Methods and formulations for treating mitochondrial dysfunction

Abstract

Provided are therapeutic methods and pharmaceutical preparations configured to treat primary and secondary mitochondrial dysfunction. The methods and treatments use agonists of AMPK. The agonists activate AMPK to activate ATP production pathways and inhibit ATP consumption pathways, thereby allowing for the alleviation of symptoms associated with mitochondrial dysfunction.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 786,970, filed on December 31, 2018, entitled “Methods and Formulations to Treat Mitochondrial Dysfunction” to Moore et al.; the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to therapeutic methods and pharmaceutical preparations for treating primary and secondary mitochondrial dysfunction. Background Art

[0004] Previously studied drug candidates are conventional AMP-dependent AMPK activators whose mechanism of action requires AMP elevation via RC inhibition (e.g., metformin, resveratrol) or conversion to an AMP mimetic (e.g., AICAR). Indirect mechanisms involving RC inhibition are not applicable in cases of underlying mitochondrial dysfunction, whereas AMP-dependent activation of AMPK leads to activation of other AMP-regulated enzymes, thereby achieving compound pleiotropic effects. In addition, previous studies have identified direct, AMP-independent AMPK agonists for the purpose of treating diabetes, obesity, and metabolic syndrome.

[0005] Primary mitochondrial diseases are a group of clinically heterogeneous conditions that are often progressive, multisystemic, and associated with high mortality in children. They are caused by inherited defects in the mitochondrial respiratory chain (RC), resulting in increased production of reactive oxygen and nitrogen species (ROS and RNS) and insufficient overall energy production. These resulting metabolic imbalances lead to cellular damage and ultimately cell death.

[0006] There is currently no curative treatment for primary mitochondrial disease. Only supportive care is available and involves treating specific symptoms (e.g., diabetes, heart disease, and ptosis) and a "mitochondrial cocktail" consisting of vitamin cofactors and antioxidants. Unfortunately, meta-analyses have shown that the available supportive interventions lack efficacy, highlighting the need for new treatments (see, e.g., Pfeffer et al., Cochrane Database Sys Rev., April 18, 2012; 4; Chinnery et al., 2006, Cochrane Database Sys Rev., January 25, 2006; (1); the disclosures of which are incorporated herein by reference in their entirety).

[0007] Secondary mitochondrial diseases also exhibit mitochondrial dysfunction, but unlike primary mitochondrial diseases, they are not caused by genes related to the mitochondrial respiratory chain. Secondary mitochondrial diseases such as Parkinson's disease or Alzheimer's disease are due to acquired mitochondrial abnormalities caused by other diseases, disorders or environmental factors that indirectly damage mitochondria. Therefore, any treatment determined for primary mitochondrial diseases is expected to be equally beneficial for disorders and conditions associated with secondary mitochondrial dysfunction, including neurodegenerative disorders, neuromuscular disorders and muscle wasting disorders. Summary of the invention

[0008] Disclosed herein are methods and formulations for treating mitochondrial dysfunction according to embodiments of the present invention.

[0009] In one embodiment, a method of treating a patient suffering from mitochondrial dysfunction comprises identifying mitochondrial dysfunction in an individual and providing an AMPK agonist to the individual.

[0010] In another embodiment, the mitochondrial dysfunction is a primary mitochondrial dysfunction.

[0011] In another embodiment, the primary mitochondrial dysfunction is selected from the group consisting of autosomal dominant optic atrophy (ADOA), Alpers-Huttenlocher syndrome (nDNA defect), ataxia neuropathy syndrome (nDNA defect), Barth syndrome / lethal infantile cardiomyopathy (LIC), coenzyme Q deficiency, complex I, complex II, complex III, complex IV and complex V deficiency (single deficiency or any combination of deficiency), chronic progressive external ophthalmoplegia (CPEO), diabetes and deafness, Kearns-Sayre syndrome (mtDNA defect), white matter disease with brainstem and spinal cord involvement and increased lactate (LBSL-leukodystrophy), Leigh syndrome (mtDNA and nDNA defects), Leber's hereditary optic neuropathy (LHON), Rafter disease, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke syndrome (MELAS) (mtDNA defect), mitochondrial enoyl-CoA reductase protein-associated neurodegeneration (MEPAN), myoclonic epilepsy with ragged red fibers (MERRF), mitochondrial recessive ataxia syndrome (MIRAS), mtDNA deletion syndrome, mtDNA depletion syndrome, mtDNA maintenance disorder, mtDNA / RNA translation defect, mitochondrial tRNA synthetase deficiency, mitochondrial myopathy, mitochondrial neurogastroenteric encephalopathy syndrome (MNGIE), neurogenic myasthenia, ataxia, and retinitis pigmentosa (NARP), Pearson syndrome, pyruvate dehydrogenase complex deficiency (PDCD / PDH), DNA polymerase gamma deficiency (POLG), pyruvate carboxylase deficiency, and thymidine kinase 2 deficiency (TK2).

[0012] In another embodiment, the mitochondrial dysfunction is secondary mitochondrial dysfunction.

[0013] In another embodiment, the secondary mitochondrial dysfunction is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and other dementias, Friedreich's ataxia (FA), Huntington's disease (HD), motor neuron disease (MND), N-glycanase deficiency (NGLY1), organic acidemias, Parkinson's disease (PD) and PD-related disorders, prion diseases, spinal muscular atrophy (SMA), spinocerebellar ataxia (SCA), Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, Emory-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular dystrophy, Charcot-Marie-Duce disease, congenital myopathies, distal myopathies, endocrine myopathies (hyperthyroid myopathy, hypothyroid myopathy), giant axonal neuropathy, hereditary spastic paraplegia, inflammatory myopathies (dermatomyositis, inclusion body myositis, polymyositis), metabolic myopathies, neuromuscular junction disorders, autism, cancer, diabetes, metabolic syndrome, chronic fatigue syndrome, inflammatory disorders, arthritis, and aging.

[0014] In another embodiment, the AMPK agonist is a direct AMPK agonist.

[0015] Again, in another embodiment, the direct AMPK agonist is selected from the group consisting of PT1, ETC-1002, salicylate, C991, C13, D561-0775, MT 63-78, A-769662, ZLN024, C24, MK-8722, PF-739 and PF-06409577.

[0016] Again, in another embodiment, the AMPK agonist is an AMP-dependent agonist.

[0017] In another embodiment, the AMP-dependent agonist is selected from the group consisting of metformin, resveratrol, and AICAR.

[0018] In another embodiment, the AMPK agonist is provided in the form of a pharmaceutical formulation.

[0019] In yet another embodiment, the pharmaceutical formulation comprises an AMPK agonist and at least one of the group consisting of a binder, a lubricant, a buffer, and a coating.

[0020] In yet another embodiment, the providing step comprises orally administering an AMPK agonist to the individual.

[0021] Again, in another embodiment, the providing step comprises administering the AMPK agonist daily for at least one week.

[0022] Again, in another embodiment, the method further comprises evaluating the efficacy of an AMPK agonist in the individual.

[0023] In another embodiment, the providing step is accomplished by administering the AMPK agonist by at least one of the group consisting of oral administration, subcutaneous administration, intravenous administration, intraperitoneal administration, intranasal administration, dermal administration, and inhalation.

[0024] In another embodiment, a method of treating a mitochondrial disorder comprises identifying a disorder in an individual and modulating AMPK activity in the individual.

[0025] Again, in yet another embodiment, the modulating step is accomplished by activating AMPK in the individual.

[0026] In yet another embodiment, the activating step is accomplished by phosphorylating AMPK or providing an agonist to AMPK.

[0027] In yet another embodiment, the modulating step is accomplished by inhibiting AMPK in the individual.

[0028] In yet another embodiment, the disorder is associated with mitochondrial dysfunction.

[0029] Again, in another embodiment, the mitochondrial dysfunction is a primary mitochondrial dysfunction.

[0030] Again, in another embodiment, the mitochondrial dysfunction is secondary mitochondrial dysfunction.

[0031] Again, in yet another embodiment, the AMPK agonist comprises the formula

[0032] The molecules,

[0033] in:

[0034] A is selected from unsubstituted or substituted with one or more C 1-6 Alkyl or fluorine-substituted 5-membered heterocyclic ring

[0035] X is CR5 or N;

[0036] R1 is H, CF3 or halo;

[0037] R2 is OR5, NHOH, NHSO2R4, OCH2OCOR4, or COR2 is C-linked tetrazole,

[0038] R3 is unsubstituted or substituted by one to three substituents selected from halo, OH and OCOR7. 1-10 Alkyl, C 3-7 Cycloalkyl, C 4-12 Alkylcycloalkyl, C 4-10 Cycloalkylalkyl, C 3-7 Heterocycloalkyl, C 4-12 Alkylheterocycloalkyl, C 4-10 heterocycloalkylalkyl, aryl or heteroaryl;

[0039] R4 is unsubstituted or substituted with one to three halogen substituents. 1-10 Alkyl, C 3-7 Cycloalkyl, C 4-12 Alkylcycloalkyl, C 4-10 Cycloalkylalkyl;

[0040] R5 is R4 or H;

[0041] R6 is H, C 1-6 Alkyl, C 3-6 Cycloalkyl or two R6 groups together with the nitrogen atom to which they are attached may form a four- to seven-membered heterocycloalkyl ring, all of which may be optionally substituted with 1 to 3 fluorine atoms; and

[0042] R7 is unsubstituted or substituted by one to three fluorine, C 1-10 Alkyl and NR6, R6 substituted C 1-10 Alkyl, C 3-7 Cycloalkyl, C 4-12 Alkylcycloalkyl.

[0043] Again, in yet another embodiment, A is selected from the group consisting of:

[0044] In yet another embodiment, A is selected from the group consisting of:

[0045]

[0046] In yet another embodiment, A is selected from the group consisting of:

[0047]

[0048] Again, in yet another embodiment, X is CH, R6 is H, R1 is Cl, and R2 is OH.

[0049] Again, in yet another embodiment, the molecule has Figures 13A-13Z and Figures 14A-14O The formula shown in one of them.

[0050] Again, in yet another embodiment, the molecule has the formula:

[0051]

[0052] Again, in yet another embodiment, the AMPK agonist comprises a molecule of the formula:

[0053]

[0054] in:

[0055] X is CH or N;

[0056] R1 is a small alkyl group (C1-C4) or a halogen (e.g., Cl, Br or F);

[0057] R2 is phenyl, alkyl C1-C10, cycloalkyl (C3-C10), hydroxyalkyl (C1-C6), heteroaromatic (e.g., pyridyl, pyrazolyl, pyrrolyl, pyrimidinyl, thienyl, furanyl or triazole) or heterocyclic C4-C6;

[0058] wherein phenyl is optionally substituted by halogen (e.g., Cl, Br or F),

[0059] wherein the alkyl group is linear or branched and is optionally substituted by OH, OMe, OEt,

[0060] wherein the cycloalkyl group is optionally substituted with one or more OH groups,

[0061] The hydroxyalkyl group is a straight chain or branched

[0062] wherein the heteroaromatic group is optionally substituted with a small alkyl group (C1-C6) or a hydroxyalkyl group (C1-C6), wherein the alkyl group and the hydroxyalkyl group are linear or branched,

[0063] wherein the heteroatom in the heterocycle is optionally O, S or NR4, wherein R4 is a linear or branched C1-C6 alkyl or hydroxyalkyl; and

[0064] R4 is a linear or branched C1-C6 alkyl or hydroxyalkyl group.

[0065] In another embodiment, the molecule has the formula:

[0066]

[0067] In another embodiment, the AMPK agonist comprises a molecule of the formula:

[0068]

[0069] in:

[0070] A is a fused ring (e.g., C3-C10 cycloalkyl);

[0071] X is CH or N;

[0072] Y is O, S, NH, or NR3;

[0073] R1 is a small alkyl group (C1-C4) or a halogen (e.g., Cl, Br or F);

[0074] R2 is phenyl, alkyl C1-C10, cycloalkyl (C3-C10), hydroxyalkyl (C1-C6), heteroaromatic (e.g., pyridyl, pyrazolyl, pyrrolyl, pyrimidinyl, thienyl, furanyl or triazole) or heterocyclic C4-C6;

[0075] wherein phenyl is optionally substituted by halogen (e.g., Cl, Br or F),

[0076] wherein the alkyl group is linear or branched and is optionally substituted by OH, OMe, OEt,

[0077] wherein the cycloalkyl group is optionally substituted with one or more OH groups,

[0078] The hydroxyalkyl group is a straight chain or branched

[0079] wherein the heteroaromatic group is optionally substituted with a small alkyl group (C1-C6) or a hydroxyalkyl group (C1-C6), wherein the alkyl group and the hydroxyalkyl group are linear or branched,

[0080] wherein the heteroatom in the heterocycle is optionally O, S or NR4;

[0081] R3 is C1-C6 alkyl (linear or branched); and

[0082] R4 is a linear or branched C1-C6 alkyl or hydroxyalkyl group.

[0083] In yet another embodiment, A is selected from a C6 benzene ring or one of the following:

[0084]

[0085] Again, in another embodiment, the molecule has the formula:

[0086] BRIEF DESCRIPTION OF THE DRAWINGS

[0087] These and other features and advantages of the present invention will be better understood with reference to the following detailed description considered in conjunction with the accompanying drawings, in which:

[0088] Figure 1A Pathways activated and inhibited by AMPK according to various embodiments are shown.

[0089] Figure 1B-1D Shown are structures of AMPK agonists according to various embodiments.

[0090] Figures 2A-2F Shown are the results of cell viability assays under various treatments according to various embodiments.

[0091] Figures 3A-3N Shown are the cell morphologies of fibroblasts under various treatments according to various embodiments.

[0092] Figure 4A Shown are pAMPK concentrations in knockdown experiments according to various embodiments.

[0093] Figure 4B Shown are cell viability results from knockdown studies with various treatments according to various embodiments.

[0094] Figures 4C-4F Shown are cell morphology results from knockdown studies with various treatments according to various embodiments.

[0095] Figure 5A Shown are pAMPK concentrations after treatment experiments according to various embodiments.

[0096] Figures 5B-5D Gene expression levels after treatment experiments according to various embodiments are shown.

[0097] Figures 6A-6H Shown are the results of cellular respiration with and without treatment on various defective fibroblast cell lines according to various embodiments.

[0098] Figures 7A-7C Shown are ATP concentrations with and without treatment of various defective fibroblast cell lines according to various embodiments.

[0099] Figure 7D-7F Shown are reactive oxygen species (ROS) concentrations with and without treatment of various defective fibroblast cell lines according to various embodiments.

[0100] Fig. 8A Shown are lipofuscin levels in control and NGLY1 -deficient fibroblast cell lines according to various embodiments.

[0101] Figure 8B Shown are lipofuscin levels in an NGLY1 -deficient fibroblast cell line and with and without treatment according to various embodiments.

[0102] Fig. 9AFundusoscopic images of the retina with and without treatment in control and induced AMD models are shown according to various embodiments.

[0103] Fig. 9B Shown are images of retinal segments of the retina with and without treatment in control and induced AMD models according to various embodiments.

[0104] Fig. 9C Shown are ONL thickness measurements of the retina with and without treatment in control and induced AMD models according to various embodiments.

[0105] Fig.9D Shown are electroretinogram images of the retina with and without treatment in control and induced AMD models according to various embodiments.

[0106] Fig.9E Shown is reduced retinal degeneration following treatment with an AMPK agonist according to various embodiments.

[0107] Fig. 10A The percentage of infarcted tissue in treated and untreated ischemic tissue according to various embodiments is shown.

[0108] Fig. 10B and Fig. 10C Shown are brain slices with infarct tissue in treated and untreated ischemic tissue according to various embodiments.

[0109] Figures 11A-11C Shown are the motor performances of treated and untreated mice according to various embodiments.

[0110] Fig.11D and Fig.11E Mitochondrial function and glycogen storage in treated and untreated mice according to various embodiments are shown.

[0111] Fig.11F and Fig.11G Shown are the results of ACC phosphorylation assays performed on treated and untreated mice according to various embodiments.

[0112] Fig. 12A The general structure of an AMPK agonist according to various embodiments is shown.

[0113] Figures 12B-12D A schematic method for synthesizing AMPK agonists according to various embodiments is shown.

[0114] Figures 13A-13Z Shown are structures of AMPK agonists according to various embodiments.

[0115] Figures 14A-14O Shown are structures of AMPK agonists according to various embodiments.

[0116] Figures 15A-15B The general structure of an AMPK agonist according to various embodiments is shown.

[0117] Fig. 15C A schematic diagram of a method of synthesizing an AMPK agonist according to various embodiments is shown.

[0118] Fig.15D Shown are structures of AMPK agonists according to various embodiments.

[0119] Fig.16A Shown are the results of an ACC phosphorylation assay performed on mitochondrial-deficient cells according to various embodiments.

[0120] Fig. 16B Shown are AMPK activities of AMPK agonists used to determine EC50 according to various embodiments.

[0121] Fig.17 Methods of treating an individual according to various embodiments are shown.

[0122] Fig.18 Methods of modulating AMPK activity in an individual according to various embodiments are presented. DETAILED DESCRIPTION

[0123] Turning now to data and instructions, methods and pharmaceutical preparations configured to treat mitochondrial disorders, diseases and / or dysfunctions are described. In various embodiments, the methods and preparations use agonists of AMP-activated protein kinase (AMPK). AMPK is a major regulator of cellular energy homeostasis and is activated to restore the level of adenosine triphosphate (ATP) when energy is insufficient. Generally speaking, AMPK restores energy homeostasis by activating ATP production pathways (e.g., glycolysis, mitochondrial biogenesis) and inhibiting ATP consumption pathways (e.g., gluconeogenesis and fatty acid synthesis). Other pathways or processes promoted by AMPK include antioxidant enzymes, autophagy, fatty acid metabolism and muscle regeneration. FIG. 1 shows a variety of pathways activated or inhibited by AMPK. AMPK's regulation of glucose and fatty acid pathways makes it an attractive therapeutic target for diabetes, obesity and metabolic syndrome, thereby determining that AMPK agonists are used for this purpose.

[0124] To overcome the deficiencies of indirect AMPK agonists, certain embodiments relate to direct, AMP-independent AMPK agonists to treat conditions and disorders with underlying mitochondrial dysfunction. AMPK direct activating compounds include PT1, ETC-1002, salicylate, C991, C13, D561-0775, MT 63-78, A-769662, ZLN024, C24, MK-8722 ( Figure 1B )、PF-739( Figure 1C ) and PF-06409577( Figure 1D ), which allosterically activates AMPK by directly binding to the α or β subunits and, in some cases, has been shown to improve lipid and glucose profiles in vivo (see, e.g., Cool et al., Cell Metab., 2006 Jun, 3(6):403-16; Pang et al., J Biol Chem., 2008 Jun 6, 283(23):16051-60; Hawley et al., Science., 2012 May 18, 336(6083):918-22; Li et al., Toxicol Appl Pharmacol., 2013 Dec 1, 273(2):325-34; Zhang et al., PLoS One., 2013 Aug 20, 8(8); Xiao et al., Nat Commun., 2013, 4:3017; Zadra et al., EMBO Mol. Med., Apr. 2014, 6(4):519-38; Hunter et al., Chem Biol., Jul. 17, 2014, 21(7):866-79; Pinkosky et al., Nat Commun., Nov. 28, 2016, 7:13457; Cameron et al., Sep. 8, 2016, 59(17):8068-81; Myers et al., Science., Aug. 4, 2017, 357(6350):507-511; Cokorinos et al., Cell Metab., May 2, 2017, 25(5):1147-1159; Xi et al., Oncotarget., Nov. 10, 2017, 8(56):96089–96102; the disclosures of each of these references are incorporated herein by reference in their entirety).

[0125] Treatment with various embodiments of direct AMPK agonists increases cellular respiration and ATP levels and reduces oxidative stress, resulting in sustained and effective improvements in cell viability in patients with primary and secondary mitochondrial diseases, including a variety of conditions and disorders associated with secondary mitochondrial dysfunction. Diseases and conditions associated with primary and secondary mitochondrial dysfunction are listed in Tables 1 and 2, respectively. In addition, direct AMPK agonists can prevent retinal degeneration in age-related macular degeneration (AMD), provide neuroprotection against ischemic stroke, and are sufficient to enhance athletic performance. In addition, direct AMPK agonists can reduce known negative effects associated with mitochondrial dysfunction in primary and secondary mitochondrial disorders, such as retinal damage, neuronal degeneration, and muscle atrophy. Thus, certain embodiments relate to the treatment of N-glycanase (NGLY1) deficiency, age-related macular degeneration (AMD), ischemic stroke, muscular dystrophy (e.g., Duchenne and Becker), Friedreich's ataxia (FA), autoimmune disorders with muscle involvement (e.g., inclusion body myositis, polymyositis, and dermatomyositis), and / or neurodegenerative disorders (e.g., amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease). In addition, AMPK activation in cardiac tissue can lead to reversible cardiac hypertrophy, and thus various embodiments relate to the treatment of diseases associated with dilated cardiomyopathy (see, e.g., Arad et al., Circ Res., Mar. 2, 2007, 100(4):474-88; Myers et al., Science, Aug. 4, 2017, 357(6350):507-511; the disclosures of which are incorporated herein by reference in their entirety).

[0126]

[0127]

[0128]

[0129] Characterization of AMPK agonist properties

[0130] The properties of selected AMPK agonists are provided below to characterize the performance of exemplary embodiments of the present invention. Although some specific agonists are discussed, it should be understood that the results are only intended to provide an overview of agonist function and are not intended to be limiting.

[0131] Effects of direct AMPK agonists on cell viability and morphology

[0132] Various embodiments utilize direct AMPK agonists to increase cell viability and morphology in individuals suffering from mitochondrial dysfunction. Figures 2A-2FThe effects of direct AMPK agonists on cell viability are shown for some embodiments. Figures 2A-2D The results show that in four cells lacking the cytochrome c oxidase assembly factor (SURF1, Figure 2A ), heme A: farnesyltransferase cytochrome C oxidase assembly factor (COX10, Figure 2B ), mitochondrial complex I (CI, Figure 2C ) and mitochondrial DNA polymerase gamma (POLG, Figure 2D ) in fibroblast cell lines. Figure 2A In the present invention, four direct AMPK agonists of various embodiments, PT1, A-769662, ZLN024 and C24, demonstrated a 35%-55% increase in viability compared to untreated cells (eg, cells treated with vehicle DMSO). Figure 2B-2D Shown are the effects of mitochondrial dysfunction on cell viability, including components of the respiratory chain and mitochondrial DNA (mtDNA) replication machinery. Figure 2B-2D , the direct AMPK agonist PT1 increased the survival of these mutants, despite the different pathogenic mechanisms of the genetic defects. In addition, the remaining compounds showed different responses between the mutants: A-769662 increased the survival of CI- and POLG-deficient cells, ZLN024 increased the survival of CI-deficient cells, and C24 increased the survival of SURF1-deficient cells but not any of the other cell lines. In addition, the indirect AMPK agonist AICAR did not increase cell viability in any of the other cell lines except SURF1 ( Figure 2A ), indicating that AICAR is not as effective as a direct AMPK agonist. The direct AMPK agonist PT1 showed similar results in fibroblasts exhibiting NGLY-1 deficiency, such as Figure 2E shown. Figure 2E Fibroblasts lacking NGLY-1 isolated from three individuals are shown. Fibroblasts treated with PT1 (+PT1) showed up to 60% increased viability compared to untreated (-PT1) cells. In addition, Figure 2F The effects of three AMPK agonists on cell viability are shown in various embodiments. Figure 2F In the present study, AMPK agonists MK-8722, PF-739, and PF-06409577 showed dose-dependent changes in the levels of cell viability in SURF1-deficient fibroblasts. Thus, direct AMPK agonists of various embodiments improve cell viability across a range of primary and secondary mitochondrial disease etiologies.

[0133] Go to Figures 3A-3N, according to many embodiments, the cell morphology of fibroblasts with various defects is shown. Specifically, Figure 3A and Figure 3B SURF1-deficient fibroblasts are shown, in which Figure 3A The morphology of untreated cells (DMSO only) is shown, while Figure 3B SURF1-deficient fibroblasts treated with PT1 showed Figure 3A Similarly, Figure 3C-3J Similar results were shown for cell morphology when treated with PT1, where Figure 3C and Figure 3D Fibroblasts lacking COX10 are shown. Figure 3E and Figure 3F Fibroblasts lacking CI are shown. Figure 3G and Figure 3H Fibroblasts lacking POLG are shown, and Fig. 3I and Figure 3J Fibroblasts lacking NGLY-1 are shown. Similarly, Figure 3C , Figure 3E , Figure 3G and Fig. 3I Untreated defective fibroblasts are shown, whereas Figure 3D , Figure 3F , Figure 3H and Figure 3J Fibroblasts treated with PT1 are shown. Figure 3K-3N The effects of the newly developed AMPK agonists MK-8722, PF-739, and PF-06409577 on SURF1-deficient fibroblasts are shown. Specifically, Figure 3K SURF1-deficient fibroblasts treated with vehicle DMSO alone are shown, whereas Figure 3L shows improved cell morphology after treatment with MK-8722, Figure 3M shows improved cell morphology after treatment with PF-739, and Figure 3N Improved cell morphology following treatment with PF-06409577 is shown. Thus, direct AMPK agonists of various embodiments improve cell morphology across a range of primary and secondary mitochondrial disease etiologies.

[0134] Now go to Figures 4A-4F , direct AMPK agonists of certain embodiments are specific for AMPK activation rather than non-specific interactions. Figure 4AWestern blot of phosphorylated AMPK (pAMPK) is shown, showing the relative levels of pAMPK and tubulin after treatment of COX10-deficient fibroblasts transfected with small interfering RNA (siRNA). siRNA specific for the AMPK alpha subunit gene PRKAA1 resulted in reduced pAMPK levels compared to cells transfected with nonspecific siRNA used as a control (siCNT). Figure 4B Cell viability of COX10-deficient fibroblasts with and without PT1 treatment and with and without siRNA transfection is shown, indicating that PT1 treatment alone (PT1) and the combination of PT1 treatment with nonspecific siCNTs (siCNT+PT1) increased viability compared to untreated cells (DMSO) and cells treated with PT1 and transfected with siAMPK (siAMPK+PT1). In addition, treatment with PT1 and selective knockdown of AMPK (siAMPK+PT1) resulted in a 63% decrease in pAMPK levels and a 41% decrease in cell viability compared to control conditions (siCNT+PT1), consistent with the concept that cellular responses to PT1 are mediated by AMPK activation. Figures 4C-4F Shown with Figure 4B The cell morphology under the same conditions. Specifically, Figure 4C Untreated COX10-deficient fibroblasts are shown; Figure 4D COX10-deficient fibroblasts treated with PT1 are shown; Figure 4E COX10-deficient fibroblasts transfected with siAMPK and treated with PT1 are shown; and Figure 4F Shown are COX10-deficient fibroblasts transfected with control siCNTs and treated with PT1.

[0135] Go to Figures 5A-5D , certain embodiments of direct AMPK agonists address metabolic perturbations caused by mitochondrial dysfunction. Figure 5A Shown are pAMPK levels normalized to actin in SURF1 -deficient fibroblasts treated with PT1 for 0, 2, 24, and 48 hours. Figure 5A It is shown that pAMPK levels increased within 2 hours after PT1 treatment and peaked at 24 hours after PT1 treatment. In addition, pAMPK levels returned to baseline levels 48 hours after PT1 treatment. Figures 5B-5D In, according to various embodiments, the effect of PT1 treatment is to increase gene expression of mitochondrial biogenesis and antioxidant genes. Figure 5B The expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) was increased by 2.4-fold, while Figure 5C and Figure 5D A 1.3-fold increase in the expression of manganese superoxide dismutase (SOD2) and a 1.5-fold increase in the expression of catalase were shown, respectively.

[0136] Effects of direct AMPK agonists on cellular respiration

[0137] Various embodiments improve cellular respiration in individuals with mitochondrial dysfunction. As described above, certain embodiments increase PGC-1α expression. PGC-1α stimulates the biogenesis of new mitochondria with enhanced respiratory function compared to aged mitochondria with accumulated secondary mtDNA mutations and reactive oxygen species (ROS). Go to Figures 6A-6H , showing improved respiratory function caused by direct AMPK agonists of some embodiments. Figures 6A-6C The basal oxygen consumption rate (OCR) of SURF1, POLG and NGLY1 deficient fibroblasts is shown, respectively. According to various embodiments, despite the etiology differences of each gene, the basal respiration of SURF1, POLG and NGLY1 deficient fibroblasts after treatment with PT1 is improved by about 30% for SURF1 and POLG deficient fibroblasts, and by 16% for NGLY1 deficient fibroblasts compared to untreated cells (DMSO). In addition, Fig.6D and Fig. 6E SURF1( Fig.6D ) and POLG( Fig. 6E In this case, treatment with PT1 showed an improvement of about 40% in SURF1-deficient fibroblasts compared to untreated (DMSO) fibroblasts ( Fig.6D ), and POLG-deficient fibroblasts improved by 30% ( Fig. 6E ), which is consistent with certain embodiments. In addition, according to some embodiments, treatment with PT1 improves maximal respiratory capacity, which reflects the ability of cells to respond to increased ATP demand, such as Figure 6F-6H As shown. Fig. 6F In the study, SURF1-deficient fibroblasts showed a maximal improvement of approximately 50% in respiration in cells treated with the direct AMPK agonist PT1 compared to untreated (DMSO) fibroblasts. Figure 6G It was shown that POLG-deficient fibroblasts showed a maximal improvement in respiration of about 20% in cells treated with the direct AMPK agonist PT1 compared to untreated (DMSO) fibroblasts. Furthermore, Figure 6HIt is shown that maximal respiration of NGLY1 -deficient fibroblasts was improved by about 40% when treated with the direct AMPK agonist PT1 compared to untreated (DMSO) fibroblasts.

[0138] Effects of direct AMPK agonists on oxidative stress and energy status

[0139] Now go to Figures 7A-7F Various embodiments increase ATP production and reduce reactive oxygen species (ROS) levels in mitochondrial dysfunction. Specifically, Fig. 7A , Figure 7B and Figure 7C When treated with the direct AMPK agonist PT1, SURF1-deficient fibroblasts showed a 35% increase in ATP production, POLG-deficient fibroblasts showed a 36% increase in ATP production, and NGLY1-deficient fibroblasts showed a 40% increase in ATP production compared to untreated (DMSO) fibroblasts. Fig.7D , Fig. 7E and Figure 7F When treated with the direct AMPK agonist PT1, ROS levels in SURF1-deficient fibroblasts were reduced by 10%, POLG-deficient fibroblasts by 15%, and NGLY1-deficient fibroblasts by 18%, respectively, compared to untreated (DMSO) fibroblasts. Mitochondrial dysfunction leads to incomplete electron transfer through the respiratory chain (RC), resulting in reduced ATP synthesis and excessive production of reactive oxygen species (ROS) (see, e.g., Atkuri et al., Proc Natl Acad Sci USA., Mar. 10, 2009, 106(10):3941-5; Enns et al., PLoS One., Jun. 18, 2014; 9(6); the disclosures of which are incorporated herein by reference in their entirety). ATP and ROS levels reflect cellular energy and oxidative status, both of which depend on efficient ATP-coupled respiration and overall mitochondrial function. The cellular response to these metabolic perturbations involves activation of pathways that improve cellular respiration (e.g., mitochondrial biogenesis) and upregulate the expression of endogenous antioxidants to neutralize ROS and reduce oxidative stress. Figures 7A-7F As shown, the increase in ATP production and the decrease in ROS levels demonstrate the ability of various embodiments to improve oxidative stress and energy status of mitochondrial dysfunction.

[0140] Effects of direct AMPK agonists on lipofuscin levels

[0141] Some embodiments reduce the accumulation of abnormal glycoproteins, such as Figures 8A-8BCertain mitochondrial dysfunction, including that caused by NGLY1 deficiency, leads to the accumulation of a protein and lipid called lipofuscin (see, e.g., Jobst et al., J Clin Pathol., 1991 May; 44(5):437–438; the disclosure of which is incorporated herein by reference in its entirety). Fig. 8A As shown, NGLY1-deficient fibroblasts exhibited approximately 60% higher lipofuscin levels compared to normal fibroblasts (control). Figure 8B It is shown that treatment with the direct AMPK agonist PT1 reduces lipofuscin levels in NGLY1-deficient fibroblasts by about 36%, according to various embodiments.

[0142] AMPK activation mitigates retinal degeneration in AMD

[0143] Now go to Figures 9A-9E , various embodiments reduce retinal damage caused by mitochondrial dysfunction. Specifically, Fig. 9A Fundusoscopic images of the retina treated with and without sodium iodate (SI) and with and without the direct AMPK agonist PT1 are shown according to certain embodiments. SI is a toxin that selectively induces oxidative stress and mitochondrial dysfunction in the retinal pigment epithelium (RPE). Thus, tissue treated with SI mimics retinal disorders caused by mitochondrial dysfunction, such as AMD. Fig. 9A As shown, tissues not treated with SI (-SI) with and without PT1 (+PT1 and -PT1) had similar normal tissues. However, tissues treated with SI but without PT1 (+SI; -PT1) showed signs of retinal degeneration, including white deposits. However, tissues treated with both SI and PT1 (+SI; +PT1) showed improved signs of retinal degeneration. Therefore, it is shown that the direct AMPK agonists of various embodiments alleviate retinal degeneration. Similarly, Fig. 9B Similar results are shown for H&E staining of retinal segments. As shown, tissues without SI treatment (-SI;-PT1 and -SI, +PT1) had a similar appearance. However, –SI; +-PT1 tissues showed signs of retinal degeneration, including thinning at the photoreceptor outer nuclear level (ONL), disorganization of the inner layer and photoreceptor outer segment / inner segment (IS / OS), and the presence of melanin debris. These indications of disease were alleviated by PT1 treatment in SI-treated tissues, including protection from ONL thinning (white bars), changes in photoreceptor morphology, and a reduction in melanin debris (black arrows). ONL thickness measurements are shown in Figure 5. Fig. 9CAs shown, tissues not treated with SI (SI-, PT1- and SI-, PT1+) and tissues treated with SI and PT1 (SI+, PT1+) showed greater thickness than tissues treated with SI without PT1 (SI+, PT1-). In addition, Fig.9D It was demonstrated that retinal tissue treated with SI and PT1 (PT1+; SI+) had improved electrical responses reaching levels similar to normal tissue (PT1-; SI- and PT1+; SI-) compared to tissue treated with SI and not PT1 (PT1-; SI+).

[0144] in addition, Fig.9E Retinal thickness of wild-type mice (WT / WT) is shown because these mice have knockout and / or knock-in alleles of the cytochrome C oxidase assembly protein SCO2, wherein the knock-in allele represents the E129K mutation on the SCO2 protein. Specifically, the mice are heterozygous knock-in and wild-type (KI / WT) mice or heterozygous knock-out and knock-in (KO / KI) mice. Fig.9E Shown is how treated (MK8722) mice exhibit less degeneration compared to mice treated with vehicle only (VEH).

[0145] Mitochondrial dysfunction in the eyes of patients with mitochondrial diseases can lead to ophthalmic manifestations such as retinopathy and optic atrophy, and impaired mitochondrial function has also been shown to be associated with age-related macular degeneration (AMD). Given that the eye is one of the most energy-dependent tissues in the human body, both primary and secondary mitochondrial diseases with ophthalmic findings are expected to benefit from AMPK activation (see, e.g., Calaza et al., Neurobiol Aging., October 2015, 36(10):2869-76; the disclosure of which is incorporated herein by reference in its entirety). Therefore, the direct AMPK agonists of various embodiments will be useful in treating patients with ophthalmic diseases associated with mitochondrial dysfunction.

[0146] Neuroprotection in ischemic stroke

[0147] Some embodiments provide neuroprotection in individuals with ischemic stroke. Impairment of mitochondrial energy metabolism in neurons is a key pathogenic factor in ischemic stroke and many neurodegenerative disorders, including certain primary mitochondrial diseases, AD, PD, and ALS (Schon et al., J Clin Invest., 2003 Feb 1; 111(3):303–312; the disclosure of which is incorporated herein by reference in its entirety). Figures 10A-10C demonstrated a direct AMPK neuroprotective effect in individuals with ischemic stroke. Specifically, Fig. 10AThe percentage of infarcted tissue in brain tissue with ischemic stroke is shown. Fig. 10A As shown, treatment with PT1 showed significant improvement compared to subjects treated with vehicle DMSO alone (vehicle). Fig. 10B and Fig. 10C , shows TCC-stained brain sections, in which individuals treated with vehicle only ( Fig. 10B ) shows high levels of ischemic tissue in white, while individuals treated with PT1 ( Fig. 10C ) showed a much smaller amount of white ischemic tissue.

[0148] Effects of AMPK agonists on exercise performance and skeletal muscle

[0149] Various embodiments will be useful for improving athletic performance in individuals suffering from mitochondrial dysfunction as well as muscle wasting diseases such as muscular dystrophy and autoimmune myositis disorders. Figures 11A-11C Improvements in muscle performance using the AMPK agonist MK-8722 according to various embodiments are shown. Fig.11A Shown is the distance traveled in the activity chamber by mice with various genotypes in the SCO2 gene treated with an AMPK agonist and with the delivery vehicle alone. Specifically, Fig.11A Shown are heterozygous knock-in and wild-type mice treated with vehicle only (1102), heterozygous knock-in and knock-out mice treated with vehicle only (1104), heterozygous knock-in and wild-type mice treated with an AMPK agonist (1106), and heterozygous knock-in and knock-out mice treated with an AMPK agonist (1108). Fig.11A As shown, heterozygous knock-in and wild-type mice showed similar activity levels whether treated with an AMPK agonist (1106) or vehicle alone (1102), while mice of the heterozygous knockout and knock-in alleles showed reduced activity when treated with vehicle alone (1104), but the activity of knockout / knock-in mice treated with an AMPK agonist (1108) was restored to levels comparable to knock-in / wild-type.

[0150] Fig. 11B The knockout / knockin mice (1108) treated with an AMPK agonist showed improved rotarod performance compared to knockout / knockin mice (1104) treated with vehicle alone. In addition, the knockout / knockin mice (1108) treated with an AMPK agonist showed higher levels than knockin / wild-type mice treated with an AMPK agonist (1106) or with vehicle (1102). Similarly, Fig. 11CResults from the wire hanging test are shown, indicating that many embodiments of the AMPK agonist improve muscle performance. Specifically, the AMPK agonist is able to improve the wire hanging time of the knockout / knock-in mouse (1108) to a higher level than the knockin / wild-type mice (1102, 1106) and the knockout / knock-in mouse (1104) treated with the vehicle. In general, Figures 11A-11C It is shown that AMPK activation by AMPK agonists from many embodiments can enhance strength, endurance, and overall motor function in muscle degenerative disorders associated with mitochondrial dysfunction.

[0151] Many embodiments can also improve mitochondrial function and / or glycogen storage in skeletal muscle, such as Figures 11D-11E shown. Fig.11D Histological staining indicating mitochondrial cytochrome C oxidase (CtyoC) and succinate dehydrogenase (SDH) activity is shown. Darker staining in embodiments treated with AMPK agonists indicates increased activity compared to lighter staining vehicle-only treated samples. Similarly, Fig.11E The darker staining of glycogen in the sample indicates an increased presence of stored glycogen in samples treated with embodiments of the AMPK agonist. The combination of increased mitochondrial function and glycogen content suggests that embodiments are able to normalize energy levels within skeletal muscle, thereby preventing degeneration and weakness.

[0152] Go to Figures 11F-11G , phosphorylation of ACC in skeletal muscle increases. Specifically, Fig.11F Shown are Western blots of phosphorylated ACC (pACC) in mice treated with vehicle only (KI / KO Veh) and in mice treated with an AMPK agonist of various embodiments (KI / KOMK8722). Fig.11G Western blots normalized to 1 in mice treated with vehicle only (KI / KO Veh) and mice treated with AMPK agonists of various embodiments (KI / KO MK8722) were summarized. Figures 11F-11G It was demonstrated that the AMPK agonists of many embodiments are capable of activating AMPK in skeletal muscle.

[0153] Embodiments of AMPK agonists

[0154] Many embodiments relate to highly effective, selective and stable small molecule agonists of AMPK. Many such embodiments relate to having Fig. 12A Many embodiments will modify the general structure of the molecule. Fig. 12A The general structure in Fig. 12AThe derivative molecules are generated by the structures in . The activity of the derivative molecules can be tested by various methods known in the art or described herein, such as measuring pACC in the sample as an indicator of increased AMPK activity. By changing certain fragments, R groups, etc., a systematic understanding of how each fragment affects AMPK activity can be formed.

[0155] Derivative molecules of certain embodiments are developed from the structure in 12A. In many embodiments, the moiety, R group or other fragment may be substituted with one or more of the following:

[0156] ●A is selected from unsubstituted or substituted with one or more C 1-6 a 5-membered heterocyclic ring substituted with an alkyl group or a fluorine group;

[0157] X is CR5 or N;

[0158] ●R1- is H, CF3- or halo;

[0159] ●R2 is OR5, NHOH, NHSO2R4, OCH2OCOR4, or COR2 is C-linked tetrazole,

[0160] ● R3 is unsubstituted or substituted by one to three substituents selected from halo, OH and OCOR7 1-10 Alkyl, C 3-7 Cycloalkyl, C 4-12 Alkylcycloalkyl, C 4-10 Cycloalkylalkyl, C 3-7 Heterocycloalkyl, C 4-12 Alkylheterocycloalkyl, C 4-10 heterocycloalkylalkyl, aryl or heteroaryl;

[0161] ●R4 is unsubstituted or substituted with one to three halogen substituents C 1-10 Alkyl, C 3-7 Cycloalkyl, C 4-12 Alkylcycloalkyl, C 4-10 Cycloalkylalkyl;

[0162] ●R5 is R4 or H;

[0163] ●R6 is H, C 1-6 Alkyl, C 3-6 Cycloalkyl or two R6 groups together with the nitrogen atom to which they are attached may form a four- to seven-membered heterocycloalkyl ring, all of which may be optionally substituted with 1 to 3 fluorine atoms;

[0164] ● R7 is unsubstituted or substituted by one to three selected from fluorine, C 1-10 Alkyl and NR6, R6 substituted C 1-10 Alkyl, C 3-7 Cycloalkyl, C4-12 Alkylcycloalkyl.

[0165] In many embodiments, A is selected from the group consisting of:

[0166]

[0167] Various embodiments may be implemented such as by using Figures 12B-12D The method shown is for manufacturing or synthesizing. Fig. 12B Shows the synthesis Fig.13A Specifically, Fig. 12B Step 1 is shown combining cyclobutanone with 2,5-dibromothiophene to produce 1-(5-bromothiophen-2-yl)cyclobutan-1-ol. At step 2, bis-pinacol boronate is added to the result of step 1 to produce 1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiophen-2-yl)cyclobutan-1-ol. Step 3 combines this product with 5-bromo-6-chloro-1H-indole-3-carboxylic acid methyl ester and K2CO3 to produce 6-chloro-5-(5-(1-hydroxycyclobutyl)thiophen-2-yl)-1H-indole-3-carboxylic acid methyl ester. Finally, step 4 generates 6-chloro-5-(5-(1-hydroxycyclobutyl)thiophen-2-yl)-1H-indole-3-carboxylic acid ( Fig.13A ).although Fig. 12B The process generated Fig.13A The compounds shown, however, should be noted that the Figures 12C-12D The process shown in FIG. 4 generates additional compounds such as Figures 13B-13Z and Figures 14A-14O The compounds shown.

[0168] Another implementation plan will be through Fig. 12C The method shown in the figure allows the boron group to be first attached to the indole ring of these embodiments and then coupled to the A ring (such as Fig. 12A shown).

[0169] Another implementation plan will be through Fig.12D The method is as follows, wherein the A ring contains four nitrogen atoms.

[0170] Other embodiments may have different structures while still maintaining the ability to activate AMPK. Some embodiments will have a general structure such as Figures 15A-15B shown.

[0171] Derivative molecules of certain embodiments are developed from the structure in 15A. In many embodiments, the moiety, R group or other fragment may be substituted with one or more of the following:

[0172] ●X is CH or N;

[0173] ●R1 is a small alkyl group (C1-C4) or a halogen (e.g., Cl, Br or F);

[0174] ●R2 is phenyl, alkyl C1-C10, cycloalkyl (C3-C10), hydroxyalkyl (C1-C6), heteroaromatic (e.g., pyridyl, pyrazolyl, pyrrolyl, pyrimidinyl, thienyl, furanyl or triazole), or heterocyclic C4-C6;

[0175] o wherein the phenyl group is optionally substituted with halogen (e.g., Cl, Br or F),

[0176] o wherein the alkyl group is linear or branched and is optionally substituted by OH, OMe, OEt,

[0177] o wherein the cycloalkyl group is optionally substituted with one or more OH groups,

[0178] ○ Wherein the hydroxyalkyl group is a straight chain or branched chain

[0179] o wherein the heteroaromatic group is optionally substituted with a small alkyl group (C1-C6) or a hydroxyalkyl group (C1-C6), wherein the alkyl group and the hydroxyalkyl group are linear or branched,

[0180] ○ wherein the heteroatom in the heterocycle is optionally O, S or NR4, wherein R4 is a linear or branched C1-C6 alkyl or hydroxyalkyl; and

[0181] ●R4 is a linear or branched C1-C6 alkyl or hydroxyalkyl group.

[0182] Derivative molecules of certain embodiments are developed from the structure in 15B. In many embodiments, the moiety, R group or other fragment may be substituted with one or more of the following:

[0183] ●A is a fused ring (e.g., C3-C10 cycloalkyl);

[0184] ●X is CH or N;

[0185] ●Y is O, S, NH, or NR3;

[0186] ●R1 is a small alkyl group (C1-C4) or a halogen (e.g., Cl, Br or F);

[0187] ●R2 is phenyl, alkyl C1-C10, cycloalkyl (C3-C10), hydroxyalkyl (C1-C6), heteroaromatic (e.g., pyridyl, pyrazolyl, pyrrolyl, pyrimidinyl, thienyl, furanyl or triazole), or heterocyclic C4-C6;

[0188] o wherein the phenyl group is optionally substituted with halogen (e.g., Cl, Br or F),

[0189] o wherein the alkyl group is linear or branched and is optionally substituted by OH, OMe, OEt,

[0190] o wherein the cycloalkyl group is optionally substituted with one or more OH groups,

[0191] ○ Wherein the hydroxyalkyl group is a straight chain or branched chain

[0192] o wherein the heteroaromatic group is optionally substituted with a small alkyl group (C1-C6) or a hydroxyalkyl group (C1-C6), wherein the alkyl group and the hydroxyalkyl group are linear or branched,

[0193] ○ wherein the heteroatom in the heterocycle is optionally O, S or NR4;

[0194] ●R3 is C1-C6 alkyl (linear or branched); and

[0195] ●R4 is a linear or branched C1-C6 alkyl or hydroxyalkyl group.

[0196] In various embodiments, A is a C6 benzene ring. In many embodiments, A is selected from the group consisting of:

[0197]

[0198] Go to Fig. 15C , showing the method of synthesizing the embodiment. Specifically, Fig. 15C Shows the synthesis Fig.15D The method of the embodiment shown (also referred to as EV8017). Fig. 15C The process generated Fig.15D The compounds shown, but it should be noted that additional compounds can be generated in a similar manner.

[0199] Go to Fig.16A , showing Fig.13A The biological effects of the AMPK agonists shown. Specifically, Fig.16A Shown using Fig.13A Western blot of phosphorylated ACC (pACC) in mitochondrial defective patient cells treated with the embodiment shown (labeled as EV8016) and cells treated with vehicle only (labeled as DMSO). Fig.11F , phosphorylation of ACC indicates AMPK activation, thus indicating that the compounds according to many embodiments are capable of activating AMPK.

[0200] Go to Fig. 16B , the EC50 of various embodiments of AMPK agonists can be determined. In the exemplary embodiment EV8016 ( Fig.13A ), the EC50 was determined to be 232 nM, while the PF-06409577 embodiment ( Figure 1D ) was determined to be 96 nM. Those skilled in the art will recognize and understand how to determine the EC50 of additional embodiments.

[0201] Methods of treating an individual with an AMPK agonist

[0202] Now go to Fig.17 , various embodiments relate to treating a mitochondrial dysfunction in an individual in process 1700. In some embodiments, at step 1702, a mitochondrial dysfunction in an individual is identified. In certain embodiments, an individual with a dysfunction is discovered by exhibiting symptoms such as N-glycanase (NGLY1) deficiency, age-related macular degeneration (AMD), ischemic stroke, muscular dystrophy (e.g., Duchenne and Becker), Friedreich's ataxia (FA), autoimmune diseases with muscle involvement (e.g., inclusion body myositis, polymyositis, and dermatomyositis), neurodegenerative disorders (e.g., amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease), diabetes, metabolic disorders, and / or obesity. In other embodiments, mitochondrial dysfunction will be identified based on molecular markers of the disease or dysfunction, such that protein blot (western blot), polymerase chain reaction (PCR), genotyping using genetic markers (e.g., single nucleotide polymorphisms (SNPs), expressed sequence tags (ESTs), simple sequence repeat markers (SSRs), etc.) will identify the specific disease or dysfunction present in an individual.

[0203] In various embodiments, at step 1704, an individual is treated with an AMPK agonist. In some embodiments, the AMPK agonist is a direct (or non-AMP-dependent) agonist, while in other embodiments, the AMPK agonist is an AMP-dependent agonist. Examples of direct agonists include PT1, ETC-1002, salicylates, C991, C13, D561-0775, MT63-78, A-769662, ZLN024, C24, MK-8722, PF-739, and PF-06409577. Examples of ATP-dependent agonists include metformin, resveratrol, and AICAR. In certain embodiments, an AMPK agonist is provided to an individual at a therapeutically effective dose, wherein during and / or after the start of treatment, the therapeutically effective dose reduces, eliminates, or mitigates the consequences of mitochondrial dysfunction. In some embodiments, the AMPK agonist is provided orally, subcutaneously, intravenously, intraperitoneally, intranasally, dermally, via inhalation, intraocularly (including intravitreally), and / or any method that provides a therapeutic effect. In certain embodiments, the AMPK agonist is formulated to provide a therapeutic effect. In some embodiments, the AMPK formulation includes a binder, a lubricant, a buffer, and / or a coating that allows the pharmacokinetic release of the AMPK agonist to provide a therapeutic effect.

[0204] In addition, the AMPK agonist and / or preparation will be provided to the individual at an appropriate dose and dosing regimen to provide a therapeutic effect. In some embodiments, the AMPK agonist will be provided in a single dose, while some embodiments will provide multiple doses over a period of time. In various embodiments, the dose will be implemented in the form of a concentration of the total volume, so that the dose will be 10nM, 30nM, 100nM, 1μM, 100μM or higher, depending on the therapeutic effect. In other embodiments, administration will be carried out at a ratio of the mass of the AMPK agonist to the mass of the treated individual, so that the dose will be 1mg / kg, 5mg / kg, 10mg / kg, 20mg / kg, 50mg / kg, 100mg / kg or higher, depending on the therapeutic effect. In the embodiment of multiple administration, the dosing regimen can be 1 dose / day, 2 doses / day, 3 doses / day or more, and can last for the necessary time, so that administration can last for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 20 weeks, or permanently exist in the life of an individual.

[0205] In other embodiments, the dosage and / or dosing regimen will vary during the course of treatment according to various factors, including reaching a threshold of enzyme activity. In some embodiments, a higher dosage will be used to treat an individual, and will be reduced after a period of time, so that in various embodiments, a daily dose of 100 μM will be used for a period of time (such as 1 week, 2 weeks, 4 weeks or longer), and then the dosage will be reduced to continue to treat the individual. In certain embodiments, the dosing regimen will change so that the time between doses will change during the course of treatment. For example, certain embodiments will provide an AMPK agonist daily dose to an individual over a period of time (e.g., 1 week, 2 weeks, 4 weeks, etc.), and then the amount of time between doses will be increased so that subsequent doses occur once every half week, weekly, or monthly during the remaining treatment.

[0206] At step 1706, various embodiments will assess the individual for AMPK agonist efficacy. In certain embodiments, this step is accomplished by assessing disease symptoms, such as those listed for step 1702, while in other embodiments, this step will be accomplished by looking at molecular profiles such as genotyping, gene expression, and other methods disclosed with reference to step 1702.

[0207] Methods for regulating AMPK activity

[0208] Now go to Fig.18 , various embodiments relate to modulating AMPK activity to treat a disease or condition in an individual, as shown in process 1800. In some embodiments, at step 1802, a condition or disease associated with mitochondrial dysfunction is identified. Examples of these diseases and / or conditions include the diseases and conditions listed in Tables 1-2, such that the disease and / or condition is associated with mitochondrial dysfunction such as primary mitochondrial dysfunction and / or secondary mitochondrial dysfunction. In various embodiments, diseases and conditions are identified based on symptoms exhibited by the individual, while in some embodiments, diseases and conditions are identified based on molecular markers of the disease or dysfunction, such as protein blots (western blots), polymerase chain reactions (PCR), genotyping using genetic markers (e.g., single nucleotide polymorphisms (SNPs), expressed sequence tags (ESTs), simple sequence repeat markers (SSRs), etc.).

[0209] At step 1804, various embodiments will regulate AMPK activity. Regulating AMPK activity can activate AMPK or inhibit AMPK activity. In certain embodiments, AMPK activity will be activated by phosphorylating AMPK or providing an AMPK agonist. In certain embodiments, regulation is accomplished by inhibiting AMPK activity. Inhibition of AMPK can be accomplished using a competitive inhibitor or an allosteric inhibitor, which prevents AMPK from catalyzing the reaction.

[0210] At step 1806, various embodiments will assess the efficacy of the treatment for the individual. In certain embodiments, this step is accomplished by assessing disease symptoms, such as those listed for step 1802, while in other embodiments, this step will be accomplished by looking at molecular profiles such as genotyping, gene expression, and other methods disclosed with reference to step 1802.

[0211] Exemplary embodiments

[0212] Experiments were performed to demonstrate the capabilities of assays and inhibitors according to embodiments. These results and discussion are not intended to be limiting, but merely to provide examples of operating devices and their features.

[0213] Materials and Methods

[0214] This study used fibroblasts previously obtained from four patients with primary mitochondrial diseases (Surf1, Complex I, Cox10, Polg), three patients with NGLY1 deficiency, and four normal controls. All samples were obtained with informed consent and approved by the Stanford IRB. Fibroblasts were maintained at 37°C, 5% CO2 in DMEM medium containing 8.3 mM glucose and supplemented with 10% fetal bovine serum (FBS) (Fisher Scientific), 1% penicillin-streptomycin (10,000 U / mL) (Life Technologies), 1% glutaMAX (Life Technologies), 1% uridine (5 mg / ml), and 1% pyruvate (11 mg / ml).

[0215] To screen various compounds, 15x10 3Cells / 500ul culture medium were seeded in quadruplicate in 24-well microtiter plates. The next day, the culture medium was removed, the wells were washed with PBS, and replaced with mitochondrial stress medium containing DMEM, 1% penicillin-streptomycin (10,000U / mL) (Life Technologies), 10% FCS (FisherScientific), 1mM galactose (SigmaAldrich) and 25μM sodium azide, with or without the following compounds: 1mM AICAR (Medchem Express), 1mM metformin (Sigma Aldrich), 100μM PT1 (Santa Cruz Biotechnology), 100μM A-769662 (Medchem Express), 100μM C24 (Medchem Express). After treatment with various compounds, the growth, oxygen consumption rate and ATP level of tissue culture were analyzed.

[0216] Example 1: Cell Growth and Viability

[0217] Methods: Cell growth was measured by fluorescence using calcein acetoacetate (Anaspec). Mitochondrial stress medium was removed, wells were washed with PBS, and then incubated with 500ul / well of 800nM calcein acetoacetate in PBS for 30 minutes at 37°C, 5% CO2. Cell viability was measured by values ​​obtained using 485nm excitation using a Flouroskan Ascent microplate fluorometer (Thermo Scientific).

[0218] Cellular ATP content is measured by LC-MS. Cells are treated with trypsin, washed with cold PBS, and lysed using NH4AC (0.05M pH6). Lysate is transferred to a molecular weight cut-off filter (Chromsystems) and spun at 4°C, 800xg for 20 minutes. After centrifugation, the supernatant is subjected to DNA quantitative analysis using NanoDropND-1000 (NanodropTechnologies), and ATP is quantified by a 6400 series triple quadrupole LC / MS system (Agilent Technologies).

[0219] Fibroblasts in tissue culture were observed by phase contrast microscopy at 10× magnification with a Leica DMIRB microscope, and images were captured with a Hamamatsu ORCA-ER camera.

[0220] Statistical significance was calculated by two-tailed Student's t-test (p<0.05).

[0221] Results: Four direct AMPK activators, PT1, A-769662, ZLN024, and C24, were tested in SURF1-deficient fibroblasts. AICAR, an AMP analog previously shown to improve mitochondrial function in vitro (Golubitzky et al., PLoS One., 2011;6(10); the disclosure of which is incorporated herein by reference in its entirety;) was included in the study design as a positive control. Mutant cells treated with direct AMPK agonists showed a 35%-55% increase in viability (P < 0.05) compared to untreated cells (i.e., cells treated with vehicle DMSO). Figure 2A A smaller but still significant effect was also observed in SURF1 cells treated with AICAR, with an average cell viability increase of 57% compared to the corresponding untreated controls (i.e., cells treated with PBS).

[0222] To assess whether these findings extend to mitochondrial disorders beyond SURF1 deficiency, similar studies were performed using fibroblasts from patients deficient in mitochondrial complex I (CI), heme A:farnesyltransferase cytochrome c oxidase assembly factor (COX10), and mitochondrial DNA polymerase gamma (POLG). Thus, these studies assessed a broad range of mitochondrial disruptions, including different components of the respiratory chain and mtDNA replication machinery. Despite the different pathogenic mechanisms, one compound, PT1, consistently and significantly improved survival in all mutant strains ( Figure 2B-2D The remaining compounds showed different responses between mutants: A-769662 improved the survival of CI- and POLG-deficient cells, ZLN024 improved the survival of CI-deficient cells, and C24 improved the survival of SURF1-deficient cells but not any of the other cell lines. Interestingly, AICAR treatment also did not improve the survival of any cell line other than SURF1. Similar results were observed with fibroblasts from patients with NLGY-1 deficiency, with viability increased by up to 60% ( Figure 2E ). In addition, the newly developed AMPK agonists MK-8722, PF-739, and PF-04609577 were tested and shown to improve the viability of SURF1 patient cells similarly to PT1, and this improvement occurred in a dose-dependent manner ( Figure 2E In addition, compared with untreated cells ( Figure 3A , Figure 3C , Figure 3E , Figure 3G and Fig. 3I ) compared to SURF1( Figure 3B )、COX10( Figure 3D )、CI( Figure 3F)、POLG( Figure 3H ) and NGLY1( Figure 3J )-deficient fibroblasts treated with PT1 showed improved cell morphology, with untreated cells showing bright apoptotic cells. Figure 3K ) compared to MK-8722( Figure 3L )、PF-739( Figure 3M ) and PF-04609577( Figure 3N ) improved the cell morphology of SURF1-deficient fibroblasts, where untreated cells showed bright apoptotic cells.

[0223] Conclusions: These results suggest that direct AMPK activation can improve cell viability across a range of primary and secondary mitochondrial disease etiologies, supported by the improved cell morphology observed in mutant cells treated with PT1 compared to cells treated with DMSO.

[0224] Example 2: Measuring AMPK agonists on AMPK

[0225] Methods: Western blotting was performed to determine the level of pAMPK in cells treated with PT1. Cells were lysed with RIPA buffer supplemented with sodium orthovanadate, PMSF, and a protease inhibitor cocktail. Whole-cell extracts were fractionated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes using a transfer apparatus according to the manufacturer's protocol (Bio-Rad). After incubation with 5% skim milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membranes were washed once with TBST and incubated with antibodies against AMPK (1:1,000), pAMPK (1:1,000), actin (1:10,000) overnight at 4°C. The membranes were washed three times for 5 min and incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies for 2 h. The blots were washed three times with TBST and developed with the ECL system (Thermo Scientific) according to the manufacturer's protocol.

[0226] To test whether the cellular response to PT1 is the result of AMPK activation itself or just a non-specific interaction, a siRNA knockdown strategy was used. PRKAA1 siRNA (s101) and negative controls (Life Technologies) were incubated with Hiperfect reagent (Qiagen) in a medium containing DMEM, 1% penicillin-streptomycin (10,000U / mL) (Life Technologies), 1% glutaMAX (Life Technologies) but without serum, and allowed to complex for 10 minutes at room temperature. The complex was then added to COX10 patient tissue culture in a 6-well microtiter plate (the final siRNA concentration for each siRNA was 10nM) and incubated for 72 hours at 37°C, 5% CO2. At the end of the incubation period, the AMPK and pAMPK levels of the cells were analyzed by Western blot analysis (see the above protocol), or the cells were incubated with PT1 (or DMSO, as an untreated control) for another six days and the viability was assessed by calcein acetyl methyl ester. COX10-deficient fibroblasts were treated with PT1 and siAMPK or siCNT, and the pAMPK protein expression in response was assessed by Western blotting ( Figure 4A ), cell viability was assessed by calcein acetyl fluorimetry ( Figure 4B ), and cell morphology was assessed by phase contrast microscopy ( Figures 4C-4F ).

[0227] Results: Cultured fibroblasts from SURF1-deficient patient cells were treated with PT1 for 0, 2, 24, and 48 hours, and pAMPK levels were measured. pAMPK levels increased by 2 hours after PT1 treatment, peaked at 24 hours, and returned to baseline at 48 hours ( Figure 5A ). Treatment with PT1 and selective knockdown of AMPK (PT1+siAMPK) resulted in a 63% decrease in pAMPK levels and a 41% decrease in cell viability compared to control conditions (PT1+siCNT), consistent with the concept that cellular responses to PT1 are mediated by AMPK activation. After 48 h of PT1 treatment, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a master regulator of mitochondrial biogenesis, increased 2.4-fold ( Figure 5B Similarly, catalase and manganese superoxide dismutase (SOD2), two major antioxidant genes regulated by AMPK, increased by 1.3-fold and 1.5-fold, respectively ( Figure 5C-5D ).

[0228] Conclusions: The mechanism of action of PT1 addresses the major metabolic disruption caused by mitochondrial dysfunction; thus stimulating mitochondrial biogenesis and triggering the oxidative stress response through AMPK activation.

[0229] Example 3: Oxygen consumption and cellular respiration

[0230] Background: To determine whether upregulation of PGC-1 by PT1 treatment is associated with improved mitochondrial respiration, the oxygen consumption rate (OCR) was assessed in fibroblasts from both SURF1 and POLG patients.

[0231] Methods: Oxygen consumption rate (OCR) was measured using an XF96 extracellular flux analyzer (Seahorse Biosciences). Fibroblasts were seeded at 10x103 cells / well in 100ul culture medium on XF 96-well plates at 37°C, 5% CO2, containing 8.3mM glucose and supplemented with 10% fetal bovine serum (FBS) (Fisher Scientific), 1% penicillin-streptomycin (10,000U / mL) (Life Technologies), 1% glutaMAX (Life Technologies), 1% uridine (5mg / ml) and 1% pyruvate (11mg / ml). The next day, the culture medium was replaced with mitochondrial stress medium (as described in detail above) containing or not containing PT1, A-769662 and AICAR. After 48 hours, the stress medium was replaced with 175μl non-buffered XF basic DMEM medium (Fisher Scientific), which had the same composition as the mitochondrial stress medium, and was incubated at 37°C for 30 minutes to reach equilibrium before measurement. OCR baseline measurements were taken three times, every five minutes. After the experiment, the cell content was estimated by calcein acetylation fluorescence intensity (FI), and OCR was calculated by dividing OCR by FI.

[0232] Results: Despite clear etiological differences, both Surf1 and POLG patient cells treated with PT1 showed an improvement in basal respiration of approximately 30% ( Figure 6A-6B ), while PT1 showed a 16% increase in basal OCR in NGLY1 patient cells ( Figure 6C ).

[0233] The fraction of basal mitochondrial oxygen consumption used for ATP synthesis (ATP-coupled respiration) was also increased by 40% in Surf1 and 30% in POLG, indicating that PT1 has a positive effect on the coupling efficiency of oxidative phosphorylation ( Figure 6D-6E ).

[0234] Maximal respiratory capacity (reflecting the cell's ability to respond to increased ATP demand) was improved in both Surf1 and POLG cells, by 50% and 20%, respectively. Figure 6F-6G ), while PT1 showed a 40% increase in maximal OCR in NGLY1 patient cells ( Figure 6H ).

[0235] Conclusions: Activation of AMPK via AMP-independent mechanisms may be a more suitable mechanism for targeting mitochondrial dysfunction.

[0236] Example 4: Measuring oxidative stress and energy status

[0237] Background: ATP and ROS levels reflect cellular energy and oxidative status, both of which depend on efficient ATP-coupled respiration and overall mitochondrial function. Mitochondrial dysfunction leads to incomplete electron transfer through the RC, resulting in reduced ATP synthesis and excessive production of reactive oxygen species (ROS). (Atkuri et al., Proc Natl Acad Sci USA., 2009 Mar 10; 106(10):3941-5; Enns et al., PLoS One., 2014 Jun 18; 9(6); the disclosures of which are incorporated herein by reference in their entirety.)

[0238] Methods: SURF1, POLG, and NGLY1 patient cells were supplemented with PT1 or DMSO (untreated control) for 48 h (n=3) and ATP levels were measured by CellTiter-Glo ATP assay or for 72 h (n=3) and ROS levels were measured by CellROX Deep Red flow cytometry assay. Results were normalized to total protein concentration. Data are presented as mean + Standard error of the mean. Statistical significance was measured at p < 0.05.

[0239] Results: Following treatment, ATP content increased by 35% in SURF1, 36% in POLG, and 40% in NGLY1 patient cells, and ROS levels decreased by 10%, 15%, and 18%, respectively, compared with DMSO-treated cells. Figures 7A-7F ).

[0240] Conclusion: The observed upregulation of ATP-coupled respiration and the expression of the antioxidants catalase and SOD2 (Example 2; Figure 5C-5D ), energy deficit and oxidative stress were ameliorated in cells from patients with primary or secondary mitochondrial dysfunction by PT1 treatment.

[0241] Example 5: Measuring effects on AMD

[0242] Methods: To generate the AMD model, mice were treated with 100 mg / kg PT1 (or vehicle) 24 hours and 12 hours before SI (or vehicle) treatment, and then every 24 hours after SI for 3 days. Both PT1 and SI were delivered by intraperitoneal (IP) injection, and animals were phenotyped 3 days after SI administration.

[0243] Mice were anesthetized and their pupils were dilated using 1% atropine sulfate, 2.5% phenylephrine hydrochloride, and 0.5% proparacaine hydrochloride. Funduscopy was performed using a Micron III Small Animal Retinal Imaging AD Camera (Phoenix Research Laboratories, INC).

[0244] Retinal function was assessed by recording dark-adapted and light-adapted ERGs (Espion E2 System, Diagnosys LLC). Prior to ERG recording, mice were dark-adapted overnight, and all procedures were performed in the dark or under dark red light. Mice were anesthetized and their pupils were dilated as described above. For ERG recording, electrodes were placed on the center of the cornea. A ground needle electrode was placed in the base of the tail, and a reference needle electrode was placed subcutaneously between the eyes. The a-wave amplitude was measured from the baseline to the trough of the a-wave, and the b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave.

[0245] Results: Fewer white precipitates were observed in PT1-treated mice (+SI; +PT1) compared with untreated mice (+SI; -PT1) (n = 5 mice per group). PT1 treatment alone (-SI; +PT1) did not alter funduscopy findings, which were very similar to those of control mice (-SI; -PT1) ( Fig. 9A ). H&E staining of retinal sections showed that PT1 treatment (+SI; +PT1) prevented ONL thinning (white bars), blocked photoreceptor IS / OS morphological changes and reduced melanin debris (black arrows) compared with untreated mice (+SI; -PT1) (n = 5 mice per group) ( Fig. 9B ONL thickness in PT1-treated mice (+SI; +PT1) was similar to that in control mice (-SI; -PT1) and significantly improved compared to untreated mice (+SI; -PT1). PT1 treatment alone did not alter ONL thickness (-SI; +PT1 vs. -SI; -PT1) (n = 3-4 mice per group) ( Fig. 9CElectroretinograms showed that rod responses (scotopic a- and b-waves) were significantly increased in PT1-treated mice (+SI; +PT1) compared with untreated mice (+SI; -PT1) (n = 3-4 mice per group) ( Fig.9D ).

[0246] Conclusions: PT1 demonstrated improvements in retinas showing AMD, thus suggesting that AMPK agonists could be actively used in the treatment of AMD.

[0247] Example 6: Neuroprotection in Ischemic Stroke

[0248] Methods: To generate an ischemic stroke model, ischemic lesions were induced by transient occlusion of the middle cerebral artery for 45 minutes followed by reperfusion. Mice were then injected intraperitoneally with 100 mg / kg of PT1 or vehicle at two doses, 1 hour and 24 hours after occlusion, and sacrificed one hour later for terminal tissue collection.

[0249] The isolated brain was placed in cold saline for 20 minutes, cut into seven coronal sections (2 mm thick), and stained in 1.0% 2,3,5-triphenyltetrazolium chloride (TTC) solution in saline at 37°C for 30 minutes. The stained brain tissue was fixed in 10% formalin in phosphate-buffered saline. Images were captured using a CCD camera (Panasonic Corporation, Japan), and the unstained damaged area was defined as infarct tissue and quantified using Image Pro Plus 4.1 software (MediaCybernetics, Silver Spring, MD).

[0250] Results: TCC-stained brain sections from mice treated with PT1 showed a reduction in the size of the infarct area (white) compared with mice treated with vehicle ( Figures 10B-10C ).

[0251] Conclusion: PT1 treatment showed a significant reduction in ischemic (stroke) area in brain sections of mice treated with PT1 compared to untreated controls.

[0252] Example 7: Enhancement of athletic performance

[0253] Background: As a high energy demand organ, the eye is particularly vulnerable to the consequences of mitochondrial damage. Similarly, skeletal muscle is also a high energy organ that relies on both oxidative phosphorylation and glycolysis for energy production.

[0254] Methods: To examine the effects of AMPK agonists on systems with mitochondrial dysfunction in vivo, a mouse model of mitochondrial disease was treated with MK8722. These mice have mitochondrial complex IV deficiency caused by deficiency of the cytochrome c oxidase assembly protein SCO2 and carry a Sco2 knockout (KO) allele and a Sco2 knockin (KI) allele expressing an E→K mutation (E129K) at position 129. The E129K mutation corresponds to the E140K mutation present in almost all human SCO2 mutation patients (see, e.g., Yang et al., Analysis of mouse models of cytochrome c oxidase deficiency owing to mutations in Sco2. Hum Mol Genet., 2010 Jan 1; 19(1): 170-80; the disclosure of which is incorporated herein by reference in its entirety). The main phenotypes of Sco2-deficient mice are reduced motor function and eye defects, both common findings in patients with primary mitochondrial diseases. Sco2 mice were divided into four treatment groups: Sco2 KI / KO+MK8722, Sco2KI / KO+DMSO (vehicle), control KI / WT+MK8722, control KI / WT+DMSO, with 7-9 mice per group. 1.5-month-old Sco2 mice were treated once daily with MK8722 (10 mg / kg) or vehicle (DMSO) by oral instillation for 14 weeks. Motor function was assessed using activity chamber, rotarod and hanging wire tests, and ocular structure was assessed using optical coherence tomography (OCT).

[0255] Results: Due to this sensitivity to mitochondrial dysfunction, ocular structure measured in vivo by optical coherence tomography (OCT) revealed retinal defects in Sco2 KI / KO as well as heterozygous KI / WT littermates. Retinal nerve fiber layer (RNFL) thickness was reduced in Sco2 KI / KO and KI / WT mice compared with WT / WT controls (KI / KO Veh: 74.1 μm and KI / WTVeh: 72.7 μm vs. WT / WTVeh: 76.2 μm). In contrast, the RNFL was thicker in MK8722-treated mice than in vehicle-treated Sco2 KO / KI and KI / WT mice (KI / KO Veh: 74.1 μm vs. KI / KO MK8722: 77 μm and KI / WTVeh: 72.7 μm vs. KI / WT MK8722: 74.7 μm) (see Fig.9E ).

[0256] In motor function studies evaluating muscle performance, Sco2KI / KO mutant mice treated with MK8722 were placed in an activity chamber (see Fig.11A ), Rotarod test (see Fig. 11B ) and the forelimb hanging wire test (see Fig. 11C ) performed better than the vehicle-treated Sco2 KI / KO mutant mice. Fig.11A As shown, vehicle-treated Sco2 KI / KO mice moved a shorter distance in the activity chamber compared with KI / WT littermate controls, and MK8722 treatment for 8 weeks restored their activity to control levels (KO / KI Veh: 1516.7 cm; KO / KI MK8722: 2337.8 cm; KI / WT Veh: 2337 cm). Fig. 11B It was shown that MK8722 treatment also improved the rotarod time of KI / KO mutant mice to a level similar to that of controls (KO / KI Veh: 176.1 sec; KO / KI MK8722: 248.1 sec; KI / WT Veh: 235.7 sec) compared to vehicle-treated mice. Fig. 11C It was shown that MK8722 treatment increased the length of time Sco2 KO / KI mutant mice could hang on their limbs to levels even exceeding those of KI / WT controls (KO / KI Veh: 173.2 sec; KO / KI MK8722: 312.5 sec; KI / WT Veh: 145.5 sec).

[0257] Fig.11D The intensity of histological staining indicating mitochondrial cytochrome C oxidase (CytoC) and succinate dehydrogenase (SDH) activity was increased in skeletal muscle tissue of Sco2 KI / KO mutant mice treated with MK8722 compared to vehicle-treated mutant mice. Fig.11E Shows that glycogen content is increased in Sco2 KI / KO mutant mice treated with MK8722 compared to untreated mice, as assessed by histology of skeletal muscle. Motor function ( Figures 11A-11C ) and mitochondrial function ( Figures 11D-11E ) is associated with increased ACC phosphorylation, such as Fig.11F shown.

[0258] Conclusion: Retinal studies ( Fig.9E ) suggest that activation of AMPK may improve and / or prevent ocular damage associated with mitochondrial dysfunction. In addition, exercise studies ( Figures 11A-11C ) showed that AMPK activation can enhance strength, endurance and overall motor function in muscle degeneration disorders associated with mitochondrial dysfunction. In addition, improved mitochondrial function ( Fig.11D ) and glycogen content ( Fig.11E) may play an integral role in the mechanism of action of normalizing energy levels in skeletal muscle, thereby preventing degeneration and weakness. These results, combined with increased ACC phosphorylation indicative of increased AMPK activation, demonstrate the ability of many embodiments of AMPK agonists to have a number of pharmacological benefits.

[0259] Example 8: Synthesis of 6-chloro-5-(5-(1-hydroxycyclobutyl)thiophen-2-yl)-1H-indole-3-carboxylic acid and its analogs thing

[0260] background: Fig. 12A The general structure of compounds capable of activating AMPK is shown. The synthesis of four derivatives is described in this example ( Figures 13A-13B and Figures 14J-14K ).

[0261] Method: This example is illustrated in Fig. 12B is described in, and this embodiment shows Fig.13A The synthesis of the compounds shown in the figure, those skilled in the art will know the use of different reagents, reactants, conditions, etc. to generate derivative compounds, including Figures 13B-13Z and Figures 14A-14O Those shown.

[0262] Step 1: 1-(5-bromothiophen-2-yl)cyclobutan-1-ol—To a stirred solution of 2,5-dibromothiophene (0.95 g, 3.922 mmol) in anhydrous THF at -78°C was added n-butyllithium (2.69 ml, 4.314 mmol) dropwise at -78°C, and the mixture was stirred at the same temperature for another 30 minutes. A solution of cyclobutanone (0.27 g, 3.922 mmol) was then added slowly over 2 minutes, and the reaction mixture was stirred at -78°C for 1 hour, then quenched with ammonium chloride. The product was extracted into ethyl acetate, dried over sodium sulfate, evaporated under reduced pressure, and then purified by flash column chromatography (230–400 mesh, 10% EA in PE) to give 1-(5-bromothiophen-2-yl)cyclobutan-1-ol (0.6 g; 66%) as a colorless liquid.

[0263] Step 2: 1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiophen-2-yl)cyclobutan-1-ol - A mixture of the product of Step 1 (2.0 g, 8.579 mmol), bispinacol boronate (3.2 g, 12.87 mmol) and KOAc (2.25 g, 25.7 mmol) in 1,4-dioxane (20 ml) was purged with N gas for 5 min, and then Pd(dppf)Cl (0.32 g, 0.429 mmol) was added. After stirring at 90 °C for 16 h, the solvent was removed by evaporation under reduced pressure and the residue was purified by column chromatography on florisil eluting with 0-10% EA / PE to give 1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiophen-2-yl)cyclobutan-1-ol (0.6 g; 25%) as a sticky solid.

[0264] Step 3: 6-Chloro-5-(5-(1-hydroxycyclobutyl)thiophen-2-yl)-1H-indole-3-carboxylic acid methyl ester - A mixture of the product of step 2 (0.275 g, 0.977 mmol), 5-bromo-6-chloro-1H-indole-3-carboxylic acid methyl ester (0.282 g, 0.977 mmol), K2CO3 (0.40 g, 2.931 mmol) in 1,4-dioxane (5 ml) and water (1 ml) was purged with N2 gas for 5 minutes. Pd(dppf)Cl2.DCM (0.04 g, 0.048 mmol) was then added and the resulting solution was stirred at 100°C for 16 hours. The solvent was removed by evaporation under reduced pressure and the residue was purified by column chromatography on florisil eluting with 0-10% EA / PE to give methyl 6-chloro-5-(5-(1-hydroxycyclobutyl)thiophen-2-yl)-1H-indole-3-carboxylate (0.15 g; 91%) as an off-white solid.

[0265] Step 4: 6-Chloro-5-(5-(1-hydroxycyclobutyl)thiophen-2-yl)-1H-indole-3-carboxylic acid - A stirred solution of the product of step 3 (0.150 g, 0.414 mmol) in ethanol and 6N NaOH solution (10 ml) was stirred at 80°C for 16 hours. The solvent was removed by evaporation under reduced pressure, and the residue was acidified with citric acid and the product was extracted into ethyl acetate. After washing the extract with water and drying over sodium sulfate, the solvent was removed under reduced pressure and the crude residue was purified by preparative HPLC to give 6-chloro-5-(5-(1-hydroxycyclobutyl)thiophen-2-yl)-1H-indole-3-carboxylic acid (0.06 g; 42%) as an off-white powder.

[0266] By changing some of the reagents, the above Fig. 12BThe method described herein produces 6-chloro-5-(5-(1-hydroxycyclobutyl)thiophen-3-yl)-1H-indole-3-carboxylic acid ( Fig. 13B )、6-chloro-5-(1-methyl-1H-pyrazol-4-yl)-1H-indole-3-carboxylic acid ( Fig.14J ) and 6-chloro-5-(1-phenyl-1H-pyrazol-4-yl)-1H-indole-3-carboxylic acid ( Figure 14K ).

[0267] Result: Step 1 produced 1-(5-bromothiophen-2-yl)cyclobutan-1-ol having the following characteristics: 1 HNMR (400MHz, CDCl3): δ6.91 (d, J = 3.6 Hz, 1H), 6.81 (d, J = 4.0 Hz, 1H), 2.49–2.36 (m, 4H), 2.21 (s, 1H), 1.96–1.91 (m, 1H), 1.76–1.69 (m, 1H). LCMS: 97.6% (217.07, M-18).

[0268] Step 3 produces methyl 6-chloro-5-(5-(1-hydroxycyclobutyl)thiophen-2-yl)-1H-indole-3-carboxylate having the following characteristics: 1 HNMR (400MHz, CDCl3): δ8.56(bs,1H),8.29(s,1H),7.93(d,J=2.8Hz,1H),7.54(s,1H),7.16(d,J=3.6Hz,1H),7.06(d ,J=3.6Hz,1H),3.92(s,3H),2.65–2.58(m,2H),2.51–2.44(m,2H),2.31(s,1H),1.99–1.91(m,1H),1.84–1.80(m,1H). LCMS: 95.63% (344.23, M-18).

[0269] Step 4 produces 6-chloro-5-(5-(1-hydroxycyclobutyl)thiophen-2-yl)-1H-indole-3-carboxylic acid having the following characteristics: 1 H NMR (400 MHz, DMSO-d6): δ 12 (br s, 1H), 8.12 (s, 1H), 8.07 (s, 1H), 7.63 (s, 1H), 7.12 (d, J = 3.6 Hz, 1H), 7.06 (d, J = 3.6 Hz, 1H), 5.96 (s, 1H), 2.45-2.31 (m, 4H), 1.91-1.82 (m, 1H), 1.78-1.68 (m, 1H). LCMS: 99.06% (465.30 [M+H]+), melting point range: 228-232 °C.

[0270] Fig. 13B 6-Chloro-5-(5-(1-hydroxycyclobutyl)thiophen-3-yl)-1H-indole-3-carboxylic acid is shown, which has 1 HNMR (400 MHz, DMSO-d6): δ11.90 (bs, 1H), 8.04 (s, 1H), 7.60 (s, 1H), 7.42 (d, J = 1.2 Hz, 1H), 7.20 (d, J = 1.6 Hz, 1H), 5.96 (s, 1H), 2.44–2.32 (m, 4H), 1.91–1.82 (m, 1H), 1.77–1.70 (m, 1H), LCMS: 97.92% (346.51 [M+H]+) and melting point range: 248-252°C.

[0271] Fig.14J 6-Chloro-5-(1-methyl-1H-pyrazol-4-yl)-1H-indole-3-carboxylic acid is shown, which has 1 HNMR (400 MHz, DMSO-d6): δ12.09 (s, 1H), 11.87 (s, 1H), 8.04 (d, J=2.4 Hz, 2H), 8.01 (s, 1H), 7.67 (s, 1H), 7.59 (s, 1H), 3.90 (s, 1H), LCMS: 99.32% (276.31 [M+H]+) and melting point range: 241-245°C.

[0272] Figure 14K 6-Chloro-5-(1-phenyl-1H-pyrazol-4-yl)-1H-indole-3-carboxylic acid is shown, which has 1 H NMR (400 MHz, DMSO-d6): δ 11.86 (br s, 1H), 8.77 (s, 1H), 8.20 (br s, 1H), 8.00 (s, 2H), 7.93 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.52 (t, 2H), 7.33 (t, 1H), LCMS: 97.20% (338.13 [M+H]+) and melting point range: 245-249°C.

[0273] Conclusion: The embodiment enables the synthesis of AMPK agonists and their derivatives, such as Figures 13B-13Z and Figures 14A-14O Those shown.

[0274] Example 9: Synthesis of EV8016 analogs ( Fig.13A )

[0275] background: Fig. 12A The general structure of compounds capable of activating AMPK is shown. The synthesis of other derivatives is described in this example ( Fig.13F and Figures 14L-14M ).

[0276] Method: This example is illustrated in Fig. 12C is described in, and this embodiment shows Figure 14L The synthesis of the compounds shown in the figure, those skilled in the art will know the use of different reagents, reactants, conditions, etc. to generate derivative compounds, including Figures 13A-13Z and Figures 14A-14O Those shown.

[0277] result: Fig.13F 6-Chloro-5-(5-(1-(hydroxymethyl)cyclopropyl)thiophen-2-yl)-1H-indole-3-carboxylic acid is shown, which has 1 HNMR (400MHz, DMSO-d6): δ11.65(bs,1H),8.24(bs,1H),7.89(bs,1H),7.55(d,J=9.2Hz,1H),705(s, 1H), 6.88 (d, J = 3.6Hz, 1H), 4.90 (s, 1H), 3.57 (s, 2H),, 1.00–0.87 (m, 4H) and LCMS: 99.06% (465.30[M+H] + ).

[0278] Figure 14L 6-Chloro-5-(2-phenyloxazol-5-yl)-1H-indole-3-carboxylic acid is shown, which has 1 H NMR (400 MHz, DMSO) δ 12.08 (s, 2H), 8.53 (s, 1H), 8.13 (s, 1H), 8.06 (d, J = 6.6 Hz, 2H), 7.81 (s, 1H), 7.72 (s, 1H), 7.59 (t, J = 7.1 Hz, 3H), LCMS: 91.95% (339.35 [M+H]+) and melting point range: 261-265°C.

[0279] Figure 14M 6-Chloro-5-(4-phenylthiophen-2-yl)-1H-indole-3-carboxylic acid is shown, which has 1 H NMR (400MHz, DMSO-d6): δ11.77(bs,1H),8.31(s,1H),7.91(s,1H),7.88(s,1H),7.76(d,J=7.6H z,2H),7.66(d,J=1.2Hz,1H),7.61(s,1H),7.43(t,2H),7.31(t,1H), LCMS: 99.55%(354.10[M+H] + ) and melting point range: 195–199°C.

[0280] Conclusion: The embodiment enables the synthesis of AMPK agonists and their derivatives, such as Figures 13A-13Z and Figures 14A-14O Those shown.

[0281] Example 10: Synthesis of other analogs of EV8016 ( Fig.13A )

[0282] background: Fig. 12A The general structure of compounds capable of activating AMPK is shown. The synthesis of other derivatives is described in this example ( Figures 14N-14O ).

[0283] Method: This example is illustrated in Fig.12D is described in, and this embodiment shows Figures 14N-14O The synthesis of the compounds shown in the figure, those skilled in the art will know the use of different reagents, reactants, conditions, etc. to generate derivative compounds, including Figures 13A-13Z and Figures 14A-14O Those shown.

[0284] result: Fig.14N 6-Chloro-5-(1H-tetrazol-5-yl)-1H-indole-3-carboxylic acid methyl ester is shown. Fig.12D The product after step 2. The compound has 1 HNMR (400MHz, DMSO-d6): δ12.03(br s,1H), 8.25(s,1H), 8.15(s,1H), 7.60(s,1H), 7.01-7.00(br s,1H), 3.81(s,3H) and LCMS: 93.9% (278.07[M+H]+).

[0285] Fig.14O 6-Chloro-5-(1H-tetrazol-5-yl)-1H-indole-3-carboxylic acid is shown. Fig.12D The product of step 3. This compound has 1 HNMR (400 MHz, DMSO-d6): δ 16.73 (s, 1H), 12.34 (s, 1H), 12.21 (s, 1H), 8.36 (s, 1H), 8.19 (d, J = 2.9 Hz, 1H), 7.78 (s, 1H) and melting point range: 268-272°C.

[0286] Conclusion: The embodiment enables the synthesis of AMPK agonists and their derivatives, such as Figures 13A-13Z and Figures 14A-14O Those shown.

[0287] Example 11: Synthesis of (3R,3aR,6R,6aR)-6-((5-([1,1'-biphenyl]-4-yl)-1H-naphtho[1,2-d] Imidazol-2-yl)oxy)hexahydrofuro[3,2-b]furan-3-ol

[0288] Method: This embodiment is illustrated as follows Fig.15A In addition, although this embodiment describes Fig. 15B The synthesis of the compounds shown is described herein, but one skilled in the art will appreciate the use of different reagents, reactants, conditions, etc. to generate derivative compounds.

[0289] Step 1: Synthesis of N-(4-bromonaphthalene-1-yl)acetamide (Compound 2)—Acetic acid (4.2 mL, 45.040 mmol) was added to a stirred solution of compound-1 (5.0 g, 22.520 mmol) in methanol at room temperature, and the reaction mixture was stirred at 70° C. for 2 hours. The reaction progress was monitored by TLC. After completion, the reaction mixture was poured into ice-cold water, and the precipitated solid was filtered off and dried under vacuum to give compound-2 (4.5 g, 75%) as a yellow solid.

[0290] Step 2: Synthesis of N-(4-bromo-2-nitronaphthalen-1-yl)acetamide (Compound 3) - To a stirred solution of Compound 2 (4.0 g, 15.150 mmol) in acetic acid (40 mL) was added fuming HNO3 (0.72 mL, 16.660 mmol) at room temperature, and the reaction mixture was stirred at 75°C for 2 hours. The reaction progress was monitored by TLC. After completion, the reaction mixture was poured into ice-cold water, the precipitated solid was filtered off, washed with plenty of water, and dried under vacuum to give Compound 3 (3.52 g, 72%) as a yellow solid.

[0291] Step 3: Synthesis of N-(4-([1,1'-biphenyl]-4-yl)-2-nitronaphthalene-1-yl)acetamide (Compound 5):

[0292] To a stirred solution of compound 3 (20.0 g, 64.720 mmol) and compound 4 (19.2 g, 97.080 mmol) in dioxane and water (9: 1) was added K2CO3 (22.30 g, 161.800 mmol), the reaction mixture was purged with nitrogen for 15 minutes, then Pd (PPh3) 4 (3.73 g, 3.230 mmol) was added, and nitrogen was purged again for 10 minutes, and then the reaction mixture was stirred at 90 ° C for 2 hours. The reaction progress was monitored by TLC. After the reaction was completed, the reaction mixture was filtered through diatomaceous earth, the filtrate was concentrated under reduced pressure, the residue was ground with water, and the precipitated solid was filtered. The solid was recrystallized from 2-propanol, and the obtained solid was filtered and dried in vacuo to obtain compound-5 (11.1 g, 40%) as a yellow solid. Compound 5 was used as it is in step 6.

[0293] Step 4: Synthesis of 4-([1,1'-biphenyl]-4-yl)-2-nitronaphthalene-1-amine (Compound 6) - To a stirred solution of compound 5 (5.0 g, 13.089 mmol) in 1,4-dioxane (500 mL) was added concentrated HCl (50 mL), and the reaction mixture was stirred at 100 ° C for 48 hours. The progress of the reaction was monitored by TLC. After the reaction was completed, dioxane was evaporated under reduced pressure, the aqueous residue was diluted with ice-cold water, and the precipitated solid was filtered. The solid was washed with a large amount of water and dried under vacuum to obtain compound-6 (4.05 g, 91.0%) as a yellow solid. The compound was used in the next step without further purification.

[0294] Step 5: Synthesis of 4-([1,1'-biphenyl]-4-yl)naphthalene-1,2-diamine (Compound 7) - To a solution of compound-6 (5.0 g, 14.662 mmol) in a mixture of THF (400 mL) and ethanol (100 mL) was added 10% w / w of wet Pd-C (1.0 g), and the resulting reaction mixture was hydrogenated at room temperature for 16 hours under a hydrogen pressure of 80 psi in a Parr apparatus. The progress of the reaction was monitored by TLC. After the reaction was complete, it was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The crude product was ground with n-pentane, and the precipitated solid was filtered and dried in vacuo to give compound-7 (3.5 g crude product) as a yellow solid. The compound was used immediately in the next step.

[0295] Step 6: Synthesis of 5-([1,1'-biphenyl]-4-yl)-1,3-dihydro-2H-naphtho[1,2-d]imidazole-2-thione (Compound 8)—To a stirred solution of compound 7 (3.5 g, 11.290 mmol) in THF (70 mL) at 0°C, DMAP (2.75 g, 22.80 mmol) was added, followed by addition of dichlorothiocarbon (0.89 mL, 11.290 mmol), and the resulting reaction mixture was stirred at room temperature for 3 hours. The reaction progress was monitored by TLC. After completion of the reaction, the solvent was evaporated under reduced pressure, and the residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give crude compound 8 (3.2 g) as a yellow solid.

[0296] Step 7: Synthesis of 5-([1,1'-biphenyl]-4-yl)-2-(methylthio)-1H-naphtho[1,2-d]imidazole (Compound 9)—To a stirred solution of Compound 8 (3.0 g, 8.522 mmol) in acetone (100 mL) at 0°C was added K2CO3 (1.41 g, 10.226 mmol), stirred for 10–15 minutes, iodomethane (0.55 mL, 8.522 mmol) was added, and the resulting reaction mixture was stirred at room temperature for 3 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the solvent was evaporated under reduced pressure, and the residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by flash column chromatography (230x400 mesh, 15%–20% EA in PE) to give 1.1 g (35.3%) of Compound 9 as a yellow solid.

[0297] Step 8: Synthesis of 5-([1,1'-biphenyl]-4-yl)-2-(methylsulfonyl)-1H-naphtho[1,2-d]imidazole (Compound 10) - To a stirred solution of compound 9 (1.0 g, 2.732 mmol) in dichloromethane (30 mL) was added 3-chloroperoxybenzoic acid (1.41 g, 8.196 mmol) at 0°C and the resulting reaction mixture was stirred at room temperature for 2 hours. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with dichloromethane, washed with 10% aqueous NaHCO3 solution, 10% sodium thiosulfate solution and then brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give 3.5 g of crude yellow solid Compound 10.

[0298] Step 9: Synthesis of 5-([1,1'-biphenyl]-4-yl)-2-(methylsulfonyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-naphtho[1,2-d]imidazole (Compound 11)—To a stirred solution of compound-10 (1.3 g, 3.266 mmol) in dichloromethane (50 mL) at 0°C, triethylamine (0.68 mL, 4.899 mmol) was added followed by SEM chloride (0.65 g, 3.919 mmol) and the resulting reaction mixture was stirred at room temperature for 2 hours. The reaction progress was monitored by TLC. After completion of the reaction, it was diluted with dichloromethane, washed with 10% aqueous NaHCO3 solution and then with brine. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography (230 x 400 mesh, 10% - 15% EA in PE) to afford compound 11 (0.35 g, 20%).

[0299] Step 10: Synthesis of 5-([1,1'-biphenyl]-4-yl)-2-(((3R,3aR,6R,6aS)-6-((tert-butyldimethylsilyl)oxy)hexahydrofuro[3,2-b]furan-3-yl)oxy)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-naphtho[1,2-d]imidazole (13) - To a stirred solution of compound 12 (0.34 g, 1.325 mmol) in tetrahydrofuran (5 mL) was added potassium tert-butoxide (0.15 g, 1.325 mmol) at 0°C, and the resulting reaction mixture was stirred at 0°C for 1 hour. To the reaction mixture was added a solution of compound 11 in tetrahydrofuran at 0°C, and the resulting reaction mixture was stirred at room temperature for 2 hours. The reaction progress was monitored by TLC. After completion of the reaction, the solvent was evaporated under reduced pressure, and the residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure to give crude compound 13 (0.6 g) as a brown sticky solid.

[0300] Step 11: Synthesis of (3R,3aR,6R,6aR)-6-((5-([1,1'-biphenyl]-4-yl)-1H-naphtho[1,2-d]imidazol-2-yl)oxy)hexahydrofuro[3,2-b]furan-3-ol (Compound EV8017)—To a stirred solution of compound 13 (0.6 g, 0.846 mmol) in formic acid (12 mL) was added a solution of KHSO4 (0.11 g, 0.846 mmol) in water (1.0 mL) and the resulting reaction mixture was stirred at 60°C for 16 h. The reaction progress was monitored by TLC and LCMS. After completion of the reaction, it was cooled to 0°C, basified with 2N NAOH aqueous solution by adjusting the pH to 12–13, the solution was stirred at 0°C for another 1 h, neutralized with 2N HCl and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography (C-18 column, 80% MeOH in water) to give compound EV8017 (0.14 g, 35.7%) as an off-white solid.

[0301] Result: Step 1 produced compound 2 with the following characteristics: LCMS: 98.8% (266.06 M+H + &264.06M-H + ); 1HNMR (400MHz, CDCl3): δ8.31 (d, J = 8.4Hz, 1H), 7.88 (d, J = 8.0Hz, 1H), 7.82–7.80 (m, 2H), 7.67–7.61 (m, 2H), 7.47 (br s, 1H), 2.36 (s, 3H).

[0302] Step 2 produced compound 3 with the following characteristics: LCMS: 94.0% (309.12 MH + ); 1 H NMR (400MHz, CDCl3): δ8.72(br s,1H),8.38(s,1H),8.30(d,J=8.4Hz,1H),8.06(d,J=8.4Hz,1H),7.82–7.78(m,1H),7.71–7.67(m,1H),2.37(s,3H).

[0303] Step 3 produced compound 5 with the following characteristics: LCMS: 91.85% (381.31 MH + ).

[0304] Step 4 produced compound 6 with the following characteristics: LCMS: 90.48% (341.29, M+H + ).

[0305] Step 5 produced compound 7 with the following characteristics: LCMS: 52.0% (311.3 [M+H] + ).

[0306] Step 6 produced compound 8 with the following characteristics: LCMS: 52.0% (353.2 [M+H] + ).

[0307] Step 7 produced compound 9 with the following characteristics: LCMS: 77.6.0% (367.29 [M+H] + ).

[0308] Step 8 produced compound 10 with the following characteristics: LCMS: 76.9% (397.29 [MH] + ).

[0309] Step 9 produced compound 11 with the following characteristics: LCMS: 88.0% (529.40 [M+H] + ); 1HNMR (400MHz, CDCl3): δ8.75(d,J=7.6Hz,1H),8.00(s,1H),7.75(d,J=6.4Hz,2H),7.71(m,3H),7.67(s,1H),7. 60(d,J=8.4Hz,1H),7.54–7.40(m,4H),6.04(s,2H),3.70(t,J=8.0Hz,2H),0.93(t,J=8.0Hz,2H),-0.47(s,9H).

[0310] Step 10 produced compound 13 with the following characteristics: LCMS: 84.8% (709.70 [M+H] + ).

[0311] Step 11 produced compound EV8017 with the following characteristics: LCMS: 99.89% (465.30 [M+H] + ); HPLC: 99.86%; melting point: 158-162°C; 1 HNMR(400MHz,DMSO-d6): δ13-12(br s,1H),8.29(br s,1H),7.87(d,J=8.4Hz,1H),7.83–7.77(m,4H),7.59–7.50(m,6H),7.42–7.35(m,2H),5.52(d,J=5.6Hz,1H),4.98(d,J=6.8Hz,1H), 4.89(t,J=4.8Hz,1H), 4.41(t,J=4.8Hz,1H), 4.23–4.16(m,2H), 3.88(q,J=6.8Hz,1H), 3.82(t,J=6.8Hz,1H), 3.48(t,J=8.4Hz,1H).

[0312] Conclusion: The embodiment enables the synthesis of AMPK agonists and their derivatives, such as Fig. 15B Those shown.

[0313] Principle of Equivalence

[0314] Although the above description contains many specific embodiments of the present invention, these should not be interpreted as limitations on the scope of the present invention, but should be interpreted as examples of one embodiment thereof. Therefore, the scope of the present invention should not be determined by the illustrated embodiments, but by the appended claims and their equivalents.

Claims

1. An AMPK agonist, which is:

2. A pharmaceutical preparation comprising the AMPK agonist according to claim 1.

3. Use of the compound of claim 1 in the manufacture of a medicament for treating mitochondrial dysfunction.

4. The use of claim 3, wherein the mitochondrial dysfunction is a primary mitochondrial dysfunction selected from autosomal dominant optic atrophy, Alpers-Huttenlocher syndrome, ataxic neuropathy syndrome, Barth syndrome, coenzyme Q deficiency, complex I, complex II, complex III, complex IV and complex V deficiency, chronic progressive external ophthalmoplegia, diabetes and deafness, Kearns-Sayre syndrome, leukoencephalopathy with brainstem and spinal cord involvement and elevated lactate, Leigh syndrome, Leber's hereditary optic neuropathy , Rafter disease, MELAS syndrome, mitochondrial enoyl-CoA reductase protein-associated neurodegeneration, myoclonic epilepsy with shattered red fibers, mitochondrial recessive ataxia syndrome, mtDNA deletion syndrome, mtDNA depletion syndrome, mtDNA maintenance disorder, mtDNA translation defect and RNA translation defect, mitochondrial tRNA synthetase deficiency, mitochondrial neurogastroenteric encephalopathy syndrome, NARP syndrome, Pearson syndrome, pyruvate dehydrogenase complex deficiency, DNA polymerase gamma deficiency, pyruvate carboxylase deficiency, and thymidine kinase 2 deficiency.

5. The use of claim 3, wherein the mitochondrial dysfunction is a secondary mitochondrial dysfunction, wherein the secondary mitochondrial dysfunction is selected from the group consisting of amyotrophic lateral sclerosis, Alzheimer's disease, Friedreich's ataxia, Huntington's disease, motor neuron disease, N-glycanase deficiency, organic acidemia, Parkinson's disease, prion disease, spinal muscular atrophy, spinocerebellar ataxia, Becker muscular dystrophy, Duchenne muscular dystrophy, Emory-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular dystrophy, Charcot-Marie-Duce disease, congenital myopathy, distal myopathy, endocrine myopathy, giant axonal neuropathy, hereditary spastic paraplegia, metabolic myopathy, neuromuscular junction disease, autism, cancer, diabetes, metabolic syndrome, chronic fatigue syndrome, inflammatory disorders, and aging.

6. The use according to claim 5, wherein the secondary mitochondrial dysfunction is arthritis.

7. The use of claim 5, wherein the secondary mitochondrial dysfunction is selected from congenital muscular dystrophy and inflammatory myopathy.

8. The use of claim 3, wherein the medicament further comprises at least one selected from the group consisting of a binder, a lubricant, a buffer and a coating.

9. The use of claim 3, wherein the medicament is formulated for at least one selected from the group consisting of oral administration, subcutaneous administration, intravenous administration, intraperitoneal administration, intranasal administration, skin administration, intravitreal injection, and inhalation.

10. Use of the compound of claim 1 in the manufacture of a medicament for treating age-related macular degeneration.

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