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

Abstract

Small molecule stimulators of steroid receptor coactivator-3 (SRC-3) and methods for their use as cardioprotectants are provided. Small molecule stimulators can be used to promote cardioprotection and repair and angiogenesis after myocardial infarction. The compounds can also be used to prevent cardiac hypertrophy and collagen deposition and improve function after myocardial infarction.

Description

[0001] Cross-reference to priority application

[0002] This application claims priority to U.S. Provisional Application No. 62 / 724,281, filed on August 29, 2018, and U.S. Provisional Application No. 62 / 825,358, filed on March 28, 2019, which are incorporated herein by reference in their entirety. Background Art

[0003] Heart failure induced by myocardial infarction (MI) is determined by progressive remodeling of cardiac tissue associated with loss of cardiomyocytes, inflammation, fibrosis, and severe reduction in cardiac ejection fraction. A promising therapeutic approach to improve cardiac function is to prevent the deleterious remodeling of native cardiac tissue by directly protecting functional myocardium. Major barriers to maintaining cardiac function after infarction include tissue destruction and the limited and restricted regenerative potential of the adult heart, which poses a hurdle for therapies aimed at promoting tissue reprogramming and repair. Summary of the invention

[0004] Small molecule stimulators of steroid receptor coactivator-3 (SRC-3) and methods of their use as cardioprotective and / or angiogenic agents are described herein. The compounds described herein can be used to promote cardioprotection and repair and angiogenesis after myocardial infarction. The methods include administering to a subject a compound as described herein.

[0005] Small molecule SRC-3 stimulators include compounds of the formula:

[0006]

[0007] and pharmaceutically acceptable salts or prodrugs thereof. In these compounds, 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 R 2 is a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted heterocycloalkyl. Optionally, the compound has the formula:

[0008]

[0009] wherein m and n are each independently 1, 2, 3, 4 or 5.

[0010] Optionally, the compound has the formula:

[0011]

[0012] wherein m and n are each independently 1, 2, 3 or 4.

[0013] In the compounds described herein, R 2 Optionally, the compound is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Optionally, the compound is selected from the group consisting of:

[0014]

[0015] Optionally, the compound is selected from the group consisting of:

[0016]

[0017] or a pharmaceutically acceptable salt or prodrug thereof.

[0018] Also described herein are methods of treating an ischemic injury (eg, myocardial infarction or stroke) in a subject comprising administering to the subject an effective amount of a compound of the formula:

[0019]

[0020] or a pharmaceutically acceptable salt or prodrug thereof. In the compound used in this 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 are each independently selected from hydrogen, substituted or unsubstituted C 1-6 The compound is selected from the group consisting of:

[0021]

[0022]

[0023] Optionally, the method may further comprise selecting a subject who has suffered a myocardial infarction or has suffered a stroke or other vascular injury to the central nervous system.

[0024] Further described herein is a method of reducing the size of a myocardial infarction in a subject who has suffered a myocardial infarction. The method may include administering to the subject an effective amount of a compound of the formula:

[0025]

[0026] 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 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 are each independently selected from hydrogen, substituted or unsubstituted C 1-6 The compound is selected from the group consisting of:

[0027]

[0028]

[0029] Optionally, the myocardial infarct size is reduced by at least 5% (eg, reduced by at least 15%) compared to the size of a myocardial infarct in an untreated subject that has suffered a myocardial infarction.

[0030] Also described herein are methods for preventing or reducing myocardial cell loss, improving cardiac vascular perfusion, and / or improving central nervous system vascular perfusion in a subject who has suffered a myocardial infarction or stroke, comprising administering to the subject an effective amount of a compound of the formula:

[0031]

[0032] or a pharmaceutically acceptable salt or prodrug thereof. In the compound used in this 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 are each independently selected from hydrogen, substituted or unsubstituted C 1-6 The compound is selected from the group consisting of:

[0033]

[0034]

[0035] Also described herein is a method of improving cardiovascular function and / or central nervous system vascular function in a subject, comprising administering to the subject an effective amount of a compound of the formula:

[0036]

[0037] or a pharmaceutically acceptable salt or prodrug thereof. In the compound used in this 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 CR1 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 hydrogen, substituted or unsubstituted C 1-6 The compound is selected from the group consisting of:

[0038]

[0039] Optionally, the subject has suffered an ischemic injury (eg, 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, comprising administering to the subject an effective amount of a compound of the formula:

[0041]

[0042] or a pharmaceutically acceptable salt or prodrug thereof. In the compound used in this 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 are each independently selected from hydrogen, substituted or unsubstituted C 1-6 The compound is selected from the group consisting of:

[0043]

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

[0045] Further described herein are methods of treating or preventing hypertrophic cardiomyopathy in a subject, comprising administering to the subject an effective amount of a compound of the formula:

[0046]

[0047] or a pharmaceutically acceptable salt or prodrug thereof. In the compound used in this 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 are each independently selected from hydrogen, substituted or unsubstituted C 1-6 The compound is selected from the group consisting of:

[0048]

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

[0050] The details of one or more embodiments are set forth in the accompanying drawings and the 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 graphs showing expression of NCOA3 in normal human heart (left panel) and muscle tissue (right panel).

[0052] Figure 2 are images of hearts from mice injected with adeno-SRC3 prior to harvest.

[0053] Figure 3AExperimental timeline describing pharmacological treatment and echocardiographic measurements after myocardial infarction (MI). Figure 3B is a graph showing the heart weight and tibia length ratio (HW / TL) of mice after myocardial infarction. Figure 3C is a graph showing the effect of MCB-613 treatment in mice following myocardial infarction. Figure 3D Images of mouse hearts harvested after myocardial infarction and stained to visualize collagen fibers.

[0054] Figure 4 is a graph showing the effect of Compound 10-1 treatment in mice after myocardial infarction.

[0055] Figure 5A Figure 2 depicts comprehensive single-cell transcriptional profiling of non-myocytes in the adult mouse heart. Figure 5B 5A is a graph showing the different cell types present in MCB-613 treated hearts following myocardial infarction. 5C is a Venn analysis of three cell clusters with endothelial markers.

[0056] Fig. 6A is a heat map showing the metabolomics of long-chain fatty acids in mouse heart after myocardial infarction. Figure 6B is a heat map showing the metabolomics of methylglutarylcarnitine in 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 Pictures contain horizontal sections of papillary muscles of hearts after myocardial infarction and after treatment with compound 10-1.

[0059] Fig. 9 Contains graphs showing the results of a progressive maximal exercise test performed in mice treated with saline ("Saline"), mice treated with MCB-613 ("MCB-613"), and non-infarcted wild-type mice ("WT"). The upper graph shows exhaled carbon dioxide, and the lower graph shows oxygen consumption.

[0060] Figures 10A-10E MCB-613 was shown to stimulate angiogenesis in chicken eggs and mouse hearts three days after MI. Fig. 10ACardiac fibroblasts were treated with DMSO or MCB-613 for 24 h. Total protein was then isolated and immunoblotted for SRC-1, -2, and -3. Hsp90 was used as a loading control. Fig. 10B Cardiac fibroblasts were transfected with GAL4 DNA binding site-luciferase reporter (pG5-luc) and GAL4-DNA binding domain-full-length SRC-1, -2 or -3 fusion (pBIND-SRC) or control pBIND expression vector. After transfection, 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). Fig. 10C Cardiac fibroblasts were treated with DMSO or MCB-613 for 24 h and then conditioned with endothelial growth medium for another 24 h in the absence of drug. The conditioned cells were then plated in Matrigel to allow tube formation overnight, and the tubes were then stained with Calcein AM dye and imaged. Fig. 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 membranes in chicken eggs. The vascular area was measured on the first and third days. Data are expressed as the percentage increase relative to the control under each condition. Six eggs were used for each condition. *P<0.05. For Fig. 10E Mice were treated with MCB-613 or control two hours after MI. Hearts were fixed and immunostained for endothelial cell-specific CD31. Fig. 10E Representative images of the infarct border zone from three control mice and three mice treated with MCB-613 are shown. Fig. 10E Also included is a bar graph showing the quantitative CD31 immunostaining density per unit area of ​​tissue for two fields per border zone *P<0.05.

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

[0062] Fig.12 Contains graphs showing the results of a progressive maximal exercise test 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 graph shows oxygen consumption, and the right graph shows carbon dioxide exhalation.

[0063] Figures 13A-13F Shown are RNA transcriptional profiling of cardiomyocytes and single-cell analysis of interstitial cells 12 weeks after MI, revealing that the MCB-613 protective response is associated with improved oxidative phosphorylation, reduced inflammation, and a decrease in immune cells. Fig.13A Schematic diagram of the isolation procedure for obtaining cardiomyocytes for total RNA sequencing and non-cardiomyocytes for single-cell RNA-seq analysis from control-treated and MCB-613-treated mice 12 weeks after MI. n = 2 hearts / group. Fig. 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 post-MI. Fig. 13Cis a gene set enrichment analysis of genes up- and down-regulated in cardiomyocytes of MCB-613 treated hearts compared to control treated hearts. Fig.13D Depicts cell populations identified by unsupervised clustering. Each dot represents a single cell. Fig.13E is a heat map indicating established cell type markers used to specifically identify each cluster. Fig.13F Representative TUNEL staining from control-treated and MCB-613-treated hearts 24 hours after MI is shown. n = 4 hearts per group. Scale bars: 2000 μm and 20 μm.

[0064] Fig.14A is a Venn analysis of gene expression in fibroblast clusters. Fig. 14B is a Venn analysis of gene expression in endothelial cell clusters. Fig. 14C is a Venn analysis of macrophage gene expression. Fig.14D is a gene set enrichment analysis of genes up- and down-regulated in granulocytes of MCB-613 treated hearts compared to control treated hearts.

[0065] Figures 15A-15D MCB-613 was shown to modulate persistent immune and endothelial cell responses 12 weeks after MI. Fig.15A Shown are the numbers of genes up- and down-regulated in non-muscle cells of control mice compared to MCB-613 treated mice. Fig. 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 pairings begins at the node and ends at the cognate receptor as indicated in the legend. The thickness of the line reflects the number of ligand-receptor pairs. Rings represent autocrine signaling circuits. Fig. 15C Figure 2 is a heat map of ligand-receptor pairings between granulocytes, fibroblast clusters, and macrophage C4. Fig.15D is a heat map showing the top 50 up- and down-regulated drug response genes from 277 and 310 granulocytes from control-treated and MCB-613-treated hearts, respectively.

[0066] Figures 16A-16C MCB-613 was shown to reduce B lymphocytes and monocytes as early as 24 hours after MI and to upregulate granulocyte genes and lysozyme. Fig.16A Shown is the quantification of cardiac immune cells from control-treated and MCB-613-treated mice by fluorescence-activated cell sorting (FACS) immunophenotyping 24 hours after MI. Fig. 16BmRNA expression in granulocytes and neutrophils isolated from bone marrow 24 hours after MI and MCB613 treatment is shown. Total RNA was isolated from neutrophil-enriched fractions and neutrophil-depleted fractions 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. Fig. 16C Representative LYZ staining from control-treated and MCB-613-treated hearts 24 hours after MI is shown. Fig. 16C The upper panel shows low magnification from endocardium to epicardium, and the lower panel shows high magnification of the subendocardial region. Arrows indicate LYZ+ cells. Fig. 16C The bar graph in Figure 2 shows quantification of LV density of LYZ+ cells. n = 3 hearts / group, >10 mm 2 Imaging / Heart, 24 hours after MI surgery *P < 0.039.

[0067] Fig.17 Graphic representation containing pharmacokinetic data for MCB-613, Compound 1, and Compound 2 obtained in CD-1 mice. DETAILED DESCRIPTION

[0068] Described herein are stimulators of steroid receptor coactivator (SRC) proteins and methods of their use. 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 stimulators of SRC-3 and can be used as cardioprotectants and / or angiogenic agents. In particular, the compounds can be used to promote cardiac protection and repair and angiogenesis after myocardial infarction or stroke. The compounds can also be used to prevent cardiac hypertrophy and collagen deposition and improve function after myocardial infarction. The compounds have been shown to increase angiogenesis, increase vascular perfusion in the heart and central nervous system, and promote cardiac beta oxidation. The administration of the compounds described herein also significantly reduces the presence of the metabolite methylglutarylcarnitine associated with dilated cardiomyopathy.

[0069] I. Compounds

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

[0071]

[0072] and pharmaceutically acceptable salts or prodrugs thereof.

[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 Each independently selected from CR 1 and N. Each R present in Formula I 1 The groups are 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 hydrogen, substituted or unsubstituted C 1-6 The group consisting of alkyl, substituted or unsubstituted cycloalkyl and substituted or unsubstituted heterocycloalkyl.

[0075] As used herein, the terms alkyl, alkenyl, and alkynyl include straight and branched monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. The range of these groups useful in the compounds and methods described herein includes C1-C 20 Alkyl, C2-C 20 Alkenyl and C2-C 20 Alkynyl. Additional ranges of these groups that can be used in the compounds and methods described herein include C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl, C2-C4 alkenyl and C2-C4 alkynyl.

[0076] Heteroalkyl, heteroalkenyl, and heteroalkynyl are similarly defined as alkyl, alkenyl, and alkynyl, but may contain O, S, or N heteroatoms, or combinations thereof, within the backbone. The range of these groups useful in the compounds and methods described herein includes C1-C 20 Heteroalkyl, C2-C 20 Heteroalkenyl and C2-C 20 Additional ranges of these groups that can be used in the compounds and methods described herein include C1-C 12 Heteroalkyl, C2-C 12 Heteroalkenyl, C2-C 12C1-C6 heteroalkyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, C1-C4 heteroalkyl, C2-C4 heteroalkenyl and C2-C4 heteroalkynyl.

[0077] The terms cycloalkyl, cycloalkenyl, and cycloalkynyl include cyclic alkyl groups having a single cyclic ring or multiple fused rings. Examples include cyclohexyl, cyclopentylethyl, and adamantyl. The range of these groups that can be used in the compounds and methods described herein includes C3-C 20 Cycloalkyl, C3-C 20 Cycloalkenyl and C3-C 20 Additional ranges of these groups that can be used in the compounds and methods described herein include C5-C 12 Cycloalkyl, C5-C 12 Cycloalkenyl, C5-C 12 cycloalkynyl, C5-C6 cycloalkyl, C5-C6 cycloalkenyl and C5-C6 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 range of these groups useful in the compounds and methods described herein includes C3-C 20 Heterocycloalkyl, C3-C 20 Heterocycloalkenyl and C3-C 20 Heterocycloalkynyl. Additional ranges of these groups that can be used in the compounds and methods described herein include C5-C 12 Heterocycloalkyl, C5-C 12 Heterocycloalkenyl, C5-C 12 C5-C6 heterocycloalkynyl, C5-C6 heterocycloalkyl, C5-C6 heterocycloalkenyl and C5-C6 heterocycloalkynyl.

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

[0080] The term alkoxy as used herein is an alkyl group bonded via a single terminal ether linkage. Likewise, the term aryloxy as used herein is an aryl group bonded via a single terminal ether linkage.

[0081] The term hydroxyl group as used herein is represented by the formula -OH.

[0082] As used herein, the term amine or amino group is represented by the formula: NZ 1 Z 2 Indicates that Z 1 and Z 2 Each may 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] Alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl molecules used herein can be substituted or unsubstituted. As used herein, the term "substituted" includes adding alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl to the position of the main chain connected to the alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl, such as hydrogen is replaced by one of these molecules. Examples of substituted groups 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 group has a full complement of hydrogens (i.e., hydrogens commensurate with its level of saturation) without substitution, for example, a straight chain decane (-(CH2)9-CH3).

[0084] In some examples, Formula I is represented by Structure IA:

[0085]

[0086] In structure IA, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , A 10 and R 2 As defined above for Formula I. In some examples of Structure IA, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A8 , A 9 and A 10 Each of them is CR 1 , where each R 1 are independently selected from the groups defined above for Formula I. For example, a compound of structure IA 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 Group. R 1 Each of the groups may be independently selected from the groups defined for Formula I above.

[0089] In some examples of Structure IA, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 and A 10 One or more of can be N. For example, the compound of structure IA can 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 Group. R 1 Each of the groups may be independently selected from the groups defined for Formula I above.

[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. 2 Selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

[0093] In some examples, Formula I is represented by Structure IB:

[0094]

[0095] In Structure IB, A 1 , A2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 , A 10 , R 3 and R 4 As defined above for Formula I. In some examples of Structure IB, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 and A 10 Each of them is CR 1 , where each R 1 are independently selected from the groups defined above for Formula I. For example, a compound of structure IB 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 may each independently include one to five R 1 Group. R 1 Each of the groups may be independently selected from the groups defined for Formula I above.

[0098] In some examples of Structure IB, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 and A 10 One or more of can be N. For example, a compound of structure IB can be represented by structure I-B2, structure I-B3, or structure I-B4:

[0099]

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

[0101] In some examples, Formula I is represented by Structure IC:

[0102]

[0103] In the structure IC, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 and A 10 As defined above for Formula I. In some examples of Structure IC, A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 , A 9 and A 10 Each of them is CR 1 , where each R 1 are independently selected from the groups defined above for Formula I. For example, a compound of structure IC 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 may each independently include one to four R 1 Group. R 1 Each of the groups may be independently selected from the groups defined for Formula I above.

[0106] In some instances of structured IC, 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 can be N. For example, a compound of structure IC can be represented by structure I-C2, structure I-C3, or structure I-C4:

[0107]

[0108]

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

[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. Various synthetic methods can be used to synthesize the compounds. 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. Optimal reaction conditions may vary with the specific reactants or solvents used, but such conditions can be determined by those skilled in the art through routine optimization procedures.

[0120] The changes in formula I include the addition, reduction or movement of various components as described for each compound. Similarly, when there are one or more chiral centers in the molecule, all possible chiral variants are included. In addition, compound synthesis may 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 in, for example, 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 that produce the compounds described herein can be carried out in a solvent that can be selected by one skilled in the art of organic synthesis. The solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products under the conditions (i.e., temperature and pressure) under which the reactions are carried out. The reactions can be carried out in one solvent or in a mixture of more than one solvent. The formation of products or intermediates can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H-NMR or 13 C-NMR), infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry or by chromatography, such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0122] Exemplary methods for synthesizing compounds as 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 Preparations

[0124] Compounds described herein or derivatives thereof can be provided in the form of pharmaceutical compositions. Depending on the intended mode of administration, pharmaceutical compositions can be in the form of solid, semisolid or liquid dosage forms (e.g., tablets, suppositories, pills, capsules, powders, liquids or suspensions), preferably in a unit dosage form suitable for single administration of precise doses. The composition will include a combination of a therapeutically effective amount of a compound described herein or derivatives thereof and a pharmaceutically acceptable carrier, and may further include other pharmaceutical agents, medicaments, carriers or diluents. So-called pharmaceutically acceptable means that it is not biologically or otherwise undesirable material, which can be administered to an individual together with a selected compound without causing unacceptable biological effects or interacting with other components of the pharmaceutical composition containing it in a harmful manner.

[0125] As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer or other substance well known in the art for pharmaceutical formulations. The selection of a carrier for use in the composition will depend on the intended route of administration of the composition. The preparation of pharmaceutically acceptable carriers and preparations containing these substances is described in, for example, 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 buffer, citrate buffer, 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 dextrins; 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 compounds described herein or derivatives thereof may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and 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. Appropriate fluidity can be maintained, for example, by using a coating agent (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant.

[0127] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispensing agents. Prevention of microbial action may be promoted by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Isotonic agents may also be included, such as sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms may be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0128] Solid dosage forms for oral administration of the compounds described herein or their derivatives include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compound or its derivative described herein is mixed with at least one inert common excipient (or carrier) such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia, (c) humectants such as glycerol, (d) disintegrants 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 glyceryl monostearate, (h) adsorbents such as kaolin and bentonite, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate or mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0129] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols.

[0130] Solid dosage forms, such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other coatings known in the art. They can contain opacifiers and can also have compositions that allow them to release one or more active compounds in a delayed manner in a certain part of the intestinal tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compound can also be in microencapsulated form, if appropriate, it can have one or more of the above-mentioned excipients.

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

[0132] Besides such inert diluents, the composition may also include additional agents such as wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, or perfuming agents.

[0133] In addition to the active compounds, suspensions may contain additional agents, for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these and the like.

[0134] Compositions of the compounds described herein or their derivatives for rectal administration are optionally suppositories which can be prepared by mixing the compounds with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ordinary temperatures but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredients.

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

[0136] The composition may include one or more compounds described herein and a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically acceptable salt refers to those salts of the compounds described herein or their derivatives, which are suitable for use in contact with the tissues of the subject within the scope of reasonable medical judgment, without undue toxicity, irritation, allergic reaction, etc., commensurate with a reasonable benefit / risk ratio, and effective for its intended use; and zwitterionic forms of the compounds described herein where possible. 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, mesylate, 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 SM Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated herein by reference in its entirety at least for the composition content taught therein.)

[0137] Administration of the compounds and compositions described herein or pharmaceutically acceptable salts thereof may be performed using 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 may be determined by one of ordinary skill in the art and includes exemplary dosages of about 0.5 to about 200 mg / kg body weight of active compound per day for mammals, which may be administered in a single dose or in separate divided doses, for example 1 to 4 times per day. Alternatively, the dosage amount may be about 0.5 to about 150 mg / kg body weight of active compound per day, about 0.5 to 100 mg / kg body weight of active compound per day, about 0.5 to about 75 mg / kg body weight of active compound per day, about 0.5 to about 50 mg / kg body weight of active compound per day, about 0.01 to about 50 mg / kg body weight of active compound per day, about 0.05 to about 25 mg / kg body weight of active compound per day, about 0.1 to about 25 mg / kg body weight of active compound per day, about 0.5 to about 100 mg / kg body weight of active compound per day, about 0.5 to about 150 mg / kg body weight of active compound per day, about 0.5 to about 200 mg / kg body weight of active compound per day, about 0.5 to about 100 mg / kg body weight of active compound per day, about 0.5 to about 150 mg / kg body weight of active compound per day, about 0.01 to about 100 mg / kg body weight of active compound per day, about 0.5 to about 150 mg / kg body weight of active compound per day, about 0.05 to about 200 mg / kg body weight of active compound per day, about 0.1 to about 200 mg / kg body weight of active compound per day, about 0.5 to about 100 mg / About 25mg / kg body weight of active compound, about 1 to about 20mg / kg body weight of active compound per day, about 1 to about 10mg / kg body weight of active compound per day, about 20mg / kg body weight of active compound per day, about 10mg / kg body weight of active compound per day, about 5mg / kg body weight of active compound per day, about 2.5mg / kg body weight of active compound per day, about 1.0mg / kg body weight of active compound per day, or about 0.5mg / kg body weight of active compound per day, or any range derivable therein. Optionally, the amount of the dosage is about 0.01mg / kg to about 10mg / kg body weight of active compound per day. Optionally, the amount of the dosage is about 0.01mg / kg to about 5mg / kg. Optionally, the amount of the dosage is about 0.01mg / kg to about 2.5mg / kg.

[0138] It will be understood by those skilled in the art that the specific dosage level and frequency of administration for any particular subject may 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, weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; and the severity of the particular condition.

[0139] The precise dose to be used in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and the circumstances of each subject. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal model test systems. Further, depending on the route of administration, one skilled in the art will know how to determine the dose that produces the plasma concentration of the desired level of response in the cells, tissues and / or organs of the subject.

[0140] IV. How to use

[0141] Provided herein are methods for treating myocardial infarction or other ischemic injuries (e.g., stroke) in a subject. The method comprises administering to the subject an effective amount of one or more compounds or compositions described herein or 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 a compound that achieves a desired pharmacological effect or other biological effect.

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

[0143] Further described herein is a method of reducing the size of a myocardial infarction in a subject who has suffered a myocardial infarction. The method comprises administering to the subject an effective amount of one or more compounds or compositions as described herein. The myocardial infarction size may 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 been administered any treatment for the myocardial infarction or a subject who has suffered a myocardial infarction and has been administered a therapeutic agent other than a compound or composition as described herein). Optionally, the myocardial infarction size may 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. The method of preventing or reducing cardiomyocyte loss in a subject who has suffered a myocardial infarction comprises administering to the subject an effective amount of one or more compounds or compositions as described herein.

[0144] Further described herein are 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 method comprises administering to the subject an effective amount of one or more compounds or compositions as described herein. Optionally, the subject has suffered from 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 exposed to secondhand smoke for a long time), an individual with elevated blood pressure, blood cholesterol or triglyceride levels, or an individual suffering from an autoimmune condition.

[0145] The method of treating myocardial infarction, reducing the size of myocardial infarction, preventing or reducing myocardial cell 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 include administering one or more additional agents to the subject. The one or more additional agents and the compounds described herein, or pharmaceutically acceptable salts or prodrugs thereof, may be administered in any order including concomitant administration, simultaneous administration, or sequential administration. Sequential administration may be sequential administration with a time interval of up to several days apart. The method may also include administering the one or more additional agents and / or the compounds described herein, or pharmaceutically acceptable salts or prodrugs thereof, more than once. The one or more additional agents and the compounds described herein, or pharmaceutically acceptable salts or prodrugs thereof, may be administered by the same or different routes and in parallel or sequentially.

[0146] Additional therapeutic agents include, but are not limited to, antiplatelet agents, statins, beta blockers, and renin-angiotensin-aldosterone system (RAAS) blockers (e.g., angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs)). Illustrative, non-limiting examples of antiplatelet agents that can 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 can 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, enakiren, 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 may be used in any combination with the compositions described herein. The combination may 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., one compound or agent is administered first, followed by the second). Thus, the term combination is used to refer to the concomitant, simultaneous, or sequential administration of two or more agents.

[0151] Optionally, a compound or therapeutic agent as described herein may be administered in combination with surgery (eg, coronary artery bypass surgery), angioplasty, stenting, or another implantation procedure.

[0152] The methods and compounds described herein can be used for preventive and therapeutic treatments. For preventive use, a therapeutically effective amount of a compound and composition 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 the occurrence of myocardial infarction. Preventive administration can be performed several days to several years before the symptoms of myocardial infarction are manifested. Therapeutic treatment involves administering a therapeutically effective amount of a compound and composition as described herein or a pharmaceutically acceptable salt thereof to a subject after the occurrence of myocardial infarction.

[0153] Methods for preventive and therapeutic treatment herein 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 the 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). The skilled person can make such a determination using a variety of prognostic and diagnostic methods, such as, for example, personal or family history of the disease or condition, clinical tests (e.g., genetic tests), etc. 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 myocardial infarction size, prevent or reduce cardiomyocyte 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. Kit

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

[0157] The kit may include oral formulations of any compound or composition described herein. The kit may include intravenous or intraperitoneal formulations of any compound or composition described herein. The kit may 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 term treatment (treatment / treat / treating) refers to a method of alleviating one or more symptoms of a disease or condition. Therefore, in the disclosed method, treatment may refer to a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in severity of one or more symptoms of a disease or condition. For example, if one or more symptoms or signs of a subject's disease (e.g., the size of a myocardial infarction) are reduced by 5% compared to a control, it is considered that the method of treating the disease is treatment. As used herein, a control refers to an untreated condition (e.g., the size of a myocardial infarction in an untreated subject who has suffered from a myocardial infarction). Therefore, reduction can be 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 to be understood that treatment does not necessarily mean cure or complete elimination of the disease, condition, or symptoms of a disease or condition.

[0159] As used herein, the terms prevent, preventing, and prevention of a disease or condition refer to actions that occur before or nearly simultaneously with a subject beginning to display one or more symptoms of the disease or condition, such as the administration of a composition or therapeutic agent, that inhibit or delay the onset or severity of one or more symptoms of the disease or condition.

[0160] As used herein, references to reduction, alleviation or inhibition include changes of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to control levels. Such terms may include but do not necessarily include complete elimination.

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

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

[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] Example

[0165] Example 1: Synthesis of compounds

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

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

[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 hours. 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 Na2SO4. 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 give 1-isoamylpiperidin-4-one as a colorless liquid (10.32 g, 61% yield). 1 H NMR (400 MHz, CDCl3): 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 piperidine-4-one (5mmol) and aldehyde (11mmol, 2.2 equivalents) were dissolved in acetic acid (10mL), to which concentrated hydrochloric acid (3mL) was added. The mixture was stirred at room temperature for 12 hours. The reaction was carefully quenched with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate (3×30mL), and the combined organic layers were washed with water and brine and dried over Na2SO4. The volatiles were removed in vacuo to give a crude oil, which was passed through column chromatography (silica gel, n-hexane to ethyl acetate from 5:1 to 1:1) to give α,β-unsaturated ketone (60-76% yield). The compounds described herein, including compounds 9-2, 9-8, 10-1 and 10-2, were prepared according to the general method. The 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. 1H NMR (400MHz, CDCl3): δ7.75(s,2H),7.36(t,J=8.0Hz,2H),7.03(d,J=8.0Hz,2H),6.96(s,2H),6.93( d,J=8.0Hz,2H),3.99(s,4H),3.85(s,6H),1.96-1.91(m,1H),0.51-0.47(m,2H) and 0.41-0.38(m,2H); 13 C NMR (100 MHz, CDCl3): 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 (400MHz, CDCl3): δ8.67(s,2H),8.59(d,J=4.8Hz,2H),7.73(s,2H),7.70(d,J=8.0Hz,2H),7. 37(dd,J=8.0,4.8Hz,2H), 3.99(s,4H), 1.99-1.94(m,1H), 0.58-0.51(m,2H) and 0.47-0.42(m,2H); 13 C NMR (100 MHz, CDCl3): 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] (3E,5E)-1-Isopentyl-3,5-bis(2-methoxybenzylidene)piperidin-4-one (10-1).

[0184]

[0185] Compound 10-1 was prepared in the form of hydrochloride (yellow powder). 1H NMR (400MHz, CDCl3): δ13.19(br,1H),8.36(s,2H),7.45(t,J=7.3Hz,2H),7.10(d,J=6.6Hz,2H),7.03(t,J=7.3Hz,2H),6.97(d,J=8.3Hz,2H),4.49(d ,J=15.6Hz,2H),4.28(d,J=15.6Hz,2H),3.88(s,6H),2.87-2.82(m,2H),1 .43 (sevent, J=6.6Hz, 1H), 1.30 (td, J=7.6, 6.6Hz, 2H) and 0.70 (d, J=6.6Hz, 6H); 13 C NMR (100 MHz, CDCl3): 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 in the form of hydrochloride (yellow powder). 1 H NMR (400MHz, CDCl3): δ13.46(br,1H),8.17(s,2H),7.40(t,J=8.0Hz,2H),7.01(dd,J=8.0,2.3Hz,2H),6.89(d,J=8.0Hz,2H),6.86(d,J=2.3Hz,2H),4.5 9(d,J=15.6Hz,2H),4.48(d,J=15.6Hz,2H),3.84(s,6H),2.89-2.84(m,2H), 1.46 (sevent, J=6.6Hz, 1H), 1.37 (td, J=7.6, 6.6Hz, 2H) and 0.72 (d, J=6.6Hz, 6H); 13 C NMR (100 MHz, CDCl3): 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 cardiac protection and repair. Representative compounds prevent, among other functions, increased infarct size, cardiac hypertrophy, and collagen deposition. The compounds significantly improve post-cardiac infarction function. The compounds also increase angiogenesis, promote cardiac beta-oxidation, and significantly reduce the level of the metabolite methylglutarylcarnitine associated with dilated cardiomyopathy.

[0191] method

[0192] All animal studies and protocols were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine and performed 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 in 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 cannulated. The heart was exposed by thoracotomy through the fourth or fifth intercostal space and an 8-0 nylon suture was tied around the LAD artery. 10ul of 9X 10 9 pfu / ml of adenovirus SRC-3 (Ad-SRC-3) or adenovirus GFP (Ad-GFP) were injected into the anterior free wall near the descending coronary artery of the left ventricle. In order to evaluate cell cycle entry, the analog 5-ethynyl-2′-deoxyuridine (EdU; 0.2g / L, Santa Cruz, SC-284628A) was added to the drinking water during the 9-day duration of the experiment. Cell proliferation was measured using the Click-iT EdU kit (Invitrogen, C10339). An initial dose of MCB-613 was administered intraperitoneally at 20mg / kg 2 hours after surgery. The same dose was then injected for another 6 days, and then repeated doses were given for 3 days at 9 weeks and 16 weeks. Mice were harvested at the specified time points for analysis.

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

[0195] Histological Analysis. Whole hearts were fixed with 10% formalin, embedded in paraffin, and sectioned at 7-μm intervals. Each slide had 3 or 10 sections (approximately 30-50 slides) starting at the apex and ending at the suture ligation site. Papillary level sections (slides 20-30) were stained with picrosirius red to identify areas of fibrosis. A length-based approach was used to determine infarct size.

[0196] Immunostaining analysis. Immunohistochemistry and immunofluorescence staining experiments were performed on FFPE (formalin-fixed and paraffin-embedded sections). TM Fluorometric TUNEL System Manufacturer's Protocol (DeadEnd TM TUNEL staining was performed using Fluorometric TUNEL System manufacturer's protocol) (Promega, GS3250) to detect apoptotic cells.

[0197] Isolation of cardiac cells for single cell transcriptional profiling. Mice were placed under anesthesia in the surgical plane before cervical dislocation. The heart was removed and cells were isolated by 15 minutes of Langendorff retrograde perfusion of a calcium-free pH 7.4 Tyrodes solution (130mM NaCl, 74.55mM KCl, 0.5mM MgCl, 0.33mM NaH2PO4, 0.25mm HEPES, 22mM glucose) containing 1mg / mL collagenase. The heart was then removed from the instrument and finely minced in the same Tyrodes buffer containing 15mg / mL bovine serum albumin (BSA), followed by grinding with a glass pipette. The cardiomyocytes were then precipitated by 300RPM differential centrifugation for 3 minutes. The supernatant containing the non-cardiomyocyte population of cells was then filtered through a 70 micron filter and precipitated at 750G, then resuspended in 1.1mL of phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS). Take out 0.1mL as "no staining control" for fluorescence activated cell sorting (FACS). Incubate the other 1mL with 4ug / mL calcein blue and 10microM DyeCycle Ruby and incubate for 10 minutes at 37°C. Then spin the cells at 600G and resuspend them in 0.5mL 2% FBS / PBS containing Sytox Green (30nM). Then use FACS Aria ii cell sorter to sort the cells for: Sytox Green-, Calcein+, DyeCycle Ruby+ into PBS containing 0.4% FBS. Then the cells are precipitated and resuspended in 100uL PBS containing 0.4% FBS, the cells are counted, and then the cells are flowed through the 10x Genomics Chromium system for single cell transcriptional profiling.

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

[0199] RER, VO2, and VCO2 measurements. The respiratory exchange ratio (RER), oxygen consumption (VO2), and expired carbon dioxide (VCO2) were measured by indirect calorimetry using a progressive maximal exercise test until mice were exhausted.

[0200] result

[0201] SRC-3 activation 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. NCOA3 expression in normal human hearts was determined and compared 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 heart (left) and muscle tissue (right). The y-axis shows fragments per kb gene length per million mapped reads (FPKM), and the data points represent samples. The data are sorted by the expression level of NCOA3.

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

[0203] Compounds described herein protect against early and progressive loss of cardiac function following myocardial infarction. Mice were treated with representative compounds described herein following myocardial infarction. Figure 3A Experimental timeline showing drug treatments and echocardiographic measurements after myocardial infarction. MCB-613 or saline was administered two hours after ligation and every 24 hours for another six days. Three repeated injections were given weekly at 8 and 16 weeks. Figure 3B Heart weight and tibia length were measured at 10 weeks as shown in . Six mice were administered saline, and seven mice were administered MCB-613. P < 0.03. Left ventricular ejection fraction was determined by echocardiography (n = 14 before 12 weeks; n = 3 at 19 weeks) ( Figure 3C ). Data were analyzed by ANOVA with repeated measures and are presented as mean + / - SEM. P < 0.001. Figure 3D Images of mouse hearts harvested after myocardial infarction and stained to visualize collagen fibers. Specifically, Figure 3DThe 4-fold magnification of the horizontal section of the papillary muscle 10 weeks after myocardial infarction and the corresponding 20-fold magnification of the horizontal section of the infarct border zone are shown. The infarct size is expressed as % length. The data show that the infarct size of mice treated with MCB-613 was reduced compared with the control mice administered with normal 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 Picrosirius red staining of horizontal sections of papillary muscles 6 weeks after myocardial infarction is shown. Infarct size is expressed as % length. The data show that infarct size was reduced in mice treated with compound 10-1 compared to control mice administered saline.

[0204] MCB-613 induces changes in major and minor non-cardiomyocyte cell types in the heart after myocardial infarction. Comprehensive single-cell transcriptional profiling of non-cardiomyocytes in the adult mouse heart. Single-cell sequencing of non-cardiomyocytes in the heart 10 weeks after myocardial infarction. Cell clusters were generated by tSNE analysis and identified by gene expression signatures. Figure 5A , which depicts comprehensive single-cell transcriptional profiling of non-myocytes in the adult mouse heart. Figure 5B Shown are the different cell types present in MCB-613 treated hearts following myocardial infarction. Figure 5C is a Venn analysis of three cell clusters with endothelial markers demonstrating that MCB-613 stimulates endothelial cell growth of two distinct endothelial cell populations in the heart 10 weeks after myocardial infarction.

[0205] MCB-613 increases cardioprotective carnitine metabolites. Fig. 6A is a heat map showing the metabolomics of long-chain fatty acids in mouse hearts 24 hours after myocardial infarction. Figure 6B is a heat map showing the metabolomics of methylglutarylcarnitine in mouse hearts after myocardial infarction (all group FDR=1). The heat map shows that MCB-613 increases cardioprotective carnitine metabolites, increases β-oxidation of long-chain fatty acids, and decreases methylglutarylcarnitine.

[0206] Compounds described herein stimulate SRC intrinsic transcriptional activity. HeLa cells transfected with a Gal4-responsive luciferase reporter (pG5-luc) and a construct 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 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.

[0207] Compound 10-1 improves cardiovascular fitness after myocardial infarction. Fig. 9 Contains graphs showing the results of a progressive maximal exercise test performed 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 graph shows exhaled carbon dioxide (VCO2), while the lower graph shows oxygen consumption (VO2). Fig. 9 As shown by representative compound 10-1, the compounds described herein improve cardiovascular and peripheral vascular fitness after myocardial infarction.

[0208] Summarize

[0209] As shown in the data provided herein, the compounds described herein stimulate angiogenesis in vivo and improve the function of damaged myocardium. Therefore, 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 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: Promoting Cardioprotection and Repair after Myocardial Infarction Using Steroid Receptor Coactivator Stimulators

[0211] Progressive remodeling of cardiac tissue with cardiomyocyte loss, inflammation, fibrosis and reduced cardiac ejection fraction is a hallmark of heart failure induced by myocardial infarction (MI). 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 steroid receptor coactivators described herein promote new blood vessel growth and improve cardiac function after MI. As shown by representative compounds, the administration of small molecule receptor coactivator stimulators can reduce infarct size, apoptosis, cardiac hypertrophy, collagen deposition, and activate cardiomyocyte energy pathways. Single cell transcriptional profiling identified different interstitial cell types and transcriptional responses associated with improved cardiac function. The compounds described herein represent novel therapeutic options for preventing early and progressive loss of cardiac function after MI.

[0212] method

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

[0214] Angiogenesis assay for treatment with small molecule stimulants. Five to seven days old fertilized chicken eggs (white leghorn) guaranteed to be free of specific pathogens (SPF) are used to enter the chorioallantoic membrane (CAM). The total vascular area on the CAM is measured using images obtained before the application of the drug. MCB-613 (100 μL) at a concentration of 0.6 μM is applied topically to the surface of the CAM. After daily application of the drug, the vascular area on the CAM is monitored daily. The vehicle control is maintained throughout the experiment. The treatment lasts for four days, at the end of which the image thresholding method (ImageJ) is used to quantify the total vascular area on the CAM. The percentage increase in vascular area of ​​the control and treated eggs is compared. n=6 eggs under each condition. This experiment is repeated three times.

[0215] Angiogenesis assay with drug-treated mouse embryonic fibroblasts (MEFs). Five to seven days old SPF-guaranteed fertilized chicken eggs (white leghorn chickens) were used to enter 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) (Corning Inc.; Corning, NY). The MEFs were then mixed thoroughly by pipetting and transferred to the surface of the CAM. The vascular area on the CAM was monitored every day. The vehicle control was maintained throughout the experiment. MEFs were allowed to grow on the CAM surface for four days, at the end of which the image thresholding method (ImageJ) was used to quantify the total vascular area on the CAM. The percentage increase in vascular area of ​​the control and treated eggs (MEF treated compared to untreated) was then compared. n=6 eggs under 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 expression vectors for GAL4 DNA binding domain (GAL4-DBD) full-length SRC-1, SRC-2, or SRC-3 fusion constructs (pBIND-SRC-1, pBIND-SRC-2, or pBIND-SRC-3) or pBIND control using Lipofectamine 3000 (Invitrogen; Carlsbad, CA). Twenty-four (24) hours after transfection, cells were treated with 6 μM MCB-613 or dimethyl sulfoxide (DMSO) and incubated overnight. Treated cells were lysed and total protein was isolated using the Promega Luciferase Assay System (Promega Life Sciences; Madison, WI). Protein concentration was measured using the Bradford assay (Bio-Rad Laboratories; Hercules, CA). Relative light units were measured and normalized to total protein concentration.

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

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

[0219] Histological Analysis. Whole hearts were fixed with 10% formalin, embedded in paraffin, and sectioned at 7 μm intervals. Each slide had three (3) to ten (10) sections starting at the apex and ending at the suture ligation site (approximately 30-50 slides). Papillary level sections (slides 20-30) were stained with picrosirius red to identify areas of fibrosis. A length-based approach was used to determine infarct size. DeadEnd TM TUNEL staining was performed using the fluorescent TUNEL system (Promega, GS3250) to detect apoptotic cells.

[0220] Electron microscopy. The animals were sacrificed, the hearts were quickly removed, and placed directly in a cold primary fixative (0.1M arsenic acid buffer containing 2% paraformaldehyde + 2.5% glutaraldehyde + 2mM CaCl2, pH 7.4), where they were sliced ​​in cross-section and then stored in a cold primary fixative for four days. After fixation, the tissues were stained with 0.1M arsenic acid buffer containing 0.1% tannic acid, rinsed and stained with osmic acid for one hour, after which the tissues were rinsed in dH2O and counterstained in aqueous uranyl acetate. The tissues were rinsed again in dH2O and then dehydrated in a series of gradient ethanol (50%, 70%, 80%, 90%, 95% and 100%). The tissues were slowly infiltrated over a four-day period by increasing dilutions of plastic resin to ethanol until 100% plastic was reached. After a full day of infiltration in three changes of 100% plastic, tissues were embedded in freshly prepared Spurr low viscosity resin and polymerized overnight at 60°C. Ultrathin sections of 55–65 nm were cut with a Diatome Ultra 45 diamond knife using a Leica UC7 ultramicrotome. Sections were collected on 150 hexagonal copper grids and viewed on a Hitachi H7500 transmission electron microscope. Images were captured using an AMT XR-16 digital camera and AMT ImageCapture, v602.600.51 software.

[0221] Isolation of cardiac cells. Place mice under anesthesia in the surgical plane before cervical dislocation. Remove the heart and separate the cells by 15 minutes of Langendorff retrograde perfusion of calcium-free pH 7.4 Tyrodes solution (130mM NaCl, 74.55mM KCl, 0.5mM MgCl, 0.33mM NaH2PO4, 0.25mm HEPES, 22mM glucose) containing 1mg / mL collagenase. Then remove the heart from the instrument and finely chop it in the same Tyrodes buffer containing 15mg / mL BSA, and then grind it with a glass pipette. Then precipitate the cardiomyocytes by 300RPM differential centrifugation for 3 minutes. Then the supernatant containing the non-cardiomyocyte population of cells is filtered through a 70 micron filter and precipitated at 750g, and then resuspended in 1.1mL phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS). Then, 0.1 mL of the mixture was taken out as a "no staining control" for fluorescence activated cell sorting (FACS). Another 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 2% FBS / PBS containing Sytox Green (30 nM). The cells were then sorted into PBS containing 0.4% FBS using a FACS Aria ii cell sorter for: Sytox Green-, Calcein+, DyeCycle Ruby+. The cells were then precipitated and resuspended in 100 μL PBS containing 0.4% FBS, and the cells were counted and passed through a 10x Genomics Chromium system.

[0222] Single-cell RNA sequencing. Raw fastq files were imported into Cell Ranger 2.1.1 (10XGenomics) for STAR alignment, data filtering, barcode counting, and UMI counting. To identify cell clusters and differentially expressed genes, 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 mapped to mitochondria were removed. 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 uses the hypergeometric distribution to identify enriched pathways (P value < 0.05).

[0223] To investigate the effects of treatment on intercellular communication in infarcted hearts, selected and putative ligand-receptor pairs in humans were obtained. For each cell type, signatures of drug treatment were obtained by applying filters with a P value < 0.05 and log2FC> 0.25 or < -0.25. Intercellular communication was established by connecting cell types A and B, where the ligand was differentially expressed in cell type A and the receptor was differentially expressed in cell type B. The network diagram was drawn using the igraph R package.

[0224] Total RNA-Seq analysis. Sequencing reads were trimmed using trimGalore software. Next, UCSC mm10 mapping reads were constructed against the human genome using HISAT and quantified against the Gencode gene model using StringTie. Gene expression (FPKM) quantiles were normalized using the R statistical system. The parametric t-test was used to determine the genes differentially expressed between tumor and normal samples with a p-value < 0.05 and a fold change of 1.25. Pathway enrichment analysis was performed using the GSEA package; the adjusted q-value reached significance (q < 0.25). Heatmaps were generated using Matplotlib, NumPy, and SciPy libraries in 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. qPCR analysis was performed using a Taqman kit with primers for Tlr7, Lcn2, and 18s.

[0226] Granulocyte separation. 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) (Ca / Mg-free) with 2% FBS. Both ends of the bone were cut open and the bone marrow was rinsed with ice-cold HBSS containing 2% FBS using a 26G needle. The clumps were broken up with an 18G needle and filtered through a 70 μm filter and centrifuged at 400 g for 10 minutes at 4 ° C. The precipitate was suspended 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 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) to 4 ml (enough 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" program was repeated, and the samples were incubated at 25°C for another 15 minutes, followed by another round of "IMPC_step2" program. Then, 400 μL of 4°C stop buffer (1x PBS, 0.1M EDTA) was added to each sample and centrifuged for one second at ~100 g to collect liquid at the bottom of the tube. The sample was filtered into a 50 mL conical tube through a mesh filter cap. The tube was then washed with 1 mL of FACs buffer, which was then also passed through the filter. The heart preparation was more viscous, so it was washed with 20 mL of cold filtered saline. The sample was centrifuged at 500 g for 6 minutes. The supernatant was discarded and the pellet was suspended in 1 mL of 4°C FACS buffer. After red blood cell (RBC) lysis and blocking, the single cell suspension was stained with an immunocytoplate and quantified using an LSR II flow cytometer. Then, 500,000 live events were counted for the spleen control, and the cardiac cells of the entire tube were recorded and analyzed.

[0228] Western blotting. The frozen whole heart was crushed using a mortar and pestle apparatus. Approximately 20 mg of powdered tissue was added to 300 μL of radioimmunoprecipitation assay (RIPA) buffer and homogenized using a tissue homogenizer. The sample was then incubated on a 4°C rotator platform for one hour, followed by centrifugation at 12,000 g for 10 minutes to remove debris. The supernatant was 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 fluoride (PVDF) membrane. Immunoblotting was performed using antibodies against SRC-1, SRC-2, SRC-3, actin, and Hsp90. HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were used at a dilution of 1:2,500. 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 drug was washed off 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 overnight tube formation. The next day, the tubes were stained with calcein AM and imaged using the Cytation imaging system.

[0230] Immunostaining. Hearts were perfused with cardioplegia 20 mM KCL-PBS and then with 10% neutral buffered formalin, followed by drop fixation and processing into paraffin. Sections (7 microns) 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 No. 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 primary antibody (1:200 rabbit anti-lysozyme, abcam catalog number AB108508; Abcam, Cambridge, UK) in blocking solution, followed by secondary antibody (1:200 donkey anti-rabbit, Alexa 647, Thermo Fisher Scientific catalog number 31573; Thermo Fisher Scientific, Waltham, MA), followed by 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 spanning the entire myocardium of 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, VO2, and VCO2 measurements. RER, VO2, and VCO2 were measured by indirect calorimetry using a progressive maximal exercise test until mice reached exhaustion.

[0232] result

[0233] MCB-613 stimulates angiogenesis. The effects of MCB-613 on angiogenesis and stromal responses were studied, particularly in adult cardiac fibroblasts. SRC-1, 2, and 3 proteins were expressed in adult cardiac fibroblasts isolated from 10-week-old mice ( Fig. 10A Cardiac fibroblasts were transfected with expression vectors for GAL4 DNA binding domain-SRC-1, 2, and 3 fusion proteins and a GAL4-responsive luciferase reporter gene to measure SRC activation after MCB-613 treatment ( Fig. 10BIn 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 stimulates tube formation in adult cardiac fibroblasts in vitro ( Fig. 10C To investigate the stimulation of angiogenesis by MCB-613 in vivo, a chicken egg angiogenesis assay was performed ( Fig. 10D ). Direct administration of MCB-613 to chicken eggs stimulated angiogenesis in vivo. In addition, the introduction of mouse embryonic fibroblasts pre-stimulated with MCB-613 may promote robust angiogenesis in a cell-non-autonomous manner. Without being bound by theory, these findings suggest that angiogenesis stimulation by MCB-613 may 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, suggesting that MCB-613 promotes angiogenesis in injured tissue and restores blood flow in the setting of vascular disease ( Fig. 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 for testing cardiovascular therapeutic interventions. To explore the effects of SRC stimulation on early and late post-infarction cardiac function and remodeling, mice undergoing MI were treated with MCB-613 or vehicle control. Mice were given 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 ( Fig.11A ). 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, ejection fraction in control-treated animals was reduced to an average of 30%. In contrast, mice treated with MCB-613 had an average ejection fraction of 43% two hours after MI, indicating that MCB-613 prevented the early reduction in ejection fraction and provided early protection to vulnerable myocardium ( Fig. 11B). Ejection fraction in control-treated mice decreased further after 24 hours and was lowest at 19 weeks post-MI, indicating a progressive loss of cardiac function over time. In contrast, following administration of MCB-613, ejection fraction was maintained above 40% from 24 hours post-MI until 19 weeks post-MI, indicating that the early cardioprotective effects of MCB-613 can prevent progressive loss of cardiac function. Repeated injections over three days at 8 and 16 weeks did not change ejection fraction, indicating that MCB-613 has no further effects on cardiac function at later time points. Maintenance of cardiac function for up to 19 weeks post-MI suggests that short-term, early intervention may be effective in preventing congestive heart failure after MI. Heart weight analysis showed that MCB-613 attenuated the compensatory response of MI-induced cardiac hypertrophy at 12 weeks post-MI ( Fig. 11C ), suggesting that protecting cardiac function is associated with preventing another key feature of heart failure. Cardiac positron emission tomography (PET) imaging was then used to spatially assess myocardial viability. 18 Improved F-FDG uptake suggests that MCB-613 protects healthy myocardium 2 weeks after MI ( Fig.11D Cardiac tissue sections were stained with picrosirius red to assess infarct size and degree of fibrosis ( Fig.11E Infarct size measured 12 weeks post-MI was larger in control-treated hearts (31% and 44%) compared with hearts from MCB-613-treated mice (3%, 14%, 20%, and 22%). In addition, cardiomyocytes were smaller and associated with less fibrosis in the infarct border zone, demonstrating that MCB-613 can prevent two additional key molecular features of progressive heart failure ( Fig.11E Cardiometabolic 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 with age-matched mice without MI as controls ( Fig.12 ). Three weeks after MI, VCO2 and VO2 were increased in MCB-613-treated animals, indicating that MCB-613 improves energy utilization during exercise in mice after MI. Thus, improved cardiac function correlates with improved energy expenditure. Electron micrographs of hearts 72 hours after MI showed that MCB-613 prevented myofibril disorganization and abnormal mitochondrial cristae architecture, indicating that MCB-613 protects the myocardium and mitochondria from MI-induced injury ( Fig.11F ). Supporting early myocardial protection, MCB-613 prevented apoptosis 24 hours after MI ( Fig.11G ). These findings suggest that MCB-613 has a direct effect in protecting functional myocardium and preventing deleterious remodeling of cardiac tissue.

[0236] MCB-613 prevents myocardial injury responses. To gain insight into the myocardial and non-myocyte-specific transcriptional functions of MCB-613 associated with reduced myocardial remodeling and improved cardiac function, transcriptome profiling of purified cardiac cells from control-treated and MCB-613-treated mice was performed 12 weeks after MI ( Fig.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 ( Fig. 13B ). Gene set enrichment analysis of differentially expressed genes showed that gene onogeny categories representing oxidative phosphorylation and adipogenesis as well as inhibition of apoptosis and inflammatory responses showed strong enrichment ( Fig. 13C ), providing further support that MCB-613 improves 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. Non-cardiomyocyte metabolically active, viable single-cell suspensions were prepared from whole hearts following Langendorff perfusion ( Fig.13A Minimal manipulation of procedures was performed to prevent loss of cell types and minimize effects on transcriptional activity. Transcriptional profiles of 21,894 cells from two saline-treated mice and 21,474 cells from two MCB-613-treated mice that passed RNA quality control were analyzed by Seurat analysis using the 10x Chromium platform. Fifteen distinct cell clusters were identified based on cellular expression patterns, unsupervised clustering, and dimensionality reduction analysis using Seurat software analysis. Fig.13D Cluster sizes ranged from 101 to 6,085 cells. Cell populations were identified based on known mouse cardiac cell type markers ( Fig.13E ). Macrophages comprised 42% of the total cells analyzed and were the major non-cardiomyocyte cell population 12 weeks post-MI, in contrast to normal adult mouse hearts, where non-cardiomyocytes consisted of only 10% hematopoietic-derived cells. Assessment of the greatest changes in cell numbers demonstrated that in hearts from MCB-613-treated mice, there was a decrease in the number of macrophage cluster 1 and B lymphocytes, while there was an increase in the number of epicardial cells, NK / T lymphocytes, fibroblasts, and endothelial cells (including lymphocytes) ( Fig.13FCardiac macrophage, fibroblast, and endothelial cell populations showed transcriptional heterogeneity and consisted of four, three, and two subclusters, respectively. Evaluation of unique gene signatures in cardiac fibroblasts revealed the presence of a population of injury-responsive fibroblasts expressing Postn (fibroblast cluster 2), and supporting the recently reported “homeostatic fibroblasts” in the remodeling heart, fibroblasts in cluster 3 uniquely expressed Comp( Fig.14A ). Genes uniquely enriched in fibroblast cluster 1 suggest the presence of fibroblast subpopulations involved in secretory functions that promote angiogenesis (Bmp4, Ecm1, Ccl11, Pgf) and extracellular matrix organization (Ecm2 and Pdgfra). Endothelial cell transcriptional signatures indicated the presence of 3 subpopulations. Compared to endothelial cluster 1, endothelial cluster 2 and lymphatic endothelial cells showed increased transcriptional activation of 218 and 308 unique genes, respectively, suggesting distinct roles in cardiac maintenance 12 weeks post-MI ( Fig. 14B ). The lymphatic endothelial cluster was defined by unique expression of the lymphatic endothelial genes Prox1 and Lyve1. In addition to the increased cell number, the concurrent expression of the pro-angiogenic regulators Hif1a and Lrg1 and the lymphangiogenic regulator Ccl121a also indicated that MCB-613 stimulated lymphangiogenesis 12 weeks after MI. The gene expression signature in endothelial cluster 2, defined by unique expression of the early cardiac genes Mkl2, Tek, and Hand2, indicated a transcriptional reversion to a more primitive cellular state, which may be due to the injury stress response. Without being bound by theory, the largest endothelial cluster (i.e., endothelial cluster 1) represents endogenous steady-state endothelial cells characterized by a small number of unique genes and the absence of any relevant GO phases or signaling pathways. The transcriptional signatures of the four macrophage subclusters were clearly separable ( Fig. 14C ). Macrophage cluster 1 is the largest subset of macrophages and is defined by the expression of an inflammatory gene signature including Ccl8, Ccl24, and Ly96, which are compatible with a role for these genes in resolving myocardial inflammation. Cluster 2 represents a population of Ccr2+ monocyte-derived macrophages that, in response to injury, express the inflammatory genes Cxcl1, Ccr2, Ccr5, and Tlr2, known to be short-lived infiltrating macrophages derived from the bone marrow. In contrast, macrophages in cluster 3 uniquely express 110 cell cycle proliferation genes, suggesting the presence of a proliferative Ccr2+ monocyte-derived macrophage population. - Small populations of macrophages, known to be maintained by local proliferation that play a role in tissue repair and myogenesis. Macrophages in cluster 4 were identified based on expression of genes involved in phagocytic activation, including Cd209 and Coro1a, suggesting the presence of a small population of phagocytic macrophages 12 weeks post-MI. Surprisingly, the relatively small changes in the number of cell populations associated with improved maintenance of cardiac function suggest that MCB-613 cardioprotection may instead be a result of changes in cellular function.

[0238] MCB-613 promotes beneficial paracrine signaling. To determine the functional responses of interstitial cell types associated with MCB-613-mediated improvements in cardiac function 12 weeks after MI, transcriptome profiles in non-cardiomyocytes from control-treated and MCB-613-treated mice were compared ( Fig.15A The large differences in transcriptional responses of the cell populations suggest that MCB-613 selective cellular responses contribute to improved cardiac function. Smaller cell populations consisting of lymphatic cell populations and immune cell populations experienced the greatest drug-induced transcriptome changes. To identify potential interstitial cell signaling interactions that contribute to improved cardiac function, the number of interactions between ligands and receptors for each cell type in control hearts compared to hearts from MCB-613-treated mice was calculated ( Fig. 15B The highest frequency interactions occurred between ligands from each fibroblast population, one macrophage subtype, and the endothelial / SMC population that propagated to granulocyte receptors. This pattern of interstitial cell signaling to granulocytes suggests extensive paracrine regulation of granulocyte function in the MCB-613 cardioprotective response 12 weeks post-MI. Ligand-receptor pairing suggests coordinated regulation of tissue architecture and anti-inflammatory signaling pathways, including MMP9-LRP1, HSP90B1-TLR7, and SERPINE1-TAUR ( Fig. 15C ). Supporting this, gene set enrichment analysis of up- and down-regulated gene signatures in granulocytes indicated that MCB-613 inhibits inflammatory granulocyte function ( Fig.14D ). The most highly up-regulated and down-regulated genes in cardiac granulocytes from MCB-613-treated mice compared to controls showed increased expression of granules involved in innate defense and decreased expression of cytokines, enzymes, and chemokines involved in inflammatory signaling ( Fig.15D These findings suggest that the myocardial response to MCB-613 is characterized by a sustained paracrine anti-inflammatory signaling landscape that underlies the improvement in cardiac function.

[0239] Since MCB-613 was administered at the time of injury, an immediate response ( Fig. 11B and Fig.11E ), so the effect of MCB-613 on immune cells was measured 24 hours after MI. The immunophenotype of single cells isolated from whole hearts was analyzed by FACS 24 hours after MI to quantify the composition of immune cells. Similar to what was observed 12 weeks after MI, there was a significant decrease in B cells, a trend toward a decrease in monocytes, and no change in the fraction of granulocytes in hearts from MCB-613-treated mice compared to controls ( Fig.16A). The acute granulocyte transcriptional response to MCB-613 and the robust transcriptional response in granulocytes due to the presence of paracrine signaling were then measured 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 extent of the inflammatory response triggered by an acute heart attack and the damage caused to the myocardium. Due to the difficulty in isolating sufficient amounts of intact granulocytes from the mouse heart, in order to study the granulocyte response, bone marrow granulocytes were isolated, which reflect the myocardial granulocyte response 24 hours after MI. The successful isolation of granulocytes was indicated by increased mRNA expression of the granulocyte marker S100A9 in granulocytes compared to granulocyte-depleted bone marrow ( Fig. 16B ). Increased expression of Tlr7 and Lcn2 in granulocytes from mice treated with MCB-613 supports single cell transcriptome analysis and reveals that modulation 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 administered control vehicle or MCB-613. No changes in cell number or gene expression were observed, suggesting that granulocyte gene expression changes were a result of MCB-613-mediated myocardial injury response. MCB-613 induces a strong transcriptome response in granulocytes, suggesting that neutrophil granules may modulate the post-MI inflammatory effects of the compound. Supporting this, LYZ1 granule expression was significantly increased in the myocardium of mice treated with MCB-613 compared to control animals 24 hours after MI ( Fig. 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 ratio of 1:9, and administered to CD-1 mice intraperitoneally (ip) or orally (po) by gavage. After administration of the compound, blood samples (3 mice per compound) were collected through the tail vein at nine time points, i.e., 5 minutes, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours. Plasma was separated from these blood samples, and 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 ), the time to reach the maximum concentration after administration of the compound (t max ), the maximum observed concentration of the compound (C max ), until the area under the curve (AUC 0-t ), the area under the curve (AUC 0-inf ) and the clearance rate (Cl) of the compound.

[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 orally administered 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 (ng / mL*h)]]> 24.3 112.6 134.6 Cl(mg) / (ng / mL) / h 34 7.3 6.1

[0248] Pharmacokinetic data are also shown in Fig.17 middle. Fig.17The graph in the middle shows the mean plasma concentration measured over time. The upper row of graphs shows pharmacokinetic data from the above study, in which MCB-613 (upper left curve), compound 10-1 (upper middle curve) and compound 10-2 (upper right curve) were administered intraperitoneally to CD-1 mice. The lower row of graphs shows pharmacokinetic data from the above study, in which MCB-613 (lower left curve), compound 10-1 (lower middle 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 the present disclosure. Various modifications of compounds and methods other than those shown and described herein are intended to fall within the scope of the appended claims. Further, although 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. Therefore, a combination of steps, elements, components or ingredients may be explicitly mentioned herein; however, all other combinations of steps, elements, components and ingredients are included even if not explicitly stated.

Claims

1. A compound selected from the group consisting of: or a pharmaceutically acceptable salt or prodrug thereof.

2. Use of the compound of claim 1 in the preparation of a medicament for treating ischemic injury in a subject, wherein the ischemic injury is myocardial infarction.

3. Use of a compound as claimed in claim 1 in the preparation of a medicament for reducing the size of myocardial infarction in a subject who has suffered a myocardial infarction.

4. The use of claim 3, wherein the myocardial infarction size is reduced by at least 5% compared to the myocardial infarction size in an untreated subject who has suffered a myocardial infarction.

5. The use of claim 3 or 4, wherein the myocardial infarction size is reduced by at least 15% compared to the myocardial infarction size in an untreated subject who has suffered a myocardial infarction.

6. Use of the compound of claim 1 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 who has suffered myocardial infarction.

7. Use of the compound of claim 1 in the preparation of a medicament for improving cardiovascular function in a subject who has suffered a myocardial infarction.

8. The method of claim 2, wherein the subject is an elderly subject.

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