not react with the starting materials (reactants), intermediates or products. 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 spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 1H-NMR or 13 13C-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.

[0122] Exemplary methods for synthesizing the compounds described herein are provided in Example 1 below and in International Patent Application Publication No. WO 2016 / 109470, which is incorporated herein by reference.

[0123] III. Pharmaceutical Formulations

[0124] The compounds or their derivatives described herein can be provided in the form of a pharmaceutical composition. Depending on the intended mode of administration, the pharmaceutical composition can be in the form of a solid, semi-solid or liquid dosage form (e.g., tablets, suppositories, pills, capsules, powders, liquids or suspensions), preferably in a unit dosage form suitable for precise dosing in a single administration. The composition will comprise a combination of a therapeutically effective amount of the compounds or their derivatives described herein with a pharmaceutically acceptable carrier, and in addition may also comprise other medicinal agents, pharmaceutical agents, carriers or diluents. By pharmaceutically acceptable is meant a substance that is not biologically or otherwise undesirable, which 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 of the pharmaceutical composition containing it.

[0125] As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer or other substances well known in the art for use in pharmaceutical formulations. The choice of carrier to be used in a composition will depend on the intended route of administration of the composition. The preparation of pharmaceutically acceptable carriers and formulations containing such substances is described, for example, in Remington′s Pharmaceutical Sciences, 21st Edition, University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia Pa, 2005. Examples of physiologically acceptable carriers include buffers such as phosphate buffers, citrate buffers and buffers with 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; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG) and PLURONICS TM (BASF; Florham Park, NJ).

[0126] Compositions suitable for parenteral injection containing the compounds or their derivatives described herein may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of 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 such as olive oil and injectable organic esters such as ethyl oleate. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0127] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Isotonic agents such as sugars, sodium chloride, etc. may also be included. Prolonged absorption of injectable drug forms can be achieved by the use of agents that delay absorption, such as aluminum monostearate and gelatin.

[0128] Solid dosage forms for oral administration of the compounds or their derivatives described herein include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds or their derivatives described herein are admixed with at least one inert conventional excipient (or carrier), such as sodium citrate or dibasic calcium phosphate, or (a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, such as glycerol, (d) disintegrating agents, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (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 glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents.

[0129] Similar types of solid compositions may also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0130] Solid dosage forms, such as tablets, dragees, capsules, pills, and granules, may be prepared with coatings and shells, such as enteric coatings and other coatings known in the art. They may contain opacifying agents and may also have compositions that release one or more active compounds in a delayed manner in a certain part of the intestine. Examples of embedding compositions that may be used are polymeric substances and waxes. The active compounds may also be in microencapsulated form, which may, if appropriate, have one or more of the above excipients.

[0131] Liquid dosage forms for oral administration of the compounds or their derivatives described herein include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of dehydrated sorbitol, or mixtures of these substances, etc.

[0132] In addition to such inert diluents, the compositions may also contain additional agents, such as wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, or fragrances.

[0133] In addition to the active compound, the suspension may also contain additional agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and tragacanth, or mixtures of these substances, etc.

[0134] The composition of the compound or its derivative described herein for rectal administration is optionally a suppository, which can be prepared by mixing the compound with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol or suppository wax, which are solid at room temperature but liquid at body temperature, and thus melt in the rectal or vaginal cavity and release the active component.

[0135] Dosage forms for topical administration of the compound or its derivative described herein include ointments, powders, sprays and inhalants. The compound or its derivative described herein is mixed with a physiologically acceptable carrier and any preservatives, buffers or propellants that may be required under sterile conditions. Ophthalmic preparations, ointments, powders and solutions are also included within the scope of the composition.

[0136] The composition may contain one or more of the compounds described herein and a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically acceptable salt refers to those salts of the compounds or their derivatives described herein that are suitable for use in contact with the tissues of a subject within the reasonable medical judgment, without undue toxicity, irritation, allergic reaction, etc., commensurate with a reasonable benefit / risk ratio, and effective for their intended use; and, where possible, the zwitterionic form of the compounds described herein. The term salt refers to 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 compound, or by reacting the purified free base form of the compound with a suitable organic or inorganic acid separately 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, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, methanesulfonate, glucoheptonate, lactobionate, methanesulfonate and laurylsulfonate, etc. These may include cations based on alkali metals and alkaline earth metals (such as sodium, lithium, potassium, calcium, magnesium, etc.), 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 content of the compositions taught therein.)

[0137] Administration of the compounds and compositions described herein, or pharmaceutically acceptable salts thereof, can be carried out with a therapeutically effective amount of the compounds and compositions described herein, or pharmaceutically acceptable salts thereof as described herein, for a period of time effective to treat the condition. The effective amount of the compounds and compositions described herein, or pharmaceutically acceptable salts thereof as described herein, can be determined by one of ordinary skill in the art and includes exemplary doses of the active compound of from about 0.5 to about 200 mg / kg body weight per day for a mammal, which can be administered as a single dose or in separate divided doses, for example, 1 to 4 times per day. Alternatively, the amount of the dose can be the active compound of from about 0.5 to about 150 mg / kg body weight per day, the active compound of from about 0.5 to 100 mg / kg body weight per day, the active compound of from about 0.5 to about 75 mg / kg body weight per day, the active compound of from about 0.5 to about 50 mg / kg body weight per day, the active compound of from about 0.01 to about 50 mg / kg body weight per day, the active compound of from about 0.05 to about 25 mg / kg body weight per day, the active compound of from about 0.1 to about 25 mg / kg body weight per day, the active compound of from about 0.5 to about 25 mg / kg body weight per day, the active compound of from about 1 to about 20 mg / kg body weight per day, the active compound of from about 1 to about 10 mg / kg body weight per day, the active compound of about 20 mg / kg body weight per day, the active compound of about 10 mg / kg body weight per day, the active compound of about 5 mg / kg body weight per day, the active compound of about 2.5 mg / kg body weight per day, the active compound of about 1.0 mg / kg body weight per day, or the active compound of about 0.5 mg / kg body weight per day, or any range derivable therefrom. Optionally, the amount of the dose is the active compound of from about 0.01 mg / kg to about 10 mg / kg body weight per day. Optionally, the amount of the dose is from about 0.01 mg / kg to about 5 mg / kg. Optionally, the amount of the dose is from about 0.01 mg / kg to about 2.5 mg / kg.

[0138] One of ordinary skill in the art will understand that the specific dosage level and frequency of administration for any particular subject can vary and will depend on a variety of factors, including the activity of the specific compound employed; the metabolic stability and duration of action of the compound; the species, age, body weight, general health, sex and diet of the subject; the mode and time of administration; the rate of excretion; drug combinations; and the severity of the particular disorder.

[0139] The exact dosage to be used in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be determined according to the judgment of the practicing physician and the circumstances of each subject. The effective dose can be extrapolated from the dose-response curve obtained from in vitro or animal model test systems. Further, depending on the route of administration, those skilled in the art will know how to determine the dose that results in a plasma concentration that produces the desired level of response in the cells, tissues, and / or organs of the subject.

[0140] IV. Methods of Use

[0141] Provided herein are methods of treating a myocardial infarction or other ischemic injury (e.g., stroke) in a subject. The methods include administering to the subject an effective amount of one or more of the compounds or compositions described herein or a pharmaceutically acceptable salt or prodrug thereof. An effective amount, when used to describe the amount of a compound in a method, refers to the amount of the compound that achieves the desired pharmacological or other biological effect.

[0142] Also encompassed are methods that include administering to a subject an amount of one or more of the compounds described herein such that an in vivo concentration corresponding to the concentration administered in vitro is achieved at the target cells of the subject.

[0143] Further described herein are methods of reducing the size of a myocardial infarction in a subject who has suffered a myocardial infarction. The methods include administering to the subject an effective amount of one or more of the compounds or compositions as described herein. The size of the myocardial infarction can be reduced by at least 5% compared to the size of the myocardial infarction in an untreated subject who has suffered a myocardial infarction (e.g., a subject who has suffered a myocardial infarction and has not received any treatment for the myocardial infarction or a subject who has suffered a myocardial infarction and has received a therapeutic agent different from the compounds or compositions described herein). Optionally, the size of the myocardial infarction 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% compared to the size of the myocardial infarction in an untreated subject who has suffered a myocardial infarction. The compounds and compositions described herein can also be used to prevent or reduce cardiomyocyte loss in a subject who has suffered a myocardial infarction. Methods of preventing or reducing cardiomyocyte loss in a subject who has suffered a myocardial infarction include administering to the subject an effective amount of one or more of the compounds or compositions as described herein.

[0144] The present invention further describes methods for improving cardiovascular function, improving cardiac vascular 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 methods comprise administering to the subject an effective amount of one or more compounds or compositions as described herein. Optionally, the subject has suffered an ischemic injury such as myocardial infarction or stroke. Optionally, the subject is an elderly individual, an obese individual, a diabetic individual, an individual suffering from metabolic syndrome, an individual exposed to smoke (e.g., a smoker and an individual chronically exposed to second-hand smoke), an individual with elevated blood pressure, blood cholesterol or triglyceride levels, or an individual suffering from an autoimmune condition.

[0145] Methods for treating myocardial infarction, reducing the size of myocardial infarction, preventing or alleviating cardiomyocyte loss, improving cardiovascular function, improving cardiac vascular 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 comprise administering to the subject one or more additional agents. The one or more additional agents and the compounds or pharmaceutically acceptable salts or prodrugs thereof as described herein may be administered in any order including concomitant administration, simultaneous administration or sequential administration. Sequential administration may be sequential administration at time intervals of up to several days apart. The methods may further comprise administering the one or more additional agents and / or the compounds or pharmaceutically acceptable salts or prodrugs thereof as described herein more than once. The one or more additional agents and the compounds or pharmaceutically acceptable salts or prodrugs thereof as described herein may be administered by the same or different routes and either in parallel or sequentially.

[0146] Additional therapeutic agents include, but are not limited to, antiplatelet agents, statins, β-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 that may be used as additional therapeutic agents as 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).

[0147] Illustrative non-limiting examples of statins that may be used as additional therapeutic agents as described herein include atorvastatin, cerivastatin, pravastatin, lovastatin, mevastatin, simvastatin, rosuvastatin, fluvastatin and pitavastatin.

[0148] Illustrative examples of beta blockers that can be used as additional therapeutic agents as 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.

[0149] Illustrative examples of renin-angiotensin-aldosterone system (RAAS) blockers (e.g., angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs)) that can be used as additional therapeutic agents as described herein include, but are not limited to, aliskiren, enalkiren, remikiren, benazepril, benazeprilat, captopril, enalapril, lisinopril, perindopril, quinapril, ramipril, trandolapril, fosinopril, moexipril, perindopril, losartan, valsartan, irbesartan, candesartan, telmisartan, tasosartan, eprosartan, spironolactone, and eplerenone.

[0150] Any of the foregoing therapeutic agents can be used in any combination with the compositions described herein. The combination can be administered concomitantly (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous catheters into the same subject), or sequentially (e.g., first administering one of the compounds or agents and then the second). Thus, the term combination is used to refer to the concomitant, simultaneous, or sequential administration of two or more agents.

[0151] Optionally, the compounds or therapeutic agents as described herein can be administered in combination with surgery (e.g., coronary artery bypass surgery), angioplasty, stent implantation, or another implantation procedure.

[0152] The methods and compounds as described herein can be used for prophylactic and therapeutic treatment. For prophylactic use, a therapeutically effective amount of the compounds and compositions as described herein or a pharmaceutically acceptable salt thereof is administered to a subject before the onset of myocardial infarction (e.g., before the signs of myocardial infarction are apparent), during the early onset of myocardial infarction (e.g., after the initial signs and symptoms of myocardial infarction appear), or after myocardial infarction has occurred. Prophylactic administration can be carried out for days to years before the manifestation of myocardial infarction symptoms. Therapeutic treatment involves administering a therapeutically effective amount of the compounds and compositions as described herein or a pharmaceutically acceptable salt thereof to a subject after myocardial infarction has occurred.

[0153] The methods for prophylactic and therapeutic treatment optionally include selecting a subject who has suffered an ischemic injury (such as myocardial infarction or stroke) or is at high risk of suffering from said ischemic injury (such as myocardial infarction or stroke) (e.g., an obese individual, an elderly individual, or an individual who has previously suffered an ischemic injury such as myocardial infarction or stroke). A person skilled in the art can make such a determination using a variety of prognostic and diagnostic methods including, for example, personal or family history of a disease or condition, clinical tests (e.g., genetic tests), and the like. Optionally, the methods herein can be used to prevent a subject who has suffered a myocardial infarction from suffering a subsequent myocardial infarction.

[0154] The compounds and compositions described herein or pharmaceutically acceptable salts thereof can be used to treat myocardial infarction, reduce the size of myocardial infarction, prevent or mitigate myocardial cell loss, improve cardiovascular function, promote wound healing, and / or prevent or treat hypertrophic cardiomyopathy in humans (including but not limited to pediatric and elderly populations) and animals (e.g., veterinary applications).

[0155] V. Kits

[0156] The present invention also provides kits for treating myocardial infarction, reducing the size of myocardial infarction, preventing or mitigating myocardial cell loss, improving cardiovascular function, promoting wound healing, and / or preventing or treating hypertrophic cardiomyopathy in a subject. The kit can include any compound or composition described herein. For example, the kit can include one or more compounds of formula I. The kit can further include one or more additional agents such as antiplatelet agents, statins, β-blockers, renin-angiotensin-aldosterone system (RAAS) blockers (e.g., angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs)), and combinations thereof.

[0157] The kit can include an oral formulation of any compound or composition described herein. The kit can include an intravenous or intraperitoneal formulation of any compound or composition described herein. The kit can additionally include instructions for use of the kit (e.g., instructions for treating a subject), a container, a device for administering the compound or composition (e.g., a syringe), and / or a carrier.

[0158] As used herein, the terms treatment / treat / treating refer to a method of alleviating one or more symptoms of a disease or medical 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 medical condition. For example, if one or more symptoms or signs of a disease in a subject (e.g., the size of a myocardial infarction) are reduced by 5% compared to a control, the method of treating the disease is considered a treatment. As used herein, a control refers to an untreated medical condition (e.g., the size of a myocardial infarction in an untreated subject suffering from a myocardial infarction). Thus, a reduction can be a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 5% and 100% compared to a native level or a control level. It is understood that treatment does not necessarily mean curing or completely eliminating a disease, medical condition, or the symptoms of a disease or medical condition.

[0159] As used herein, the terms prevent / preventing / prevention of a disease or medical condition refer to an action, such as the administration of a composition or therapeutic agent, that inhibits or delays the onset or severity of one or more symptoms of a disease or medical condition, for example, before or almost simultaneously with a subject starting to exhibit one or more symptoms of the disease or medical condition.

[0160] As used herein, references to a reduction, alleviation, or inhibition include a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater change compared to a control level. Such terms can include, but do not necessarily include, complete elimination.

[0161] As used herein, the term subject refers to both mammals and non-mammals. Mammals include, for example, humans; non-human primates, such as apes and monkeys; cattle; horses; sheep; rats; mice; pigs; and goats. Non-mammals include, for example, fish and birds.

[0162] Throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference in their entireties into this application.

[0163] 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.

[0164] Examples

[0165] Example 1: Synthesis of a Compound

[0166] All chemicals used for synthesis were purchased from Alfa Aesar (Ward Hill, MA) or Aldrich (Milwaukee, WI). The compounds were characterized by 1 1H NMR on a Varian (Palo Alto, CA) 400-MR spectrometer. The purity of the synthesized compounds was determined by Shimadzu Prominence HPLC with a Zorbax C18 (or C8) column (4.6 x 250 mm) and UV monitoring at 254 nm. The reported compound purity was found to be > 95%.

[0167] Synthesis of 1-cyclopropylpiperidin-4-one:

[0168]

[0169] Cyclopropylamine (6.9 mL, 100 mmol) and ethyl acrylate (22.3 mL, 210 mmol, 2.1 equiv) were dissolved in anhydrous 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, hexane to ethyl acetate from 10:1 to 1:1) to afford diethyl 3,3′-(cyclopropylazanediyl)dipropionate as a colorless liquid (15.68 g, 61%).

[0170] Sodium hydride (60% dispersion in oil, 3.0 g, 75 mmol, 1.5 equiv) and tetrahydrofuran (THF, 30 mL) were placed in a dried flask, and a solution of diethyl 3,3′-(cyclopropylazanediyl)dipropionate (12.8 g, 50 mmol) in THF (20 mL) was added dropwise thereto. Then anhydrous ethanol (2.9 mL, 50 mmol, 1.0 equiv) was added, and the resulting mixture was stirred at reflux for 24 h. The reaction was quenched with saturated ammonium chloride (50 mL). The mixture was extracted with ether (3 x 100 mL), and the combined organic layers were washed with water and brine and dried over Na 2 SO 4 4. The volatiles were removed in vacuo to give a crude oil, which was purified by column chromatography (silica gel, hexane to ethyl acetate from 10:1 to 2:1) to afford 1-cyclopropylpiperidin-4-one as a colorless liquid (4.52 g, 65% yield). 1 1H NMR (400 MHz, CDCl 3 3): δ 2.92 (t, J = 6.2 Hz, 4H), 2.42 (t, J = 6.2 Hz, 4H), 1.80 - 1.70 (m, 1H), and 0.55 - 0.47 (m, 4H).

[0171] Synthesis of 1-isopentylpiperidin-4-one:

[0172]

[0173] 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 h. The mixture was cooled to room temperature, extracted with ether (3 × 100 mL), and the combined organic layers were washed with water and brine and dried over Na 2 SO 4 . Volatiles were removed in vacuo to give a crude oil, which was purified by column chromatography (silica gel, hexane / ethyl acetate from 10:1 to 2:1) to give 1-isopentylpiperidin-4-one as a colorless liquid (10.32 g, 61% yield). 1 H NMR (400 MHz, CDCl 3 3): 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).

[0174] General method for the synthesis of α,β-unsaturated ketones:

[0175]

[0176] 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 thereto. 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 Na 2 SO 4 . Volatiles were removed in vacuo to give a crude oil, which was purified by column chromatography (silica gel, hexane / ethyl acetate from 5:1 to 1:1) to give 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. Characterization of each of these compounds is provided below.

[0177] (3E,5E)-1-Cyclopropyl-3,5-bis(3-methoxybenzylidene)piperidin-4-one (9-2).

[0178]

[0179] Yellow powder. 1 H NMR (400 MHz, CDCl 3 ): δ 7.75 (s, 2H), 7.36 (t, J = 8.0 Hz, 2H), 7.03 (d, J = 8.0 Hz, 2H), 6.96 (s, 2H), 6.93 (d, J = 8.0 Hz, 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); 13 C NMR (100 MHz, CDCl 3 ): 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.

[0180] (3E,5E)-1-Cyclopropyl-3,5-bis(pyridin-3-ylmethylene)piperidin-4-one (9 - 8).

[0181]

[0182] Orange powder. 1 H NMR (400 MHz, CDCl 3 ): δ 8.67 (s, 2H), 8.59 (d, J = 4.8 Hz, 2H), 7.73 (s, 2H), 7.70 (d, J = 8.0 Hz, 2H), 7.37 (dd, J = 8.0, 4.8 Hz, 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 3 ): 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.

[0183] (3E5E)-1-Isopentyl-3,5-bis(2-methoxybenzylidene)piperidin-4-one (10 - 1).

[0184]

[0185] Compound 10-1 was prepared as the hydrochloride salt (yellow powder). 1 H NMR(400MHz, CDCl 3 ): 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(septet, J = 6.6Hz, 1H), 1.30(td, J = 7.6, 6.6Hz, 2H) and 0.70(d, J = 6.6Hz, 6H); 13 C NMR(100MHz, CDCl 3 ): 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。

[0186] (3E,5E)-1-isopentyl-3,5-bis(3-methoxybenzylidene)piperidin-4-one (10-2).

[0187]

[0188] Compound 10-2 was prepared as the hydrochloride salt (yellow powder). 1 H NMR(400MHz, CDCl 3 ): δ13.46(br, 1H), 8.17(s, 2H), 7.40(t, J = 8.0Hz, 2H), 7.01(dd, J = 8.0, 2.3Hz, 2H), 6.89(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 = 6.6Hz, 6H); 13 C NMR(100MHz, CDCl 3):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.

[0189] Example 2: Cardioprotection promotion and repair after myocardial infarction

[0190] The data herein show that administration of the compounds described herein after myocardial infarction promotes cardioprotection and repair. Among other functions, representative compounds also prevent infarct size enlargement, cardiac hypertrophy, and collagen deposition. The compounds significantly improve cardiac function after infarction. The compounds also increase angiogenesis, promote cardiac β-oxidation, and significantly reduce the level of the metabolite methylglutaryl carnitine associated with dilated cardiomyopathy.

[0191] Methods

[0192] All animal studies and protocols were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine and were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult (8 - 10 weeks old) ICR (CD1) mice were used for all studies.

[0193] Adult mouse heart failure model. To induce myocardial infarction (MI) in 8 - 10 week old mice, the left anterior descending (LAD) artery was permanently ligated. Briefly, mice were anesthetized with 2% isoflurane and then intubated. Thoracotomy was performed by passing through the fourth or fifth intercostal space to expose the heart, and an 8 - 0 nylon suture was tied around the LAD artery. 10 ul of a titer of 9X10 9Adenovirus SRC-3 (Ad-SRC-3) or adenovirus GFP (Ad-GFP) at pfu / ml was injected into the anterior free wall near the left ventricular descending coronary artery. To assess cell cycle entry, during the 9-day duration of the experiment, the analog 5-ethynyl-2′-deoxyuridine (EdU; 0.2 g / L, Santa Cruz, SC-284628A) was added to the drinking water. Cell proliferation was measured using the Click-iT EdU kit (Invitrogen, C10339). The initial dose of MCB-613 was administered intraperitoneally at 20 mg / kg 2 hours after surgery. Subsequently, the same dose of injection was given on another 6 days, and then repeated doses were given for 3 days at 9 weeks and 16 weeks. Mice were harvested at the designated time points for analysis.

[0194] Echocardiography. Cardiac function was determined by echocardiography (VisualSonics, Vevo 2100, 40Mhz - 550S probe). After alignment with the papillary muscle in the transverse B-mode, cardiac function was measured on the M-mode image.

[0195] Histological analysis. The whole heart was fixed with 10% formalin, embedded in paraffin, and sectioned at 7-μm intervals. Each slide had 3 or 10 sections (about 30 - 50 slides) starting from the apex and terminating at the suture ligation site. Sections at the papillary level (slides 20 - 30) were stained with Sirius red to identify fibrotic areas. The infarct size was determined using a length-based approach.

[0196] Immunostaining analysis. Immunohistochemical and immunofluorescence staining experiments were performed on FFPE (formalin-fixed and paraffin-embedded sections). The DeadEndT M Fluorescent assay TUNEL system manufacturer's protocol (DeadEndT M Fluorometric TUNEL System manufacturer’s protocol) (Promega, GS3250) was used for TUNEL staining to detect apoptotic cells.

[0197] Isolation of cardiac cells for single-cell transcriptional profiling. Mice were placed under anesthesia at the surgical plane before cervical dislocation. The heart was removed and digested with calcium-free pH 7.4 Tyrodes solution containing 1 mg / mL collagenase (130 mM NaCl, 74.55 mM KCl, 0.5 mM MgCl, 0.33 mM NaH 2 PO 4, 15-minute Langendorff retrograde perfusion of isolated cells with 0.25 mM HEPES and 22 mM glucose. The heart was then removed from the apparatus and minced finely in the same Tyrode's buffer containing 15 mg / mL bovine serum albumin (BSA), and then ground with a glass pipette. The cardiomyocytes were then precipitated by differential centrifugation at 300 RPM for 3 minutes. The supernatant containing the non-cardiomyocyte population with cells was filtered through a 70-micron filter and pelleted at 750 G, and then resuspended in 1.1 mL of phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS). 0.1 mL was taken as the "unstained control" for fluorescence-activated cell sorting (FACS). The additional 1 mL was incubated with 4 μg / mL calcein blue and 10 μM DyeCycle Ruby and incubated at 37 °C for 10 minutes. The cells were then spun down at 600 G 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 for: Sytox Green-, Calcein+, DyeCycle Ruby+ into PBS containing 0.4% FBS. The cells were then pelleted and the cells were resuspended in 100 μL of PBS containing 0.4% FBS, the cells were counted, and then the cells were flowed through a 10x Genomics chromium system for single-cell transcriptional profiling.

[0198] Metabolomic profiling. Hearts were isolated from 4 control and 4 MCB-613-treated mice 24 hours after MI, perfused with 10 mM KCl, snap-frozen in liquid nitrogen, and stored at -80 °C. Fatty acids and carnitines in 10 mg of tissue were analyzed and normalized relative to 3 normal liver control samples. Fatty acids were normalized with the internal standard L-tryptophan, and carnitines were normalized with the internal standard L-zeatin.

[0199] RER, VO 2 and VCO 2 Measurement. Respiratory exchange ratio (RER), oxygen consumption (VO 2 ), and carbon dioxide exhalation (VCO 2 ) were measured by indirect calorimetry using a progressive maximal exercise test until the mice were exhausted.

[0200] . Results

[0201] Activation of SRC-3 has low toxicity. Nuclear receptor coactivator 3 (NCOA3) is expressed at low levels in the adult heart, suggesting that activation of SRC-3 will have few side effects. In addition, MCB-613 has a low in vitro and in vivo toxicity profile. Determine NCOA3 expression in normal human hearts and compare it to muscle tissue analyzed from the GTEx database from approximately 1,000 autopsy donors. Figure 1 Contains a graph showing the expression of NCOA3 in normal human hearts (left panel) and muscle tissue (right panel). The y-axis shows fragments per kilobase of gene length per million mapped reads (FPKM), and the data points represent samples. The data are categorized by the expression level of NCOA3.

[0202] SRC-3 expression induces proliferation of non-myocardial cells in the heart. Adenovirus-derived SRC-3 (Adeno-SRC3) was injected into the left ventricular free wall of the hearts of wild-type adult mice. Mice were fed water treated with 5-ethynyl-2'-deoxyuridine (EdU water) as a cell proliferation marker. Hearts were harvested nine days later and stained with 4',6-diamidino-2-phenylindole (Dapi) to identify cell nuclei, the PCM1 cardiomyocyte marker, the cell surface marker wheat germ agglutinin (WGA), and the cell proliferation marker EdU. Figure 2 Is an image of the heart and shows that SRC-3 expression induces proliferation of non-myocardial cells in the hearts of wild-type adult mice.

[0203] The compounds described herein prevent the early and progressive loss of cardiac function after myocardial infarction. Mice were treated with a representative compound described herein after myocardial infarction. Figure 3A Shows the experimental timeline for drug treatment and echocardiogram measurements after myocardial infarction. MCB-613 or saline was administered two hours after ligation and every 24 hours for an additional six days. At 8 weeks and 16 weeks, three repeated injections were given weekly. As Figure 3B shown, heart weight and tibia length were measured at 10 weeks. Six mice were administered saline and seven mice were administered MCB-613. P < 0.03. Left ventricular ejection fraction was determined by echocardiogram (n = 14 before 12 weeks; n = 3 at 19 weeks) ( Figure 3C ). Data were analyzed by ANOVA with repeated measures and expressed as mean + / - SEM. P < 0.001. Figure 3D Contains an image of a mouse heart harvested after myocardial infarction and stained to visualize collagen fibers. Specifically, Figure 3DSirius red staining of a 4x magnified cross-section of the papillary muscle at 10 weeks after myocardial infarction, and corresponding Sirius red staining of a 20x magnified cross-section of the infarct border zone. Infarct size is expressed as % length. Data show that the infarct size was reduced in mice treated with MCB-613 compared to control mice treated with saline. The left ventricular ejection fraction of mice treated with compound 10-1 was determined by echocardiography after myocardial infarction (see Figure 4 ). Figure 8 Sirius red staining of a cross-section of the papillary muscle at 6 weeks after myocardial infarction is shown. Infarct size is expressed as % length. Data show that the infarct size was reduced in mice treated with compound 10-1 compared to control mice treated with saline.

[0204] MCB-613 induces changes in major and minor non-myocyte cell types in the heart after myocardial infarction. Comprehensive single-cell transcriptional profiling of non-muscle cells in the adult mouse heart was performed. Single-cell sequencing of non-myocardial cells in the heart was performed at 10 weeks after myocardial infarction. Cell clusters were generated by tSNE analysis and identified by gene expression markers. See Figure 5A , which depicts comprehensive single-cell transcriptional profiling of non-muscle cells in the adult mouse heart. Figure 5B Shows different cell types present in the heart treated with MCB-613 after myocardial infarction. Figure 5C Is a Venn analysis of three cell clusters with endothelial markers, which shows that MCB-613 stimulates the growth of endothelial cells in two different endothelial cell populations in the heart at 10 weeks after myocardial infarction.

[0205] MCB-613 increases cardioprotective carnitine metabolites. Figure 6A Is a heatmap showing metabolomics of long-chain fatty acids in the mouse heart at 24 hours after myocardial infarction. Figure 6B Is a heatmap showing metabolomics of methylglutaryl carnitine in the mouse heart after myocardial infarction (whole group FDR = 1). The heatmap shows that MCB-613 increases cardioprotective carnitine metabolites, increases β-oxidation of long-chain fatty acids, and decreases methylglutaryl carnitine.

[0206] 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 DNA-binding domain of Gal4 (pBIND-SRC-1, pBIND-SRC-2, or pBIND-SRC-3) were exposed to treatment with compounds including MCB-613, compound 10-1, and compound 10-2 as described herein. Figure 7 The upper figure shows that MCB-613 selectively stimulates the intrinsic transcriptional activity of SRC.Figure 7 The middle figure shows that compound 10-1 selectively stimulates the intrinsic transcriptional activity of SRC. Figure 7 The following figure shows that compound 10-2 selectively stimulates the intrinsic transcriptional activity of SRC.

[0207] Compound 10-1 improves cardiovascular adaptability after myocardial infarction. Figure 9 A graph showing the results of a progressive maximal exercise test conducted in mice treated with saline ("saline"; n = 3), mice treated with MCB-613 ("MCB-613"; n = 3), and non-infarcted wild-type mice ("WT"; n = 2). The upper figure shows the volume of carbon dioxide exhaled (VCO 2 ), while the lower figure shows the volume of oxygen consumed (VO 2 ). As Figure 9 shown by representative compound 10-1, the compounds described herein improve cardiovascular and peripheral vascular adaptability after myocardial infarction.

[0208] Summary

[0209] As shown by the data provided herein, the compounds described herein stimulate angiogenesis in vivo and improve the function of damaged myocardium. Thus, the compounds described herein are excellent therapeutic agents that can be used to repair and prevent chronic wounds, promote angiogenesis, restore blood flow in vascular diseases, and inhibit the harmful structural remodeling of vulnerable myocardium by preventing metabolic remodeling. The compounds can be used for cardiac repair after coronary artery insufficiency.

[0210] Example 3: Promotion of Cardioprotection and Repair after Myocardial Infarction Using Steroid Receptor Coactivator Stimulators

[0211] Progressive remodeling of heart tissue with cardiomyocyte loss, inflammation, fibrosis, and reduced cardiac ejection fraction is a hallmark of myocardial infarction (MI)-induced heart failure. The key therapeutic goals after MI are to protect the myocardium, minimize infarct size, prevent progression to heart failure, and support functional recovery. The data herein show that small molecule stimulators of the steroid receptor coactivators described herein promote new blood vessel growth and improve heart function after MI. As shown by 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 heart function. The compounds described herein represent novel therapeutic options for preventing the early and progressive loss of heart function after MI.

[0212] Methods

[0213] Animals. All animal studies and protocols were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine and were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult (8 - 10 weeks old) ICR (CD1) mice were used for all studies.

[0214] Angiogenesis assay with small molecule stimulant treatment. Fertilized chicken eggs (White Leghorn) of specific pathogen - free (SPF) status at 5 - 7 days old were used for accessing the chorioallantoic membrane (CAM). The total vascular area on the CAM was measured using images obtained before drug application. MCB - 613 (100 μL) at a concentration of 0.6 μM was topically applied to the surface of the CAM. After drug application every day, the vascular area on the CAM was monitored daily. Vehicle control was maintained throughout the experiment. The treatment lasted for four days, at the end of which the total vascular area on the CAM was quantified using the image thresholding method (ImageJ). The percentage increase in vascular area of control and treated eggs was compared. n = 6 eggs for each condition. This experiment was repeated three times.

[0215] Angiogenesis assay with drug - treated mouse embryonic fibroblasts (MEFs). Fertilized chicken eggs (White Leghorn) of SPF status at 5 - 7 days old were used for accessing the chorioallantoic membrane (CAM). Mouse embryonic fibroblasts (MEFs) were treated with 0.6 μM MCB - 613 for 24 hours. After treatment, two million MEFs were suspended in 60 μL of PBS containing magnesium and calcium and 40 μL of Matrigel (2M / egg) (Coming Inc.; Coming, NY). Then the MEFs were thoroughly mixed by pipetting and transferred to the surface of the CAM. The vascular area on the CAM was monitored daily. Vehicle control was maintained throughout the experiment. The MEFs were allowed to grow on the CAM surface for four days, at the end of which the total vascular area on the CAM was quantified using the image thresholding method (ImageJ). Then the percentage increase in vascular area of control and treated eggs (MEF - treated vs. untreated) was compared. n = 6 eggs for each condition.

[0216] Reporter gene assay. Cardiac fibroblasts were plated in six-well plates and transfected with a GAL4-responsive luciferase reporter gene (pG5-luc) and an expression vector for the full-length SRC-1, SRC-2, or SRC-3 fusion construct (pBIND-SRC-1, pBIND-SRC-2, or pBIND-SRC-3) for the GAL4 DNA-binding domain (GAL4-DBD) or a pBIND control using Lipofectamine 3000 (Invitrogen; Carlsbad, CA). Twenty-four (24) hours after transfection, the cells were treated with 6 μM MCB-613 or dimethyl sulfoxide (DMSO) and incubated overnight. The 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 the Bradford assay (Bio-Rad Laboratories; Hercules, CA). Relative light units were measured and normalized relative to the total protein concentration.

[0217] Heart failure model in adult mice. To induce MI in 8- to 10-week-old mice, the left anterior descending (LAD) artery was permanently ligated. Briefly, mice were anesthetized with 2% isoflurane and then intubated. Thoracotomy was performed by passing through the fourth or fifth intercostal space to expose the heart, and an 8-0 nylon suture was tied around the LAD. An initial dose of MCB-613 at 20 mg / kg was administered intraperitoneally two hours after surgery. Subsequently, injections were given at the same time of day and at the same dose for six more days, and then repeated doses were given at 9 weeks and 16 weeks for three days. Mice were harvested at the designated time points for analysis.

[0218] Echocardiography. Cardiac function was determined by echocardiography (VisualSonics, Vevo 2100, 40Mhz-550S probe). After alignment with the papillary muscle in the transverse B mode, cardiac function was measured on M-mode images. Figure 11A Animal numbers for cardiac function were control on day 0 (17); MCB-613 (15), control on day 1 (10); MCB-613 (12), vehicle on day 14 (19); control (19), control on day 56 (12); MCB-613 (15), control on day 70 (8); MCB-613 (10), control on day 80 (8); MCB-613 (11), control on day 133 (3); and MCB-613 (3).

[0219] Histological analysis. The entire heart was fixed in 10% formalin, embedded in paraffin, and sectioned at 7 μm intervals. Each slide had three (3) to ten (10) sections (approximately 30 - 50 slides) starting at the apex and terminating at the site of suture ligation. Sections at the papillary level (slides 20 - 30) were stained with Sirius red to identify fibrotic areas. Infarct size was determined using a length-based approach. TUNEL staining was performed using the DeadEnd TM Fluorescent Assay TUNEL System (Promega, GS3250) to detect apoptotic cells.

[0220] Electron microscopy. Animals were sacrificed, the hearts were quickly removed, and placed directly in cold primary fixative (0.1 M cacodylate buffer containing 2% paraformaldehyde + 2.5% glutaraldehyde + 2 mM CaCl 2 2 at pH 7.4), where they were sectioned cross-sectionally and then stored in the cold primary fixative for four days. After fixation, the tissues were stained with 0.1 M cacodylate buffer containing 0.1% tannic acid, rinsed, and stained with osmium tetroxide for one hour, after which the tissues were rinsed in dH 2 2O and counterstained in uranyl acetate aqueous solution. The tissues were rinsed again in dH 2 2O, then dehydrated in a series of graded ethanols (50%, 70%, 80%, 90%, 95%, and 100%). The tissues were slowly infiltrated over four days with dilutions of ethanol in increasing plastic resin until 100% plastic was reached. After infiltrating in 100% plastic three times for one day, the tissues were embedded in freshly made Spurr low-viscosity resin and polymerized overnight at 60 °C. Using a Leica UC7 ultramicrotome, ultra-thin sections of 55 - 65 nm were cut with a Diatome Ultra 45 diamond knife. The sections were collected on 150 mesh copper grids and observed on a Hitachi H7500 transmission electron microscope. Images were taken using an AMT XR-16 digital camera and AMT ImageCapture, v602.600.51 software.

[0221] Isolation of cardiac cells. Mice were placed under anesthesia at the surgical plane before cervical dislocation. The hearts were removed and passed through a calcium-free pH 7.4 Tyrodes solution (130 mM NaCl, 74.55 mM KCl, 0.5 mM MgCl, 0.33 mM NaH 2 2PO 4, 15-minute Langendorff retrograde perfusion of isolated cells with 0.25 mM HEPES and 22 mM glucose. The heart was then removed from the apparatus and minced finely in the same Tyrode's buffer containing 15 mg / mL BSA, and then ground with a glass pipette. The cardiomyocytes were then precipitated by differential centrifugation at 300 RPM for 3 minutes. The supernatant containing the non-cardiomyocyte population with cells was filtered through a 70-μm filter and pelleted at 750 g, and then resuspended in 1.1 mL of phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS). Then, 0.1 mL of the mixture was taken as the "unstained control" for fluorescence-activated cell sorting (FACS). The additional 1 mL of the mixture was incubated with 4 μg / mL calcein blue and 10 μM DyeCycle Ruby and incubated at 37 °C for 10 minutes. The cells were then spun down at 600 g 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 for: Sytox Green−, Calcein+, DyeCycle Ruby+ into PBS containing 0.4% FBS. The cells were then pelleted and resuspended in 100 μL of PBS containing 0.4% FBS, the cells were counted and passed through the 10x Genomics Chromium system (10x Genomics Chromium system).

[0222] Single-cell RNA sequencing. The raw fastq files were imported into Cell Ranger 2.1.1 (10X Genomics) for STAR alignment, data filtering, barcode counting, and UMI counting. To identify cell clusters and differentially expressed genes, the Cell Ranger results were analyzed using the Seurat suite version 3.0.0 implemented in R (version 3.4.3). As a quality control measure, cells expressing <200 or >5,000 unique genes or cells with >25% of reads mapping to mitochondria were removed. The 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. Genes differentially expressed 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 that utilized the hypergeometric distribution to identify enriched pathways (P value < 0.05).

[0223] To study the effect of treatment on intercellular communication in infarcted hearts, curated and putative ligand-receptor pairs in humans were obtained. For each cell type, drug-treated markers were obtained by applying filters with a P-value < 0.05 and log2FC > 0.25 or < -0.25. Intercellular communication was established by linking cell type A and B, where the ligand was differentially expressed in cell type A and the receptor was differentially expressed in cell type B. Network diagrams were drawn using the igraph R package.

[0224] Total RNA-Seq analysis. Sequencing reads were trimmed using the trimGalore software. Next, HISAT was used to build UCSC mml0 mapped reads against the human genome, and StringTie was used for quantification against the Gencode gene model. Gene expression (FPKM) was quantile-normalized using the R statistical system. Genes differentially expressed 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; the adjusted q-value reached significance (q < 0.25). Heatmaps were generated using the Matplotlib, NumPy, and SciPy libraries under python.

[0225] RNA isolation and qPCR. Total RNA was isolated from cells using the Qiagen RNA isolation kit. cDNA was prepared using the VILO master mix reagent. qPCR analysis was performed using a Taqman kit with primers for Tlr7, Lcn2, and 18s.

[0226] Granulocyte isolation. Bone marrow cells were isolated from the hind legs of mice treated with control or MCB-613 for 24 hours. The hind legs were removed and placed in ice-cold Hanks balanced salt solution (HBSS) (without Ca / Mg) supplemented with 2% FBS. The ends of the bone were cut, and the bone marrow was flushed with ice-cold HBSS supplemented with 2% FBS using a 26G needle. The clumps were broken up using an 18G needle and filtered through a 70 μm filter, and centrifuged at 400 g for 10 minutes at 4°C. The pellet was resuspended in RBC lysis buffer (BD Biosciences Pharmigen; San Diego, CA) and incubated at room temperature for two minutes. HBSS buffer (8 mL) was added, and the mixture was centrifuged 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).

[0227] Flow cytometry and cardiac immunophenotyping. Hearts and spleens isolated from control and MCB-613-treated mice 24 hours after MI or sham surgery were digested in digestion buffer: 500 μL DNase I (10 mg / ml), 500 μl Collagenase II (50 mg / ml) in 4 ml (sufficient for approximately 12 spleens @ 1x) RPMI 1640. Cells were placed on a GentleMacs dissociator, run twice on "IMPC_step2", and incubated at 25 °C for 15 minutes. The "IMPC_step2" procedure was repeated, and the samples were incubated at 25 °C for another 15 minutes, followed by another round of the "IMPC_step2" procedure. Then, 400 μL of 4 °C termination buffer (1x PBS, 0.1 M EDTA) was added to each sample, and the samples were centrifuged at ~100 g for one second to collect the liquid at the bottom of the tube. Samples were filtered through a mesh filter cap into 50 mL conical tubes. The tubes were then washed with 1 mL of FAC buffer, and the buffer was also passed through the filter. The heart preparation was more viscous and was washed with 20 mL of cold filtered saline. Samples were centrifuged at 500 g for 6 minutes. The supernatant was discarded, and the pellet was resuspended in 1 mL of 4 °C FACS buffer. After red blood cell (RBC) lysis and blocking, the single cell suspension was stained with an immunocytometry panel and quantified using an LSR II flow cytometer. Then, 500,000 live events were counted for spleen controls, and cardiac cells from the entire tube were recorded and analyzed.

[0228] Western blot. Frozen whole hearts were pulverized using a mortar and pestle device. 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 4 °C rotator platform for one hour, followed by centrifugation at 12,000 g for 10 minutes to clear debris. Supernatants were collected and stored at -80 °C for future use. Protein concentration was determined using a bicinchoninic acid assay (BCA) reagent system. For cell lysates, cells were lysed and total protein was isolated using NETN buffer containing 10% glycerol. All lysis buffers were supplemented with protease and phosphatase inhibitors. Tissue lysate protein (30 - 50 μg) or cell lysate protein (50 - 70 μg) was loaded onto a 4 - 15% gradient gel (Bio-Rad) and transferred to a polyvinylidene difluoride (PVDF) membrane. Immunoblot assays were 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. Pierce ECL was used for chemiluminescent detection.

[0229] Tube formation assay. Cardiac fibroblasts were treated with DMSO or 6 μM MCB-613. Twenty-four hours after treatment, the drugs were washed away by rinsing the cells twice with PBS. The cells were then conditioned with endothelial cell growth medium for 24 hours. After conditioning, the cells were plated on growth factor-reduced Matrigel (10 mg / ml) to allow tube formation overnight. The next day, the tubes were stained with calcein AM and imaged using a Cytation imaging system.

[0230] Immunostaining. The heart was perfused with cardioplegic 20 mM KCL-PBS and then with 10% neutral buffered formalin, followed by drip fixation and then processed into paraffin. Sections (7 μm) were then cut and placed on slides. Immunofluorescence was performed by first removing the paraffin and then rehydrating the sections. Thereafter, antigen retrieval was performed (antigen unmasking solution, Tris-based, Vector Labs catalog number H-3301; Vector Labs, Burlingame, CA). The sections were permeabilized with 0.1% tween20-PBS, blocked with 1% tween20-PBS containing 10% donkey serum, and then incubated with a primary antibody (1:200 rabbit anti-lysozyme, abcam catalog number AB108508; Abcam, Cambridge, UK) in the blocking solution, followed by a secondary antibody (1:200 donkey anti-rabbit, Alexa 647, Thermo Fisher Scientific catalog number 31573; Thermo Fisher Scientific, Waltham, MA), then with rhodamine-conjugated WGA (1:250 Vector Labs catalog number RL-1022) and DAPI (1:500 Thermo Fisher Scientific catalog number 62248). Images were taken on a Zeiss LSM780 confocal microscope. LYZ+ cells were manually counted from random images of the entire myocardium spanning the left ventricle below the left anterior descending coronary artery occlusion surgery. n = 3 hearts / group, >10 mm 2 Imaging / heart, 24 hours after MI surgery.

[0231] RER, VO 2 and VCO 2 Measurement. RER, VO 2 and VCO 2 were measured by indirect calorimetry using a progressive maximal exercise test until the mice reached exhaustion.

[0232] Results

[0233] MCB-613 stimulates angiogenesis. The effects of MCB-613 on angiogenesis and the stromal response were investigated, particularly in adult cardiac fibroblasts. SRC-1, 2, and 3 proteins were expressed in adult cardiac fibroblasts isolated from 10-week-old mice ( Figure 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 gene to measure SRC activation after treatment with MCB-613 ( Figure 10B ). In response to MCB-613, SRC-3 activity was induced to a greater extent than SRC-1 and SRC-2, indicating that MCB-613 preferentially stimulates SRC-3 activity in cardiac fibroblasts. Functionally, MCB-613 stimulated tube formation in adult cardiac fibroblasts in vitro ( Figure 10C ). To investigate the stimulation of angiogenesis by MCB-613 in vivo, a chicken egg angiogenesis assay was performed ( Figure 10D ). Direct application of MCB-613 to chicken eggs stimulated angiogenesis in vivo. Additionally, the introduction of mouse embryonic fibroblasts pre-stimulated with MCB-613 had the potential to promote robust angiogenesis in a cell-non-autonomous manner. Without being bound by theory, these findings suggest that the angiogenic stimulation by MCB-613 can occur through multiple mechanisms.

[0234] To determine whether MCB-613 improves recovery after ischemia-induced myocardial injury, mice were administered MCB-613 or vehicle control two hours after myocardial injury induced by permanent surgical ligation of the left anterior descending coronary artery. Three days after MI, increased angiogenesis was observed in the infarct border zone, indicating that MCB-613 promotes angiogenesis in injured tissue and restores blood flow in a vascular disease setting ( Figure 10E ).

[0235] MCB-613 prevents loss of cardiac function after MI. Surgical ligation of the left anterior carotid artery in mice is a commonly used preclinical MI model to test cardiovascular therapeutic interventions. To explore the role of SRC stimulation on early and late post-infarct cardiac function and remodeling, mice undergoing MI were treated with MCB-613 or vehicle control. Mice were administered 20 mg / kg MCB-613 or control vehicle by intraperitoneal injection two 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. At 24 hours after MI, the ejection fraction in sham-treated animals decreased to an average of 30%. In contrast, the average ejection fraction in mice treated with MCB-613 two hours after MI was 43%, indicating that MCB-613 prevented the early decrease in ejection fraction and provided early protection to the vulnerable myocardium( Figure 11B )。The ejection fraction in sham-treated mice further decreased after 24 hours and was lowest at 19 weeks after MI, indicating a progressive loss of cardiac function over time. In contrast, after administration of MCB-613, the ejection fraction remained above 40% from 24 hours after MI until 19 weeks after MI, indicating that the early myocardial protective effect of MCB-613 prevented the progressive loss of cardiac function. Three-day repeated injections at 8 and 16 weeks did not change the ejection fraction, indicating that MCB-613 had no further effect on cardiac function at later time points. Maintenance of cardiac function for up to 19 weeks after MI indicates that short-term early intervention can effectively prevent congestive heart failure after MI. Analysis of heart weight showed that MCB-613 attenuated the MI-induced cardiac hypertrophy compensatory response at 12 weeks after MI( Figure 11C ),indicating that protection of cardiac function is associated with another key feature in preventing heart failure. Then cardiac positron emission tomography (PET) imaging was used to spatially evaluate myocardial viability. In the infarct area 18 Improved F-FDG uptake indicates that MCB-613 protects healthy myocardium two weeks after MI( Figure 11D )。Heart tissue sections were stained with Sirius red to evaluate infarct size and degree of fibrosis( Figure 11E )。The infarct size measured at 12 weeks after MI was greater in sham-treated hearts (31% and 44%) compared to hearts from MCB-613-treated mice (3%, 14%, 20%, and 22%). In addition, the cardiomyocytes were smaller and associated with less fibrosis in the infarct border zone, demonstrating two additional key molecular features by which MCB-613 prevents progressive heart failure( Figure 11E )。Cardiac metabolic dysfunction is a common feature of heart failure. SRC coordinates different metabolic demands in tissues including skeletal and cardiac muscle. Indirect calorimetry with exercise was performed to determine the effect of MCB-613 on energy expenditure three weeks after MI compared to age-matched mice without MI as controls( Figure 12 )。At three weeks after MI, VCO 2 and VO 2Increased, indicating that MCB-613 can improve energy utilization during exercise in mice after MI. Therefore, improved cardiac function is associated with improved energy consumption. Electron micrographs of the heart 72 hours after MI showed that MCB-613 could prevent disorganization of myofibrillar structure and abnormal mitochondrial cristae architecture, indicating that MCB-613 can protect the myocardium and mitochondria from MI-induced damage( Figure 11F ). Supporting early myocardial protection, MCB-613 could prevent apoptosis 24 hours after MI( Figure 11G ). These findings suggest that MCB-613 has a role in directly protecting functional myocardium and preventing harmful remodeling of cardiac tissue.

[0236] MCB-613 prevents cardiomyocyte injury responses. To gain insight into cardiomyocyte- and non-cardiomyocyte-specific MCB-613 transcriptional functions associated with reduced myocardial remodeling and improved cardiac function, transcriptome profiling was performed on cardiac cells purified from control-treated and MCB-613-treated mice 12 weeks after MI( Figure 13A ). Differential gene expression analysis of cardiomyocytes showed that 122 upregulated genes and 107 downregulated genes were associated with improved cardiac function 12 weeks after MI( Figure 13B ). Gene set enrichment analysis of differentially expressed genes showed strong enrichment of gene ontology categories representing oxidative phosphorylation and adipogenesis as well as inhibition of apoptosis and inflammatory responses( Figure 13C ), which provides further support for MCB-613 improving cardiac energy utilization in addition to preventing signaling associated with cardiomyocyte injury.

[0237] MCB-613 reduces inflammatory macrophages. Single-cell transcriptome profiling was performed 12 weeks after MI to identify cell types and cell type-specific signaling responses associated with improved cardiac function in MCB-613-treated mice. After Langendorff perfusion, a metabolically active and viable single-cell suspension of non-cardiomyocytes was prepared from the whole heart( Figure 13A ). Minimal procedural manipulations were performed to prevent loss of cell types and minimize effects on transcriptional activity. The transcriptional profiles of 21,894 cells from two saline-treated mice and 21,474 cells from two MCB-613-treated mice passing RNA quality control were analyzed by Seurat analysis using the 10x chromium platform. Fifteen distinct cell clusters were identified based on cell expression patterns, unsupervised clustering, and dimensionality reduction analysis using Seurat software( Figure 13D ). Cluster sizes ranged from 101 to 6,085 cells. Cell populations were confirmed based on known mouse cardiac cell type markers( Figure 13E) Macrophages accounted for 42% of the total cells analyzed and were the major non - myocardial cell population at 12 weeks post - MI, which was different from the normal adult mouse heart where non - myocardial cells consisted of only 10% hematopoietic - derived cells. Evaluation of the largest changes in cell numbers showed that in the hearts from MCB - 613 - treated mice, macrophage cluster 1 and B lymphocyte numbers decreased, while the cell numbers of epicardial cells, NK / T lymphocytes, fibroblasts, and endothelial cells (including lymphocyte - containing) increased( Figure 13F ) The cardiac macrophage, fibroblast, and endothelial cell populations showed transcriptional heterogeneity and were composed of four, three, and two sub - clusters, respectively. Evaluation of unique gene markers in cardiac fibroblasts revealed the presence of a population of injury - responsive fibroblasts expressing Postn (fibroblast cluster 2), and in support of the recently reported "homeostatic fibroblasts" in the remodeling heart, fibroblasts in cluster 3 uniquely expressed Comp( Figure 14A ) Genes uniquely enriched in fibroblast cluster 1 indicated the presence of a sub - population of fibroblasts with secretory functions involved in promoting angiogenesis (Bmp4, Ecm1, Ccl11, Pgf) and extracellular matrix organization (Ecm2 and Pdgfra). Endothelial cell transcriptional markers indicated the presence of 3 sub - populations. Compared to endothelial cluster 1, endothelial cluster 2 and lymphatic endothelial cells showed increased transcriptional activation of 218 and 308 unique genes, respectively, indicating different roles in cardiac maintenance at 12 weeks post - MI( Figure 14B ) The lymphatic endothelial cluster was defined by the unique expression of the lymphatic endothelial genes Prox1 and Lyvel. In addition to the increased cell numbers, the parallel expression of the pro - angiogenic regulators Hif1a and Lrg1 and the lymphangiogenic regulator Ccl121a also indicated that MCB - 613 stimulated lymphangiogenesis at 12 weeks post - MI. Gene expression markers in endothelial cluster 2, defined by the unique expression of the early cardiac genes Mkl2, Tek, and Hand2, indicated transcriptional reversion to a more primitive cell state, likely due to the injury stress response. Without being bound by theory, the largest endothelial cluster (i.e., endothelial cluster 1) represented 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 markers of the four macrophage sub - clusters were clearly separable( Figure 14C) Macrophage cluster 1 is the largest subset of macrophages and is defined by the expression of inflammatory gene markers, including Ccl8, Ccl24, and Ly96, which are compatible with the role of these genes in resolving myocardial inflammation. Cluster 2 represents a population of Ccr2+ monocyte-derived macrophages that express the inflammatory genes Cxcl1, Ccr2, Ccr5, and Tlr2, known to be short-lived infiltrating macrophages derived from the bone marrow, in response to injury. In contrast, macrophages in cluster 3 uniquely express 110 cell cycle proliferation genes, indicating the presence of a small population of proliferative Ccr2− macrophages, known to be maintained by local proliferation that functions in tissue repair and myogenesis. Macrophages in cluster 4 were identified based on the expression of genes involved in phagocytic activation, including Cd209 and Corola, indicating the presence of a small population of phagocytic macrophages 12 weeks after MI. Unexpectedly, a rather small change in the number of cell populations associated with improved cardiac function suggests that MCB-613 cardioprotection may instead be the result of changes in cell function.

[0238] MCB-613 promotes beneficial paracrine signaling. To determine the functional responses of interstitial cell types associated with MCB-613-mediated improvement in cardiac function 12 weeks after MI, transcriptome profiles were compared in non-myocardial cells from control-treated and MCB-613-treated mice ( Figure 15A ). Large differences in the transcriptional responses of cell populations suggest that MCB-613-selective cell responses contribute to improved cardiac function. Smaller cell populations consisting of lymphatic and immune cell populations underwent the largest drug-induced transcriptome changes. To identify potential interstitial cell signaling interactions contributing to improved cardiac function, the number of ligand–receptor interactions between each cell in the control heart compared to the heart from MCB-613-treated mice was calculated ( Figure 15B ). The highest frequency of interactions occurred between ligands from each fibroblast population, one macrophage subtype, and the endothelial / SMC population that transmits to granulocyte receptors. This pattern of interstitial cell-to-granulocyte signaling implies extensive paracrine regulation of granulocyte function in the MCB-613 cardioprotective response 12 weeks after MI. Ligand–receptor pairings suggest coordinated regulation of tissue architecture and anti-inflammatory signaling pathways, including MMP9-LRP1, HSP90B1-TLR7, and SERPINE1-TAUR ( Figure 15C ). Supporting this, gene set enrichment analysis of upregulated and downregulated gene markers in granulocytes indicates that MCB-613 inhibits inflammatory granulocyte function ( Figure 14D)。Compared with the control, the most upregulated and downregulated genes in cardiac granulocytes from MCB-613-treated mice showed increased expression of granules involved in innate defense and decreased cytokines, enzymes, and chemokines involved in inflammatory signaling ( Figure 15D )。These findings suggest that the myocardial response to MCB-613 is characterized by a persistent paracrine anti-inflammatory signaling landscape that underlies the improvement in cardiac function.

[0239] Since administration of MCB-613 at the time of injury results in an immediate response at 24 hours ( Figure 11B and Figure 11E ), the effect of MCB-613 on immune cells was measured at 24 hours after MI. The immunophenotype of single cells isolated from the whole heart by FACS analysis at 24 hours after MI was used to quantify the composition of immune cells. Similar to what was observed at 12 weeks after MI, B cells were significantly reduced and monocytes trended lower in the hearts from MCB-613-treated mice compared with the control, while the fraction of granulocytes did not change ( Figure 16A )。The transcriptional response of acute granulocytes to MCB-613 and the robust transcriptional response in granulocytes due to the presence of paracrine signaling were then measured at 12 weeks after MI. Granulocytes are the first innate immune cells to arrive at the myocardium after acute ischemic injury and are key mediators of the degree of inflammatory response triggered by an acute heart attack and the damage caused to the myocardium. Since it is difficult to isolate sufficient amounts of undamaged granulocytes from the mouse heart, bone marrow granulocytes were isolated to study the granulocyte response, which reflects the myocardial granulocyte response at 24 hours after MI. Increased mRNA expression of the granulocyte marker S100A9 in granulocytes compared with granulocyte-depleted bone marrow indicated successful isolation of granulocytes ( Figure 16B )。Increased expression of Tlr7 and Lcn2 in granulocytes from MCB-613-treated mice supported the single-cell transcriptome analysis and revealed 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 due to tissue trauma caused by the surgical procedure in the absence of MI, granulocytes were isolated from mice 24 hours after sham surgery and either control vehicle or MCB-613 was administered. No changes in cell number or gene expression were observed, indicating that the granulocyte gene expression changes are a result of the MCB-613-mediated myocardial injury response. MCB-613 induced a strong transcriptome response in granulocytes, suggesting that neutrophil granules may regulate the post-MI inflammatory effects of the compound. Supporting this, LYZ1 granule expression was significantly increased in the myocardium of MCB-613-treated mice compared with control animals at 24 hours after MI ( Figure 16C )。

[0240] Example 4: Pharmacokinetic (PK) Study of MCB-613, Compound 10-1, and Compound 10-2

[0241] The pharmacokinetics of MCB-613, Compound 10-1, and Compound 10-2 were tested in CD-1 mice. Each of the three compounds was dissolved in DMSO (20 mg / mL), mixed with 30% hydroxypropyl-β-cyclodextrin at a 1:9 ratio, and administered to CD-1 mice by intraperitoneal (ip) or oral (po) gavage. After administration of the compounds, blood samples (3 mice for each compound) were collected via the tail vein at nine time points, namely 5 minutes, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours. Plasma was isolated from these blood samples, and the compound plasma concentrations were determined by HPLC-MS / MS. Pharmacokinetic parameters were calculated using the PKSolver program (an additional program for analyzing 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 include the half-life (t 1 / 2 ), terminal half-life (terminal t 1 / 2 ), time to reach the maximum concentration after administration of the compound (t max ), maximum concentration of the compound observed (C max ), area under the curve until the last measurable concentration (AUC 0-t ), area under the curve to infinite time (AUC 0-inf ), and clearance rate of the compound (Cl).

[0242] 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.

[0243] Table 1

[0244] MCB - 613 10-1 10-2 <![CDATA[t 1 / 2 (h)]]> 0.86 0.64 0.25 <![CDATA[End-stage t 1 / 2 (h)]]> 6.5 18.1 15.9 <![CDATA[t max (h)]]> 0.08 0.25 0.08 <![CDATA[C max (ng / mL)]]> 49.2 356.7 463.3 <![CDATA[AUC 0-t (ng / mL*h)]]> 122.2 622.6 333.8 <![CDATA[AUC 0-inf (ng / mL*h)]]> 129.6 851.1 481.5 Cl (mg) / (ng / mL) / h 6.4 0.97 1.7

[0245] 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.

[0246] Table 2

[0247] MCB - 613 10-1 10-2 <![CDATA[t 1 / 2 (h)]]> 0.94 1.2 0.78 <![CDATA[End-stage t 1 / 2 (h)]]> 16.1 10.2 27.1 <![CDATA[t max (h)]]> 0.25 0.5 0.25 <![CDATA[C max (ng / mL)]]> 5.6 53.2 181.9 <![CDATA[AUC 0-t (ng / mL*h)]]> 15.7 98.4 131.1 <![CDATA[AUC 0-inf (nanograms per milliliter per hour)]]> 24.3 112.6 134.6 Cl (mg) / (ng / mL) / h 34 7.3 6.1

[0248] Pharmacokinetic data are also shown in Figure 17 . Figure 17 The graphs in show the mean plasma concentration measured over time. The upper row of graphs shows the pharmacokinetic data from the above study in which MCB-613 (upper left curve), Compound 10-1 (middle upper curve), and Compound 10-2 (upper right curve) were administered intraperitoneally to CD-1 mice. The lower row of graphs shows the pharmacokinetic data from the above study in which MCB-613 (lower left curve), Compound 10-1 (middle lower curve), and Compound 10-2 (lower right curve) were administered orally to CD-1 mice.

[0249] The compounds and methods of the appended claims are not limited in scope by the specific compounds and methods described herein, which are intended to illustrate several aspects of the claims, and any compounds and methods that are functionally equivalent are within the scope of this disclosure. Various modifications of the compounds and methods other than those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compounds, methods, and aspects of these compounds and methods are specifically described, other compounds and methods are intended to fall within the scope of the appended claims. Accordingly, combinations of steps, elements, components, or ingredients may be expressly recited herein; however, all other combinations of steps, elements, components, and ingredients are included even if not expressly stated.

Claims

1. A compound of the following formula: 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 selected from CR 1 and N, where each R 1 is hydrogen or an alkoxy group; and X is NR 2 or CR 3 R 4 , wherein R 2 , R 3 and R 4 are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1-6 alkyl, and substituted or unsubstituted cycloalkyl, provided that R 2 is not cyclohexyl; Wherein A 3 、A 4 、A 5 、A 8 、A 9 and A 10 are CR 1 wherein each R 1 is hydrogen.

2. The compound according to claim 1, wherein R 2 is selected from cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl and cyclooctyl.

3. A compound of the following formula: or a pharmaceutically acceptable salt or prodrug thereof, wherein: ■A 2 and A 7 each independently selected from CR 1 wherein each R 1 is halogen, alkoxy, cyano, trifluoromethyl or substituted or unsubstituted C 1-6 alkyl; ■A 5 and A 10 each independently selected from CR 1’ , where R 1’ is hydrogen, cyano or substituted or unsubstituted C 1-6 alkyl; and ■R 2 is a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocycloalkyl, wherein R 2 is not cyclohexyl.

4. The compound according to claim 3, wherein R 2 is selected from cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl and cyclooctyl.

5. A compound selected from the group consisting of:

6. Use of the compound according to any one of claims 1 to 5 in the preparation of a medicament for treating ischemic injury in a subject.

7. Use according to claim 6, wherein the ischemic injury comprises myocardial infarction or stroke.

8. Use according to claim 6, which further comprises selecting a subject having suffered ischemic injury, wherein the ischemic injury comprises myocardial infarction or stroke.

9. Use of the compound according to any one of claims 1 to 5 in the preparation of a medicament for reducing the size of myocardial infarction in a subject having suffered myocardial infarction.

10. Use according to claim 9, wherein the size of the myocardial infarction is reduced by at least 5% compared to the size of the myocardial infarction in an untreated subject having suffered myocardial infarction.

11. Use according to claim 9, wherein the size of the myocardial infarction is reduced by at least 15% compared to the size of the myocardial infarction in an untreated subject having suffered myocardial infarction.

12. Use of the compound according to any one of claims 1 to 5 in the preparation of a medicament for preventing or reducing myocardial cell loss, improving cardiac vascular perfusion and / or improving central nervous system vascular perfusion in a subject having suffered myocardial infarction or stroke.

13. Use of the compound according to any one of claims 1 to 5 in the preparation of a medicament for improving the cardiovascular function and / or central nervous system vascular function of a subject.

14. Use according to claim 13, wherein the subject has suffered ischemic injury.

15. Use according to claim 14, wherein the ischemic injury is myocardial infarction or stroke.

16. Use according to claim 13, wherein the subject is an elderly subject.

17. Use of the compound according to any one of claims 1 to 5 in the preparation of a medicament for promoting wound healing in a subject.

18. Use of the compound according to any one of claims 1 to 5 in the preparation of a medicament for treating or preventing hypertrophic cardiomyopathy in a subject, which.

19. Use according to claim 18, wherein the subject has suffered ischemic injury.

20. Use according to claim 19, wherein the ischemic injury is myocardial infarction or stroke.

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