Compounds and methods for promoting mitophagy

Abstract

Mitophagy is a selective autophagic process, essential for cellular homeostasis, that eliminates dysfunctional mitochondria. Embodiments include (1-phenyl-2-heteoaryl)ethyl-guanidine compounds and methods of treating a subject with impaired mitophagy and / or a mitochondrial disorder. In aspects, one or more of the compounds described herein are used therapeutically to modulate the turnover of mitochondria and / or prevent accumulation of dysfunctional mitochondria which can lead to cellular degeneration. In aspects, the compounds described herein can induce or promote mitophagy by modulating membrane potential of a cell. The compounds and methods described herein can be used to treat ailments related to mitophagy such as, for example, Parkinson's disease, Alzheimer's and Huntington's disease.

Description

PATENT SR3-005WOCOMPOUNDS AND METHODS FOR PROMOTING MITOPHAGYRELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application number 63 / 740,268 filed on December 30, 2024. The contents of the aforementioned application are incorporated herein by reference.FIELD OF THE INVENTION

[0002] The invention relates to the field therapeutics and more specifically, to compositions and methods of promoting mitophagy by removing dysfunctional mitochondria to treat mitochondrial diseases.BACKGROUND

[0003] A mitochondrion is a double-membrane-bound organelle found in most eukaryotic organisms. Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own genome ("mitogenome") that is similar to bacterial genomes. Mitochondrial proteins (i.e., proteins transcribed from mitochondrial DNA) vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria, whereas in rats, 940 proteins have been reported. The mitochondrial proteome is thought to be dynamically regulated.

[0004] Mitochondria use aerobic respiration to generate most of the cell's supply of adenosine triphosphate (ATP), which is subsequently used throughout the cell as a source of energy. Oxidative phosphorylation (OXPHOS), the Krebs's cycle, the urea cycle, heme biosynthesis and fatty acid oxidation take place within the mitochondria. In addition to supplying cellular energy, mitochondria are involved in signaling, cellular differentiation, and cell death, as well as maintaining control of the cell cycle and cell growth. Mitochondrial biogenesis is in turn temporally coordinated with these cellular processes. Mitochondria have been implicated in several human disorders and conditions, such as mitochondrial diseases, cardiac dysfunction, heart failure and sarcopenia. Mitochondria dysfunction have also been implicated in neurologicalPATENT SR3-005WOdisorders such as autism, Parkinson’s and Alzheimer’s disease.

[0005] The majority of mitochondrial proteins are encoded by the nuclear genome, translated in the cytosol, and imported into the mitochondrion. However, 13 core subunits of respiratory complexes are encoded by the reduced mitochondrial genome and synthesized within the mitochondrial matrix. Mutations in these 13 genes (or their associated non-protein-coding genes) tend to be especially severe, as all 13 proteins are core subunits of the oxidative phosphorylation pathway. Any disruption to subunit structure, stability, or function can have major biochemical and physiological consequences. For example, mtDNA mutations that cause structural changes in OXPHOS subunits disrupt the electron transfer relay, resulting in inefficient energy production. Inefficient transfer can generate superoxide byproducts, resulting in increased reactive oxygen species (ROS) and reactive nitrogen species (RNS), causing a chronic state of cellular stress.

[0006] Mitochondrial dysfunction is also a hallmark of aging and cellular senescence. There are changes in mitochondrial metabolism with aging that are linked to changes in mitochondrial organization. Studies suggest that the dynamic equilibrium between fusion and fission is essential for healthy mitochondrial function. In senescent cells, fusion exceeds fission and large mitochondria form that contribute to cell senescence. The fission process is coupled with ER-microtubule function; therefore, interaction of mt-ER-Lysosome affects the fission process. There is evidence of decreased senescence with exercise and findings indicate that mechanical forces alter MT-ER junction protein complexes through unknown processes in the fission and fusion functions.

[0007] Mitophagy can be defined as the selective degradation of mitochondria by autophagy. It often occurs to defective mitochondria following damage or stress. It promotes turnover of mitochondria and prevents accumulation of dysfunctional mitochondria which can lead to cellular degeneration. Mitophagy is required to adjust mitochondrial numbers to changing cellular metabolic needs, for steady-statePATENT SR3-005WOmitochondrial turnover, and during certain cellular developmental stages, such as during cellular differentiation of red blood cells. Damaged mitochondria can cause a depletion in ATP and a release of cytochrome c, which leads to activation of caspases and onset of apoptosis. Mitochondrial damage is not caused solely by oxidative stress or disease processes; normal mitochondria will eventually accumulate oxidative damage hallmarks overtime, which can be deleterious to mitochondria as well as to the cell. These faulty mitochondria can further deplete the cell of ATP, increase production of ROS, and release proapoptopic proteins such as caspases.

[0008] Because of the danger of having damaged mitochondria in the cell, the timely elimination of damaged and aged mitochondria is essential for maintaining the integrity of the cell. Mitochondrial depletion reduces a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Accordingly, mitophagy can be considered a “quality control” process for cellular homeostasis and to maintain the health of a cell. In addition to the selective removal of damaged mitochondria, mitophagy is also required to adjust mitochondrial numbers to changing cellular metabolic needs, for steady-state mitochondrial turnover, and during certain cellular developmental stages, such as during cellular differentiation of red blood cells.

[0009] If mitophagy is impaired, the process of removing damaged mitochondria from cells is defective. This can lead to an accumulation of damaged mitochondria and negative consequences. Impaired mitophagy is associated with tissue dysfunction and disease as a result of cell death. Common diseases associated with mitophagy include neurodegenerative diseases (e.g., Alzheimer's disease and Parkinson's), cardiovascular diseases, cancers and kidney disease. Impaired mitophagy has also been linked to premature aging and age-associated disorders.

[0010] There are currently no cures for impaired mitophagy in mitochondrial disorders. The few treatment options available are generally limited to managing symptoms of the disorders. Conventional treatments are generally ineffective andPATENT SR3-005WOlimited to nutraceutical supplements, dietary restriction and exercise. Thus, there is a need for reliable methods of treating mitochondrial disorders such as impaired mitophagy. The present invention includes compounds and methods that can be used therapeutically to promote mitophagy and treat mitochondrial diseases and age-related conditions.SUMMARY OF THE INVENTION

[0011] The inventions described and claimed herein have many attributes and embodiments including those set forth or described or referenced in this brief summary. The inventions described and claimed herein are not limited to, or by, the features or embodiments identified in this summary, which is included for purposes of illustration only and not restriction.

[0012] The present disclosure solves the problems described above by providing compounds and methods for inducing or promoting mitophagy. In aspects, the compounds and methods are useful for treating mitochondrial diseases and diseases related to aging.

[0013] Specifically, the present disclosure relates to small-molecule compounds and pharmaceutical compositions that can promote mitophagy through modulation of mitochondrial bioenergetics. In particular embodiments, the invention provides compounds that inhibit ATPase inhibitory factor 1 (ATPIF1 ), thereby altering mitochondrial membrane potential and promoting selective clearance of dysfunctional mitochondria.

[0014] Accordingly, embodiments include small-molecule mitophagy-promoting compounds of Formula (II), including (1-phenyl-2-heteroaryl)ethyl-guanidine analogs, and pharmaceutically acceptable salts thereof.

[0015] Embodiments include (1-phenyl-2-heteoaryl)ethyl-guanidine compounds and analogs.PATENT SR3-005WO

[0016] Embodiments also include a compound of Formula (II):Formula (II)or a pharmaceutically acceptable salt thereof, wherein R is selected from:

[0017] Embodiments also include isomers of a compound of Formula (II):or a pharmaceutically acceptable salt.

[0018] In embodiments, the compound is selected from a compound of Formula A, Formula B, Formula C, Formula D, Formula E, Formula F, Formula G, Formula H,PATENT SR3-005WO Formula I, Formula J, Formula K, Formula L or Formula M:PATENT SR3-005WOFormula I Formula JFormula K Formula LFormula Mor a pharmaceutically acceptable salt.

[0019] In embodiments, one or more of the compounds described herein are used therapeutically, for example, to treat a mitophagy-related disease or disorder. The mitophagy-related disease or disorder can be, for example, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), Kearns-Sayre syndrome (KSS), Leigh syndrome or Leber’s hereditary optic neuropathy (LHON).

[0020] In embodiments, one or more of the compounds described herein are used therapeutically to modulate the turnover of mitochondria and / or prevent accumulation ofPATENT SR3-005WOdysfunctional mitochondria which can lead to cellular degeneration.

[0021] The compounds can modulate ATPIF1 -mediated inhibition of F1F0-ATP synthase, increase mitochondrial membrane potential, restore ATP production and promote selective removal of damaged mitochondria.

[0022] In embodiments, one or more compounds described herein can be administered to treat a mitochondrial disorder associated with pathogenic mitochondrial DNA (mtDNA) mutation.

[0023] In embodiments, one or more of the compounds described herein are administered to treat diseases associated with mitophagy including neurodegenerative diseases (e.g., Alzheimer's disease and Parkinson's), cardiovascular diseases, cancers and kidney diseases. The compounds can also be administered to treat premature aging and age-associated disorders (i.e., senescence-associated diseases, sarcopenia and disorders).

[0024] Embodiments also include methods of promoting longevity, slowing the aging process or reducing signs of aging by administering one or more compounds described herein.

[0025] The compounds can reduce mutant mitochondrial DNA levels and increase wild-type mitochondrial DNA copy number in cells harboring mitochondrial mutations, including Kearns-Sayre syndrome (KSS) and ATP6-mutant Leigh syndrome models.

[0026] Pharmaceutical compositions comprising the compounds and methods of increasing mitophagy, restoring mitochondrial function, and treating mitochondrial DNA mutation disorders are also provided. The compounds can be used to treat mitochondrial diseases and other conditions associated with mitophagy impairment.

[0027] In aspects, the methods described herein include administering one or more additional therapeutic agents (e.g., anti-apoptoic and metabolism enhancing agents). InPATENT SR3-005WOaspects, the compounds described herein are administered in combination with a senolytic agent or senomorphic agent.

[0028] In aspects, the compounds described herein are administered orally or parenterally. In aspects, the compounds are formulated as a solid oral dosage form or injectable formulations.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings illustrate aspects of the invention. In such drawings:

[0030] FIG. 1 shows the chemical structure of Formula (II). Formula (II) can include a combination of the two mirror-image isomers (“a” and “b”). The isomers can have different pharmacological, pharmacokinetic and off-target properties.

[0031] FIG. 2A depicts the process of mitophagy which occurs in response to alterations in mitochondrial membrane potential. Proteins are activated that initiate autophagosome engulfment primarily via the Parkin-PINK1 pathway and other alternative receptor-mediated pathways.

[0032] FIG. 2B depicts the process with normal membrane potential. The mitochondria is not tagged for mitophagy.

[0033] FIG. 3A is a bar graph of BH2 mtDNA content after 42 days of treatment. The results demonstrate that treatment with Formula (II) reduces mutant mtDNA load in the Common deletion (BH2) cell line (A) and the ATP6 mutant cell line (B) after 42 days. n=3 / group, Unpaired student’s t test. ****p<0.0001. RII: Rock II inhibitor.

[0034] FIG. 3B is a bar graph of ATP6 mtDNA content after 42 days of treatment.

[0035] FIG. 4A is a bar graph showing ATP production after 42 days of treatment. The results show Formula (II) incomplete functional recovery of mitochondria in mtDNA disease models. Measurement of ATP production ratios after 42 days of treatment showPATENT SR3-005WOrecovery of BH2 Formula (II) and RII treated cells (yellow middle bar) compared to control and 143B wildtype cells (red and green bars). Mutant ATP6 cells showed reduced rescue (purple bar). n=8 wells / cell line normalized to cell count within well. One-way ANOVA with Tukey’s post hoc, ****p<0.0001

[0036] FIG. 5 depicts the mitochondrial genome of the BH2 common deletion cell line. The BH2 line is heteroplasmic for normal mtDNA and mtDNA with the deletion of genes shown above (red section).

[0037] FIG. 6A is a heatmap of TPM values from complete RNA seq data that shows a unique transcriptional signature of treated cells (middle) compared to controls. Darker color indicates higher expression.

[0038] FIG. 6B - 6D show Go enrichment analysis of genes upregulated in treated cells compared to controls.

[0039] FIG. 6E shows transcript levels of mtDNA protein coding genes. Bold gene names are genes found in the common deletion region.

[0040] FIG. 6F shows mtDNA encoded tRNAs. Bold gene names are genes found in the deletion.

[0041] FIG. 7 shows the steps in a method of synthesizing a compound of Formula (II) and analogs.

[0042] FIG. 8 is a table of compounds used in a study (LRI001-012 DNA).

[0043] FIG. 9A is a bar graph that shows mtDNA content after 28-day treatment with different compounds.

[0044] FIG. 9B is a bar graph that shows mtDNA content after 28-day treatment with different compounds and Rock II inhibitor.

[0045] FIG. 10 shows results of in vivo studies carried out with n=3 mice per group (female) starting injection at 56 mg / kg. The plot of plasma concentration (ng / ml) overPATENT SR3-005WOtime (One way ANOVA Tukey’s Multiple Comparison) shows time from 15 minutes to 24 hours.

[0046] FIG. 11A is a plot of plasma concentration (ng / ml) at 24 hours (brain tissue).

[0047] FIG. 11 B is a plot of plasma concentration (ng / ml) at 24 hours (liver tissue).

[0048] FIG. 11C is a plot of plasma concentration (ng / ml) at 24 hours (kidney).

[0049] FIG. 11 D is a plot of plasma concentration (ng / ml) at 24 hours (spleen).Definitions

[0050] Reference in this specification to "one embodiment / aspect" or "an embodiment / aspect" means that a particular feature, structure, or characteristic described in connection with the embodiment / aspect is included in at least one embodiment / aspect of the disclosure. The use of the phrase "in one embodiment / aspect" or "in another embodiment / aspect" in various places in the specification are not necessarily all referring to the same embodiment / aspect, nor are separate or alternative embodiments / aspects mutually exclusive of other embodiments / aspects. Moreover, various features are described which may be exhibited by some embodiments / aspects and not by others. Similarly, various requirements are described which may be requirements for some embodiments / aspects but not other embodiments / aspects. Embodiment and aspect can be in certain instances be used interchangeably.

[0051] The terms used in this specification generally have their ordinary meanings in the art, within the context of the disclosure, and in the specific context where each term is used. Certain terms that are used to describe the disclosure are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the disclosure. It will be appreciated that the same thing can be said in more than one way.

[0052] Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. Nor is any special significance to be placedPATENT SR3-005WOupon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only and is not intended to further limit the scope and meaning of the disclosure or of any exemplified term. Likewise, the disclosure is not limited to various embodiments given in this specification.

[0053] Without intent to further limit the scope of the disclosure, examples of instruments, apparatus, methods and their related results according to the embodiments of the present disclosure are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the case of conflict, the present document, including definitions, will control.

[0054] The term “mitochondrial diseases” refers to chronic, genetic, often inherited disorders that occur when mitochondria fail to produce enough energy for the body to function properly. Mitochondrial diseases can affect almost any part of the body, including the cells of the brain, nerves, muscles, kidneys, heart, liver, eyes, ears or pancreas. Mitochondrial diseases can be caused by mitochondrial DNA (mtDNA) disorders or nuclear DNA (nDNA) disorders.

[0055] Mitochondrial DNA (mtDNA) disorders include, for example, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy with ragged-red Fibers (MERRF), Leber's hereditary optic neuropathy (LHON), Kearns-Sayre syndrome (KSS), Leigh syndrome, leukodystrophy w / complex II deficiency, cardiomyopathy & encephalopathy (complex I deficiency), optic atrophy and ataxia (complex II deficiency), hypokalemia and lactic acidosis, hepatopathy & ketoacidosis, hypertrophic cardiomyopathy, liver failure, renal tubulopathy (w / complex III deficiency) and encephalopathy (w / complex V deficiency), autosomal progressive external ophthalmoplegia, mitochondrial neurogastrointestinal encephalomyopathy,PATENT SR3-005WOAlpers-Huttenlocher syndrome, ataxia neuropathy syndromes, infantile myopathy / spinal muscular atrophy and hypotonia.

[0056] The term “primary mitochondrial disorders” refers to a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. The mitochondrial respiratory chain is the essential final common pathway for aerobic metabolism. Tissues and organs that are highly dependent on aerobic metabolism are usually most affected by mitochondrial disorders. Many genetic and non-genetic disorders involve mitochondrial mechanisms as a secondary feature. However, "primary mitochondrial disorders" are considered to be known or presumed genetic disorders caused by pathogenic variants in genes coding for the mitochondrial respiratory chain and related proteins.

[0057] Nuclear DNA (nDNA) disorders include, for example, Leigh syndrome, leukodystrophy w / complex II deficiency, cardiomyopathy & encephalopathy (complex I deficiency), optic atrophy & ataxia (complex II deficiency), hypokalemia & lactic acidosis (complex III deficiency), hepatopathy and ketoacidosis, cardiomyopathy and encephalopathy, leukodystrophy & renal tubulopathy, hypertrophic cardiomyopathy, liver failure, renal tubulopathy (w / complex III deficiency), encephalopathy (w / complex V deficiency), coenzyme Q10 deficiency, Barth syndrome, autosomal progressive external ophthalmoplegia, mitochondrial neurogastrointestinal encephalomyopathy, Alpers-Huttenlocher syndrome, ataxia neuropathy syndromes, infantile myopathy / spinal muscular atrophy, hypotonia, reversible hepatopathy, myopathy with cataract and combined RC deficiency.

[0058] The term “secondary mitochondrial dysfunction” refers to any abnormal mitochondrial function other than a primary mitochondrial disorders. Secondary mitochondrial dysfunction (SMD) can be caused by genes encoding neither function nor production of the oxphos proteins and accompanies many hereditary non-mitochondrial diseases. Secondary mitochondrial dysfunction can also be due to nongenetic causes such as environmental factors. Secondary mitochondrial dysfunction is seen in many different genetic disorders, including ethylmalonic aciduria (caused by mutation ofPATENT SR3-005WOETHE1), Friedreich ataxia (FXN), hereditary spastic paraplegia 7 (SPG7), and Wilson disease (ATP7B), and is also seen as part of the aging process.

[0059] The term “mitophagy” referers to the process by which a cell degrades mitochondira that has no or has lost its ability to function. Mitophagy is activated through many mechanisms including PINK1 activating PARKIN, MUL1 ubiquitination, cardiolipin ejection into the cytoplasm and others. Mitophagy is induced by the mitochondria losing its membrane potential and its membrane integrity and therefore its function.Mitochondria are enveloped in vesicles and targeted to lysosomes and perioxosomes in order to break them down. Mitophagy has been shown to be reduced in multiple diseases as well as during the aging process, leading to a buildup of damaged mitochondria, reduction in ATP production and an increase in reactive oxygen species that can further damage the cell.

[0060] The term “mitophagy related disease or disorder” refers to an ailment that is associated with dysfunctional mitochondria and can include, for example, neurodegenerative diseases (e.g., Alzheimer's disease and Parkinson's), cardiovascular diseases, cancers and kidney diseases.

[0061] The term “neurodegenerative disorder” or “neurodegenerative disease” refers to a disease that causes nerve cells in the brain and spinal cord to deteriorate and die. Common symptoms of neurodegenerative disorders include problems with movement (e.g., muscle wasting, ataxia and parkinsonism) and mental functioning problems (e.g., dementia). Neurodegenerative disorders include, for example, Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS), motor neuron disease, multiple system atrophy, progressive supranuclear palsy and post-stroke cognitive impairment.

[0062] The term “senescence” refers to gradual deterioration of functional characteristics in living organisms. Cellular senescence is often defined as a stress-induced, durable cell cycle arrest of previously replication-competent cells. The effects of senescent cells can be thought of as beneficial or detrimental with regard to hostPATENT SR3-005WOphysiology and disease, although in some contexts, senescent cells affect a disease state in a complex manner both promoting and opposing certain conditions.

[0063] The term “senescence-associated disease or disorder” refers to an ailment that is associated with age and can include, for example, atherosclerosis, osteoarthritis, osteoporosis, hypertension, arthritis, cataracts, cancer, Alzheimer’s disease, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis. Other ailments (including age-related conditions) associated with age or senescence include hair graying, sarcopenia, adiposity, neurogenesis, fibrosis and glaucoma.

[0064] Still other ailments associated with age or senescence include cardiovascular disease (e.g., atherosclerosis, angina, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary thrombosis, myocardial infarction, hypertension, aortic aneurysm, cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapsed, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis, brain aneurysm, and stroke). A senescence-associated disease or disorder can also be an inflammatory or autoimmune disease or disorder (e.g., osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease or kyphosis). A senescence-associated disease or disorder can also be a neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, mild cognitive impairment or motor neuron dysfunction). A senescence-associated disease or disorder can also be a metabolic disease (e.g., diabetes, diabetic ulcer, metabolic syndrome or obesity). A senescence-associated disease or disorder can also be a pulmonary disease (e.g., pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis or age-related loss of pulmonary function). A senescence-associated disease or disorder can also be an eye disease or disorder (e.g., macular degeneration, glaucoma, cataracts, presbyopia or vision loss). A senescence-associated disease or disorder can also be renal disease, renal failure, frailty, hearing loss, muscle fatigue, skin conditions, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucosa fibrosis or sarcopenia. A senescence-associated disease or disorder can also be a dermatological disease or disorder (e.g., eczema, psoriasis,PATENT SR3-005WOhyperpigmentation, nevi, rashes, atopic dermatitis, urticaria, diseases or disorders related to photosensitivity or photoaging).

[0065] The term “mitochondrial dysfunction” refers generally to the failure of mitochondria which can lead to a plethora of symptoms and disorders. Mitochondrial dysfunction is a driving force behind cellular senescence, a process where cells permanently lose the ability to divide. Senescent cells have mitochondria that are elongated and produce more reactive oxygen species (ROS) while producing less ATP. The mitochondrial genome can accumulate damage and mutations over time, which can lead to loss of function. Further, mitophagy is disrupted in senescence.

[0066] The term “Senescence-Associated Mitochondrial Dysfunction” or “SAMD” is a result of mitochondrial dysfunction and is associated with metabolic dysfunction and tissue decline. Similarly, “Senescence-Associated Secretory Phenotype” or “SASP” refers to a distinct secretory phenotype of senescent cells. SAMD is responsible for at least part of SASP.

[0067] The term “mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes” or “MELAS” refers to one of the most severe mtDNA related disorders with mutations such as in the tRNA-Leu gene leading to inefficent protein translation in the mitochondria. The stroke-like episodes result from neuronal impairment caused by reduced ATP production and fragmented mitochondrial buildup. This reduction in ATP production corresponds with a reduction in membrane potential. tRNA mutations are especially debilitating because they alter the production of every protein requiring leucine. There are other cases with MELAS that contain a COX3 mutation affecting Complex III activity but with the same deleterious symptoms leading to the MELAS diagnosis.

[0068] The term “Kearns-Sayre Syndrome” or “KSS” refers to a mtDNA disorder that results from a 5 kb deletion in the mtDNA in a heteroplasmic manner. The genes that are rendered non-functional or missing are: ND3-6, ND4L, A8, A6, and COX3. ThePATENT SR3-005WOpatient (where the cell line BH2s are derived from) experienced difficulty walking and was deaf, with high levels of lactate in CSF and blood and ragged red fibers indicating mitochondrial network collapse. 72% of the mtDNA contain this deletion therefore the ETC cannot function properly. These patients are diagnosed in childhood and often have cognitive and mobility issues and shortened lifespans. Mitochondria with these deletions have reduced ATP production and mitochondrial membrane potential as well as an increase in mitochondiral mass indicating that these mitochondria are unable to be targeted to the autophagosome.

[0069] The term “Leigh Syndrome” refers to a classification of syndromes resulting from mutations in the ATPV complex. These mutations originate in the ATPV subunit encoded by MT-ATP6 generating the ATP6 protein. There are mutations possible in the gene and range in severity and mutation load. There are many commonalities between these diseases as well further complicating their definitions and diagnosis parameters. One example is the buildup of lactic acid throughout the CSF and blood as well as concurrent electrophysiological issues (i.e. seizures).

[0070] The term “Leber’s hereditary optic neuropathy” or “LHON” refers to another syndrome of mitochondrial origin. LHON symptoms are focused around the retina and the neurons that connect the optic nerve. Three mutations in the genes that create the NADH Dehydrogenase complex are responsible for most of the cases, a trait that is similar to other mtDNA diseases such as LS and KSS. Men are more susceptible to LHON than women and the mtDNA haplotype has an effect on the clinical presentation. The loss of Complex I function reduces the ability for mitochondria to properly coordinate membrane potential and mitophagy targeting. This leads to both a reduction in ATP production and an escape of the normal mitophagy signaling. Studies have shown in LHON primary fibroblasts and cybrids that mitophagy is increased to try and remove the damaged mitochondria but this isn’t clearing the ones with the mutation loads. Current treatments manage symptoms and there are some therapies in development for gene therapy therefore requiring other approaches targeting dysfunctional mitochondria to mitophagy.PATENT SR3-005WO

[0071] The term “senolytic” or “senolytic agent” refers to a therapeutic such as a small molecule that can selectively or preferentially induce death of senescent cells. A senolytic agent may kill senescent cells by inducing (i.e. , activating, stimulating or removing inhibition of) an apoptotic pathway that leads to cell death. Senolytic agents may be useful for treatment of senescence-associated diseases or disorders. The drugs dasatinib, quercetin, fisetin and navitoclax have potential senolytic activities.

[0072] The term “senomorph” or term “senomorphic agent” refers to one of a range of agents that can modulate the phenotypes of senescent cells (SCs) to those of young cells through interfering with senoinflammation / inflammaging, senescence-related signal pathways and SASP, without induction of SC apoptosis.

[0073] The term “SASP inhibitor” refers to an agent that can inhibit development of a senescence-associated secretory phenotype (SASP). Suppressing the SASP without eliminating senescent cells is an alternative therapeutic approach for alleviating cellular senescence-related phenotypes or diseases. SASP inhibitors (i.e., senomorphics) can directly or indirectly attenuate the SASP of senescent cells by inhibiting transcription factor nuclear factor (NF)-KB, the JAK-STAT signal transduction pathway, the serine / threonine protein kinase mTOR, mitochondrial complex-1 -related or4-related targets, or other pathways involved in the induction and maintenance of the SASP

[0074] The term “apoptosis” refers to a caspase-mediated programmed cell death characterized by formation of membrane-enveloped apoptotic bodies that are rapidly phagocytosed by macrophages or neighboring cells. There is evidence of apoptotic mechanisms in animal models of several neurodegenerative diseases, but evidence in human tissues is limited. Caspase-1, -3, -8, and -9 activation and cytochrome c release seen in models of Huntington’s disease (HD) were also demonstrated in human striatal brain tissue. Similarly, caspase activation and neuronal apoptosis have been demonstrated in ALS and HIV-associated neurodegeneration.

[0075] The term “necroptosis” refers to a form of programmed cell death that is independent from the caspase activation and involves loss of plasma membranePATENT SR3-005WOintegrity. Two main effector proteins of necroptosis are receptor-interacting serine / threonine-protein kinase 1 (RIPK1) and mixed-lineage kinase domain-like (MLKL). Astrocytes release TNF-a, FasL, and TRAIL, which can trigger necroptosis through RIPK1 and MLKL activation, and this mechanism has been demonstrated in murine models of ALS. RIPK1 -mediated axonal pathology was observed in pathological specimens from ALS patients. Necroptotic mechanisms were also observed in MS pathological samples.

[0076] The term “oxidative phosphorylation” or “OXPHOS” refers to the process by which ATP synthesis is coupled to the movement of electrons through the mitochondrial electron transport chain and the associated consumption of oxygen. In eukaryotes, this takes place inside mitochondria. In eukaryotes, redox reactions are catalyzed by a series of protein complexes within the inner membrane of the cell's mitochondria.These linked sets of proteins are called the electron transport chain. In eukaryotes, five main protein complexes are involved that use a variety of electron donors and acceptors.

[0077] More than 70 different polypeptides interact on the inner mitochondrial membrane to form the electron transport chain, also known as the respiratory chain. Thirteen essential subunits are encoded by mitochondrial DNA (mtDNA) located within mitochondria, along with the ribosomal and transfer RNAs required for intra-mitochondrial protein synthesis. The remaining respiratory chain polypeptides, and proteins essential for the assembly of the respiratory chain, mitochondrial structure, and the maintenance and expression of mtDNA are encoded by the nuclear genome (nDNA).

[0078] The term “ATPIFi” or “ATPase inhibitory factor 1” refers to a mitochondrial enzyme that in humans is encoded by the ATPIF1 gene. This gene encodes a mitochondrial ATPase inhibitor. Alternative splicing occurs at this locus and three transcript variants encoding distinct isoforms have been identified. It prevents ATPase from switching to ATP hydrolysis during collapse of the electrochemical gradient, for example during oxygen deprivation. ATP synthase inhibitor forms a one-to-onePATENT SR3-005WOcomplex with the F1 ATPase, possibly by binding at the alpha-beta interface. It is thought to inhibit ATP synthesis by preventing the release of ATP. The inhibitor has two oligomeric states, dimer (the active state) and tetramer. At low pH, the inhibitor forms a dimer via antiparallel coiled coil interactions between the C-terminal regions of two monomers. At high pH, the inhibitor forms tetramers and higher oligomers by coiled coil interactions involving the N terminus and inhibitory region, thus preventing the inhibitory activity.

[0079] The term “treating” or “treatment” refers to one or more of (II) inhibiting the disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e. , arresting further development of the pathology and / or symptomatology); and (2) ameliorating the disease condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and / or symptomatology) such as decreasing the severity of disease.

[0080] The term anti-apoptotic agent refers to a substance that prevents apoptosis, or programmed cell death. Examples of anti-apoptotic agents include Bcl-2 proteins, lAPs, FLICE-like inhibitory protein (c-FLIP) and nuclear factor-KB (NF-KB). Apoptosis has been linked to the elimination of potentially malignant cells, hyperplasia, and tumor progression. Reduced apoptosis or resistance to apoptosis plays a vital role in carcinogenesis.

[0081] The term "administration" refers to the introduction of an amount of a predetermined substance into a patient by a certain suitable method. The compositions disclosed herein may be administered via any of the common routes, as long as it is able to reach a desired tissue, for example, inhaling, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, or intrarectal administration.

[0082] The term “small molecule” or “micromolecule” refers to a low molecular weight (< 1000 daltons) organic compound that may regulate a biological process, withPATENT SR3-005WOa size on the order of 1 nm. Larger structures such as nucleic acids and proteins, and many polysaccharides are not small molecules, although their constituent monomers (e.g., ribo- or deoxyribonucleotides, amino acids, and monosaccharides, respectively) are often considered small molecules.

[0083] The term “ROCK (Rho-associated protein kinase) inhibitor” refers to a drug or compound that blocks the activity of the Rho kinase pathway, which is involved in regulating the cell's internal cytoskeleton, cell shape, and movement. These inhibitors are used in cell culture to improve stem cell survival, and they have potential therapeutic applications for conditions such as glaucoma, cardiovascular diseases like hypertension and stroke, and various cancers. Examples of approved ROCK inhibitors include Fasudil and Ripasudil.

[0084] The term "subject" refers to those who a susceptible to an ailment (e.g., a disease related to senescence) or who are suspected of having or diagnosed with the ailment. However, any subject to be treated with the therapeutic methods described herein is included without limitation.

[0085] The term “aryl” refers to a monocyclic or polycyclic aromatic hydrocarbon ring (e.g., phenyl, naphthyl), optionally substituted.

[0086] The term “heteroaryl” refers to an aromatic ring containing 1-4 heteroatoms selected from N, O, and S.

[0087] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are to be understood as approximations in accordance with common practice in the art. When used herein, the term “about” may connote variation (+) or (-) 1%, 5% or 10% of the stated amount, as appropriate given the context. It is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0088] Many known and useful compounds and the like can be found inPATENT SR3-005WORemington’s Pharmaceutical Sciences (13thEd), Mack Publishing Company, Easton, PA — a standard reference for various types of administration. As used herein, the term “formulation(s)” refers to a combination of at least one active ingredient with one or more other ingredient, also commonly referred to as excipients, which may be independently active or inactive. The term “formulation” may or may not refer to a pharmaceutically acceptable composition for administration to humans or animals and may include compositions that are useful intermediates for storage or research purposes.

[0089] Other technical terms used herein have their ordinary meaning in the art that they are used, as exemplified by a variety of technical dictionaries. The particular values and configurations discussed in these non-limiting examples can be varied and are cited merely to illustrate at least one embodiment and are not intended to limit the scope thereof.DETAILED DESCRIPTION

[0090] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the subject technology as claimed. Additional features and advantages of the subject technology are set forth in the description below, and in part will be apparent from the description, or may be learned by practice of the subject technology. The advantages of the subject technology will be realized and attained by the structure particularly pointed out in the written description and claims hereof.

[0091] Mitochondria produce ATP as the fuel to execute all the body’s physiological processes. They also act as sensors of metabolites and the overall state of the cell. Optimal mitochondrial function is essential for peak physical and mental performance. A decline in mitochondrial function is one of the hallmarks of accelerated aging. Age-related changes in mitochondria may take several forms including decreased numbers of mitochondria within a cell, lowered mitochondrial DNA (mtDNA) copy numbers concomitant with a reduction in their oxidative phosphorylation (OxPHOS) subunits production, reduced ATP generation, and impaired mitophagy.PATENT SR3-005WO

[0092] Quality and quantity of mtDNA has been linked to mitochondrial function. Inadequate function can cause mitochondrial diseases. Senescence is also linked to mitochondrial function. Age-related changes in mitochondrial metabolism can lead to, for example, a decreased number of mitochondria, declining rates of ATP synthesis, or reduced oxidative capacity.Mitophagy

[0093] The removal of damaged mitochondria through mitophagy is critical for maintaining proper cellular functions. Impaired mitophagy is linked to diminished mitochondrial fitness, accelerated aging, and a disruption of multiple physiological processes. It is also observed in several age-related neurodegenerative diseases such as Alzheimer’s, and Parkinson’s Disease. Mitophagy can be triggered by cellular stress, cellular aging and / or senscence often due to mtDNA mutations that compromise mitochondrial function.

[0094] Removal of damaged mitochondria through autophagy requires two steps: (a) induction of general autophagy and (b) priming of damaged mitochondria for selective autophagic recognition. Recent progress in mitophagy studies reveals that mitochondrial priming is mediated either by the Pinkl -Parkin signaling pathway or the mitophagic receptors Nix and Bnip3. It can be triggered by cellular stress, often due to mtDNA mutations that compromise mitochondrial function.

[0095] PINK1, a protein kinase, and PARKIN, an E3 ubiquitin ligase, control the specific elimination of dysfunctional or superfluous mitochondria, thus fine-tuning mitochondrial network and preserving energy metabolism. PINK1 regulates PARKIN translocation in impaired mitochondria and drives their removal via mitophagy. FIG. 2A and 2B show a model for the multifunctional role of PINKI / Parkin in mitochondrial quality control.

[0096] Mitophagy occurs in response to alterations in mitochondrial membrane potential. As shown in FIG. 2A, PINK1 localizes on the outer membrane of thePATENT SR3-005WOmitochondria. With dysregulated membrane potential, it remains on the outer membrane. Next, parkin can bind to PINK1 and p62 can ubiquitinate the mitochondria which tags it for mitophagy. FIG. 2B depicts the process with normal membrane potential. As above, PINK1 localizes on the outer membrane. With normal membrane potential, PINK1 is internalized and degraded. In this case, the mitochondria is not tagged for mitophagy.

[0097] Based on this mechanism, Applicants propose that inhibiting abnormal ATP hydrolysis can be exploited to target dysfunctional mitochondria for mitophagy by sensing the change in membrane potential. This approach - optimizing ATP production and reducing reactive oxygen species (ROS) - can be beneficial for stemming both accelerated aging due to stress and environmental exposure and chronic diseases associated with natural aging. This will also be beneficial for heteroplasmic mtDNA mutation diseases acquired with age or inherited at birth.Small molecule mitophagy inducers

[0098] Embodiments include compounds and methods of protecting mtDNA to prevent age-related mitochondrial decline by maintaining the fitness of mitochondria. As described herein, small molecule mitophagy inducers are an innovative approach to treat mitophagy-related disease or disorders as well as to slow the progression of aging and aging-related diseases.

[0099] Applicants have discovered that small molecule analogs of N-[1-aryl-2-(1-imidazolo)ethyl]-guanidine can act as mitophagy-inducing compounds. FIG. 1 shows the chemical structure of isomers of Formula (II). In previous studies, it has been identified as a cardioprotective agent with good pharmacokinetics (PK) and bioavailability in rats. As shown below, data indicate that treating heteroplasmic mtDNA mutation cell lines with Formula (II) reduces mutation and restores lost OxPHOS function concomitantly.

[0100] Without being bound by theory, Applicants propose that Formula (II) induces mitophagy of damaged mitochondria by using the cell’s own mechanisms. In a healthyPATENT SR3-005WOcell, the F1F0-ATP synthase complex controls the mitochondrial membrane potential by pumping hydrogen ions from the intermembrane space to the matrix for the synthesis of ATP molecules. Under pathophysiological conditions, the F1F0-ATP synthase complex switches from generating ATP to hydrolyzing ATP, thus generating the “artificial” membrane potential. Mitophagy, a response to alterations in mitochondrial membrane potential, activates proteins that initiate autophagosome engulfment primarily via the Parkin-PINK1 pathway and other alternative receptor-mediated pathways as depicted in FIG. 2A and FIG. 2B.

[0101] ATPase inhibitory factor 1 (ATPIF1) is phosphorylated into active dimers in response to changing ATP content. When the membrane potential changes, ATPIF1 binds to the F1 subunit of the F1F0-ATP synthase complex and prevents further ATP hydrolysis and, ultimately, mitophagy. Applicants have identified small molecules that can inhibit ATPIF1 to prevent its binding to the F1F0-ATP synthase complex, thereby driving cellular mitophagy to proceed in cells with dysfunctional / membrane potential-impaired mitochondria.

[0102] As described below, Applicants have identified Formula (II) as a compound selective for the soluble F1F0-ATP subcomplex that preserves ATP production in ischemic models and promotes clearance of deletion-bearing mitochondria in model cell lines. Formula (II) has good oral availability (volume of distribution (Vss) at 2.37 L / kg and the Cmax at 21 mM) in rats and has a low IC50 (0.018 mM) to ATPIF19.11.EXAMPLES

[0103] The following non-limiting examples are provided for illustrative purposes only in order to facilitate a more complete understanding of representative embodiments now contemplated. These examples are intended to be a mere subset of all possible contexts in which the components of the formulation may be combined. Thus, these examples should not be construed to limit any of the embodiments described in the present specification, including those pertaining to the type and amounts of components of the formulation and / or methods and uses thereof.PATENT SR3-005WO Example 1Synthesis of Compound Formula (II) Analogs.

[0104] FIG. 7 shows the steps in a method of synthesizing a compound of Formula (II) and analogs. The approach produces both (R) and (S) entantiomers (i.e., a racemic). The enantiomers can be separated from one another. Structural analogs of Formula (II) are then synthesized as shown below.

[0105] Solubility modifications included conversion of parent compounds into appropriate salt formulations and / or the inclusion of solubilizing functional groups.Three areas were interrogated by adding electron withdrawing and donating groups at the R position: (II) meta-phenyl substituents, (2) ortho-phenyl substituents, (3) heterocycle and aliphatic R groups. The purity of analogs was >95% as confirmed by LCMS. Compounds meeting the metrics for solubility (soluble 33 - 100 mg / ml to sparingly soluble drugs 10 - 33 mg / ml) and permeability (>50% penetrance in the efflux assay) were further studied for activity as discussed below.Example 2Screening Test Compounds

[0106] Solubility and permeability of test compounds was determined using standard industry methods as described in detail below.

[0107] Kinetic solubility: Stock solutions of test compounds were made with neat dimethyl sulfoxide (DMSO). Duplicate dilutions (200 pM) were prepared in phosphatePATENT SR3-005WObuffered saline (PBS; 0.1 M, pH 7.4). Test compound calibration standards were prepared at 200 pM and 10 pM concentrations in neat DMSO. The PBS and DMSO dilutions were equilibrated by shaking at room temperature for two hours before filtration (Multiscreen HTS solubility filter plate). Filtrates were analyzed by LC-UV-MS. The concentration of compound in PBS filtrate was determined by comparing the UV absorbance peak with that of the DMSO calibration standards. Mass spectrometry was used to confirm the presence of the expected molecular ion in the UV peak measured.

[0108] Permeability: Wild-type Madin-Darby Canine Kidney (MDCK) and MDR1-MDCK cells were seeded into 96 well Transwell plates and cultured for three days to form monolayers. Test compound (10 pM in Hanks’ Balanced Salt Solution containing 25 mM HEPES buffer) was loaded into the donor compartments of the Transwell plates (pH 7.4 for both donor and receiver compartments). Lucifer Yellow was added to the apical buffer in all wells to assess integrity of the cell monolayer. Duplicate wells were prepared and incubated at 37 °C in a CO2 incubator. Samples were removed at time 0 and 60 minutes and test compound analyzed by LCMS / MS. Concentrations of Lucifer Yellow in the samples were measured using a fluorescence plate reader. The apparent permeability (Papp) values were determined for both the apical to basal (A>B) and basal to apical (B>A) permeation and the efflux ratio (B>A: A>B) determined in both the wild-type MDCK and MDR1-MDCK cells. The effective efflux ratio was also determined from the ratio in MDR1-MDCK cells relative to the ratio observed in wild-type cells. Substrates for human MDR1 typically display effective efflux ratios of greater than two.

[0109] A library of 12 compounds was generated including the enantiomers of Formula (II). Based on preliminary studies, Applicants demonstrated that modifying Formula (II) resulted in the identification of “hits” with improved aqueous solubility and permeability (at physiological pH) for in vitro efficacy testing.Example 3Screening to assess In Vitro Efficacy, Toxicity and Viability

[0110] In this example, the objective was to identify the compounds most capable of1PATENT SR3-005WOinducing mitophagy through the F1F0-ATP synthase complex. Studies from the current embodiment has shown that treatment with Formula (II) results in a modest increase in ATP production from mitochondria in both the common deletion cell line and the point mutation cell line for Leigh’s disease. This is hypothesized to be the result of the reduction in mutated mtDNA and an increase in gene expression of previously missing ETC genes.

[0111] Applicants conducted the qPCR-mtDNA content assay to determine mutant and wt mtDNA composition on the common deletion cell line (BH2). Further studies also used the BH2 cell line to determine ATP production recovery, induction of mitophagy and membrane potential. The proposed method of action for these compounds is to promote mitophagy in damaged cells by using the cells own mechanisms. As described herein, mitophagy is a response to alterations in mitochondrial membrane potential which activates proteins that initiate autophagosome engulfment primarily via the Parkin-PINK1 pathway and other alternative receptor-mediated pathways. Therefore, these experiments evaluated the compounds for their impact on mitochondrial membrane potential and mitophagy initiation. The methods used in the in vitro experiments determined that the compounds are effective in removing mutated mtDNA (qPCR), mitochondrial function recovery (ATP production assay), and re-establishing the membrane potential.

[0112] qPCR-mtDNA content assays were used to determine mutant and wt mtDNA composition using the common deletion cell line BH2. This common deletion cell line is a stable cybrid model for Kearn’s-Sayre Syndrome, caused by the deletion of ~5 kb in human mtDNA (coding regions ND5 to ATP8), with ~60% mutation load. The BH2 cells were dosed at varying concentrations (1 pM to 10 pM) for two months in the presence or absence of RII inhibitors (500 nM). Samples were collected at 0-, 30- and 60-day intervals. The timeline allowed for at least two rounds of mitophagy, with one round of mitophagy occurring approximately every 15 days.

[0113] To measure the mtDNA, whole cell DNA (nuclear DNA + mtDNA) was isolated from cell pellets (~1.0 x 106cells) using a genomic DNA isolation kit. Genomic DNAPATENT SR3-005WO(100 ng) was used in qPCR reactions along with a multiplexing assay master mix and custom-designed primer / probe sets for the following genes (GAPDH; normalization control for nuclear genes and cell number, CYTB; normalization control for mitochondrial DNA genes (in a region away from the mutation impact) ; primer probes distinguishing wt and mutant mtDNA for the common deletion DNA). This allowed for the measurement of normal vs. mutated mtDNA. The return of wt mtDNA was measured by measuring the amount of COX3, a gene deleted in the common deletion region (FIG. 5). The AACT values obtained after normalizing to the control nuclear and mitochondrial genes were used to quantify the mutant versus wild type mtDNA content. Genomic DNA from a wild type cell line (such as the 143B osteosarcoma cell lines) was used as a positive control.

[0114] The ATP production assay determined the regain in OxPHOS function in the BH2 cells through the elimination of mutant mitochondria. BH2 cells treated with the library of compounds (1-10 pM) at 0-, 30- and 60-day time intervals were used in this assay. Subsequent ATP production was assessed using the Seahorse xFe96 platform. Plates were primed and run in the Seahorse xFe96 analyzer using the ATP Production Assay. This assay uses OxPHOS complex-specific inhibitors to determine the oxygen consumption and ATP synthase capability in mitochondria. Cells were plated at a density of 20,000 cells per well. Oligomycin (Complex V inhibitor) rotenone (Complex I inhibitor) and antimycin (Complex III inhibitor) were sequentially added to inhibit the activities of complexes V, I, and III respectively. The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) was used to determine the mitochondrial ATP production versus glycolysis. Previous data has shown that with Formula (II) treatment in BH2 cells, ATP production from mitochondria increases by 50%.

[0115] Applicants also evaluated efficacy in another model of mitochondrial mutation, the Leigh’s Disease model with a mutation in the ATP6 gene, m.8993T>G (80% heteroplasmy for the mutation). Applicants evaluated the ability of the compounds in reducing the mtDNA mutation load and in the recovery of OxPHOS function, particularly the ATP generating capacity.PATENT SR3-005WO

[0116] As noted above, mitochondria can induce mitophagy and degradation through the signaling pathway Parkin-PINK1 , which is initiated by the presence of PINK1 on the outer membrane. With the gene mutations present in the model cell lines, there is either a reduced level of the electron transport chain (ETC) protein components or an impairment in the function of those genes, thus increasing the possibility for those mitochondria to escape this mechanism. Applicants used two methods to evaluate mitochondrial membrane potential. First, tetramethylrhodamine methyl ester (TMRM), an electron gradient sensor, was used that allows calculation of the flux across the mitochondrial membrane with and without the ETC functioning. This allows one to understand the function of the compounds and their effects on the hydrogen flow through the ATP synthase complex.

[0117] Second, mitophagy was assessed with a fluorescence protein-based assay. This involved a stable introduction of the Mito-SRAI construct in our model cell lines. Several of the dye-based assays including TMRM have the drawback of inconsistency and can be implemented only for live cells. Mito-SRAI is a pH-based system to measure mitophagy flux in cells and animal models. This uses a system involving a fusion of two fluorescent proteins to measure the entrance of mitochondria into lysosomes. A blue (TOLLES) fluorescent protein and a yellow (Ypet) fluorescent protein are conjugated and targeted to the mitochondrial matrix resulting in green puncta under normal conditions in the mitochondria. In cells undergoing active mitophagy, the Ypet fluorophore disintegrates and dissolves upon contact with the acidic environment of the lysosomes. However, the blue (TOLLES) fluorophore is resistant to the acidic lysosomal environment. Cells can be fixed or analyzed by flow cytometry and other high throughput assays unlike TMRM studies. The ratio of blue to yellow can be quantified via flow cytometry or images analyzed with Cell P rofi ler to determine the mitophagic flux. Treated and untreated BH2 cells can be assessed for mitophagy and membrane potential perturbations. Membrane potential dissipators such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) can be used as controls to validate the assay in wt 143B osteosarcoma cells. Successful implementation of this assay can serve as a semi-high-throughput system in real-time monitoring of the drug screen.PATENT SR3-005WO

[0118] To clear the mutated mtDNA and mitochondria harboring these mutations, both mitophagy and mitochondrial membrane potential increase. The increase in TMRM signal in cells during and after treatment is indicative of the compound’s function on ATP synthase complex activity.Example 4Formula (II) reduces the mtDNA mutation load in two different mtDNA mutation models.

[0119] The results demonstrate the influence of Formula (II) and analogs on the BH2 cells. FIG. 3A is a bar graph that compares the fold change expression (relative to CYB) of mitochondrial ATP production normalized to the control.

[0120] Data from a 42-day treatment test showed that mutated mtDNA levels decreased (-60%; p<0.0001) at 4 pM Formula (ll)and 500 nM Rock II (RII) inhibitors. With this decrease in mutated mtDNA, an increase in cytochrome c oxidase subunit III (COX3) (~xx125%) was also observed. This is a gene that is otherwise absent in the deleted region of the mutated mtDNA. OxPHOS analysis showed a 45 % increase in the ratio of mitochondrial ATP production in treated cells (Fig. 4 Yellow Bar).

[0121] Similarly, there was a significant decrease in mutant mtDNA in the heteroplasmic ATP6 mutant cell line (80 %; pc****, FIG. 3B) as well as a recovery of 200% of the wildtype mtDNA (p<0.0001_. There was an almost significant increase in the ratio of mitochondrial ATP production (20%; p=0.066) after 42 days of treatment (Fig. 4, Purple Bar) compared to control ATP6 cells. However, this was not the same ratio of ATP production observed in wt-143B cells. It was hypothesized that the ATP6 mutant took longer to show a functional difference because the mitochondria need to (II) replace the ATP6 protein with functioning ATP6 from the wildtype DNA, and (2) take the non-functioning ATP6 out of the complex, which can take up to one month in some tissues. Cells maintained the reduction in mutated mtDNA and increased mitochondrial ATP production for three weeks following dosage. Based on the genomic and functional recovery it was hypothesized that Formula (II) and analogs present a novelPATENT SR3-005WOtherapy for mtDNA mutations.

[0122] The approach described herein for treating mitochondrial dysfunction is innovative because it takes a mutation-agnostic approach and focuses on using the cell’s own mitophagy mechanisms to clear damaged or mutated mitochondria. By modulating membrane potential, mitophagy can be activated independently of any mutation present, creating a mutation agnostic, broad-spectrum treatment option for mitochondrial disease and accelerated aging.Example 5RNA-seq Analysis

[0123] RNA-seq was used to study gene expression. In summary, samples were analyzed as whole cell pellets and total RNA was isolated onsite with polyA selection to isolate tRNA and mRNA transcripts. Samples were sequenced with 20M paired end reads and aligned with hg38 and counts were generated with hit-counts and DESeq2. R statistical analysis heatmap was used to generate FIG. 6A and ENRICHR was used to perform the GO analysis. TPM Values for individual genes were graphed using GraphPad Prism Version 10.2.3, n=3 per group. Two-way-AN OVA with Tukey’s multiple comparisons.

[0124] Sequencing of LRI001+RII treated BH2 cells vs. DMSO treated cells and wildtype (wt) 143B controls show a recovery of critical mtDNA transcripts as well as potential mechanisms of action. FIG. 6A shows a heatmap of TPM values from complete RNA seq data shows a unique transcriptional signature of treated cells (middle) compared to controls. Darker color indicates higher expression. (FIG. 6B -6D) Go analysis of genes upregulated in treated cells compared to DMSO controls, genes had to have + / - two-fold and q<0.05. ENRICHR Combined Score: done by taking the log of the p-value of the Fisher Exact Test, and multiplying it by the z-score of the deviation from the expected rank. (FIG. 6E) Transcript levels of mtDNA protein coding genes. Bold gene names are genes found in the common deletion region. (FIG. 6F) mtDNA encodied tRNAs. Bold gene names are genes found in the deletion. Two-WayPATENT SR3-005WOANOVA with Tukey’s Multiple Comparisons in between cell lines in each gene. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. n=3 per group.Example 6LRI001-012 DNA Results

[0125] In the next experiment, cells carrying known mutant mtDNA were treated with (and without) ROCK II inhibitor.

[0126] Cells with mutant mtDNA were dosed for 28 days with compounds with and without 500 nM Rock II Inhibitor with media being changed every other day to keep the concentration high. The study entailed 4 pM of each tested compound, the concentration selected based on prior LRI001 experiments. FIG. 8 is a table of compounds used in a study (LRI001-012 DNA).

[0127] Media (and thus compound concentration) was refreshed every other day to maintain consistent exposure. Co-treatment variable: ± 500 nM ROCK II inhibitor to determine whether ROCK II inhibition influences compound activity. Data were normalized to DMSO vehicle control. Mutant mtDNA load was quantified relative to total mtDNA.

[0128] FIG. 9A is a bar graph that shows mtDNA content after 28-day treatment with different compounds. FIG. 9B is a bar graph that shows mtDNA content after 28-day treatment with different compounds and Rock II inhibitor. Individual Students two-tailed t-tests were performed comparing compound to DMSO control treated cells.Example 7In Vivo Studies

[0129] FIG. 10A is a dot bar graph showing in vivo data of compound presence in blood over time. Wildtype female C57 / BL6 mice of five months of age were dosed with 56mg / kg using corn oil and DMSO as vehicles for intraperitoneal (IP) injection. Plasma was collected and analyzed using LC-MS / MS to detect compound in ng / pl up to 24PATENT SR3-005WOhours post injection. Data shows it is detectable 15 minutes after administration and highest at two hours after injection. LRI002 remains detectable at six hours post injection (one-way ANOVA, n=3 with a Tukey’s Post Hoc Test).

[0130] At 24 hours organs were harvested to detect compound penetration beyond the vascular system. FIG. 11A - 11 D are plots of plasma concentration (ng / ml) at 24 hours. LRI002 is detectable in the liver, spleen, kidney and brain 24 hours after IP injection, n=3 per sample. This demonstrates bioavailability and organ presence for future studies.Methods of Use

[0131] Embodiments include methods of treating pathogenesis or defects in the mitochondrial respiratory chain or the oxidative phosphorylation system. The methods can include administration of a therapeutic amount of a compound described herein.

[0132] The compounds of Formula (II) which contain a basic moiety such as, for example, an amine or a pyridine or imidazole ring, may form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid), hydrobromides (formed with hydrogen bromide), hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates (formed with maleic acid), methanesulfonates (formed with methanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (such as those formed with sulfuric acid), sulfonates (such as those mentioned herein), tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates, and the like.PATENT SR3-005WO

[0133] The compounds of Formula (II) which contain an acidic moiety such as, for example, a carboxylic acid, may form salts with a variety of organic and inorganic bases. Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines [formed with N,N-bis(dehydro-abietyl)ethylenediamine], N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogencontaining groups may be quatemized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others.

[0134] Compounds of the Formula (II), and salts thereof, may exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the present invention.

[0135] All stereoisomers of the present compounds, such as those, for example, which may exist due to asymmetric carbons, including enantiomeric forms (which may exist even in the absence of asymmetric carbons) and diastereomeric forms, are contemplated and within the scope of this invention. Individual stereoisomers of the compounds of this invention may, for example, be substantially free of other isomers, or may be admixed, for example, as racemates or with all other or other selected, stereoisomers. The chiral centers of the present invention can have the S or R configuration as defined by the IUPAC 1974 Recommendations.

[0136] In addition, compounds of Formula (II) may have prodrug forms. Any compound that will be converted in vivo to provide the bioactive agent (i.e. , a compound of formula I) is a prodrug within the scope and spirit of the invention.

[0137] It is understood that the present invention encompasses the use, wherePATENT SR3-005WOapplicable, of stereoisomers, diastereomers and optical stereoisomers of the compounds of the invention, as well as mixtures thereof. Additionally, it is understood that stereoisomers, diastereomers, and optical stereoisomers of the compounds of the invention, and mixtures thereof, are within the scope of the invention. By way of example, the mixture may be a racemate or the mixture may comprise unequal proportions of one particular stereoisomer over the other. Additionally, the compounds can be provided as substantially pure stereoisomers, diastereomers and optical stereoisomers (such as epimers).

[0138] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended to be included within the scope of the invention unless otherwise indicated. Compounds that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods of preparation of optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds are also included within the scope of the invention and can be isolated as a mixture of isomers or as separated isomeric forms. Where a compound is capable of stereoisomerism, all such isomers are contemplated.

[0139] Compounds may also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Examples of prototropic tautomers include, but are not limited to, ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamineimine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system including, but not limited to, 1 H- and 3H-imidazole, 1 H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.PATENT SR3-005WO

[0140] Compounds also include hydrates and solvates, as well as anhydrous and non-solvated forms. Compounds can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[0141] In some embodiments, the compounds, or salts thereof, are substantially isolated. Partial separation can include, for example, a composition enriched in the compound of the invention. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the invention, or salt thereof. Methods for isolating compounds and their salts are routine in the art.

[0142] One or more of the compounds described herein can be administered by any method known in the art, including, for example, intranasal, oral, transdermal, ocular, intraperitoneal, inhalation, intravenous, ICV, intracistemal injection or infusion, subcutaneous, implant, vaginal, sublingual, urethral (e.g., urethral suppository), subcutaneous, intramuscular, intravenous, rectal, sub-lingual, mucosal, ophthalmic, spinal, intrathecal, intra-articular, intra-arterial, sub-arachinoid, bronchial and lymphatic administration. A topical formulation can be in the form of gel, ointment, cream, aerosol, etc. An intranasal formulation can be delivered as a spray or in a drop. A prodrug formulationcan be administered via a transdermal patch or iontophoresis. A formulation for inhalation can be delivered using a nebulizer or similar device. Compositions can also take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols or any other appropriate compositions.

[0143] In another aspect, certain embodiments are directed to a sustained release drug delivery platform that releases a therapeutic compound or compounds disclosed and made as a formulation described herein over a period of, without limitation, about 3 days after administration, about 7 days after administration, about 10 days afterPATENT SR3-005WOadministration, about 15 days after administration, about 20 days after administration, about 25 days after administration, about 30 days after administration, about 45 days after administration, about 60 days after administration, about 75 days after administration, or about 90 days after administration. In other aspects of this embodiment, a sustained release drug delivery platform releases a therapeutic compound or compounds disclosed herein with substantially first order release kinetics over a period of, without limitation, at least 3 days after administration, at least 7 days after administration, at least 10 days after administration, at least 15 days after administration, at least 20 days after administration, at least 25 days after administration, at least 30 days after administration, at least 45 days after administration, at least 60 days after administration, at least 75 days after administration, or at least 90 days after administration.

[0144] A pharmaceutical composition comprising an agent in accordance with the present disclosure can be formulated in any pharmaceutically acceptable carrier(s) or excipient(s). As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutical compositions can include suitable solid or gel phase carriers or excipients. Exemplary carriers or excipients include calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Exemplary pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the therapeutic agents.

[0145] Dosing can be single dosage or cumulative (serial dosing), and can bePATENT SR3-005WOreadily determined by one skilled in the art. For example, treatment of a mitochondrial deficiency an include a one-time administration of an effective dose of a pharmaceutical composition disclosed herein. Alternatively, treatment can include multiple administrations of an effective dose of a pharmaceutical composition carried out over a range of time periods (e.g., once daily, twice daily, trice daily, once every few days, or once weekly). The timing of administration can vary from individual to individual, depending upon such factors as the severity of an individual's symptoms. For example, an effective dose of a pharmaceutical composition disclosed herein can be administered to an individual once daily for an indefinite period of time, or until the individual no longer requires therapy. A person of ordinary skill in the art will recognize that the condition of the individual can be monitored throughout the course of treatment and adjusted accordingly.

[0146] Combination treatments are also contemplated. For example, a subject can be treated with a compound of Formula II in addition to one or more additional compounds, biologies and cell therapies to provide improved mitochondrial function and clearance of dysfunctional mitochondria through mitophagy.

[0147] In one embodiment, a compound disclosed herein induces mitophagy be, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% rate higher than an untreated subject. In other aspects of this embodiment, a method disclosed herein leads to increased levels of mitophagy of, e.g., about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%,PATENT SR3-005WOabout 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[0148] In one embodiment, a therapeutic disclosed herein is capable of reducing the signs / symptoms of a mitochondrial disease, age-related disease or mitophagy-associated disease or disorder by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a patient not receiving the same treatment. In other aspects of this embodiment, a therapeutic is capable of reducing the number of signs / symptoms of a mitochondrial disease, age-related disease or mitophagy-associated disease or disorder in an individual by, e.g., about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% as compared to a patient not receiving the same treatment.

[0149] In one embodiment, a therapeutic disclosed herein is capable of reducing signs / symptoms in an individual suffering from a mitochondrial disease, age-related disease or mitophagy-associated disease or disorder by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a patient not receiving the same treatment. In other aspects of this embodiment, a therapeutic is capable of reducing signs / symptoms in an individual suffering from a mitochondrial disease, age-related disease or mitophagy-associated disease or disorder by, e.g., about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, aboutPATENT SR3-005WO40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% as compared to a patient not receiving the same treatment.

[0150] In one embodiment, a therapeutic disclosed herein is capable of reducing signs / symptoms of aging (or an age-related condition) in an individual by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a patient not receiving the same treatment. In other aspects, a therapeutic is capable of reducing signs / symptoms of aging in a subject by, e.g., about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% as compared to a patient not receiving the same treatment.

[0151] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend forPATENT SR3-005WOthe present invention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0152] Groupings of alternative embodiments, elements, or steps of the present invention are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0153] Unless otherwise indicated, all numbers expressing a characteristic, item, quantity, parameter, property, term, and so forth used in the present specification and claims are to be understood as being modified in all instances by the term “about.” As used herein, the term “about” means that the characteristic, item, quantity, parameter, property, or term so qualified encompasses a range of plus or minus ten percent above and below the value of the stated characteristic, item, quantity, parameter, property, or term. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of numericalPATENT SR3-005WOranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the present specification as if it were individually recited herein.

[0154] The terms “a,” “an,” “the” and similar referents used in the context of describing the present invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the present invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the present specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0155] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s).Embodiments of the present invention so claimed are inherently or expressly described and enabled herein.

[0156] Groupings of alternative embodiments, elements, or steps of the present invention are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in thePATENT SR3-005WOappended claims.

[0157] All patents, patent publications, and other publications referenced and identified in the present specification are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely fortheir disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0158] In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular methodology, protocol, and / or reagent, etc., described herein. As such, various modifications or changes to or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of the present specification. Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

PATENT SR3-005WOCLAIMSWhat is claimed is:

1. A compound of Formula (II):or a pharmaceutically acceptable salt thereof, wherein R is selected from:

2. A method of treating an ailment, the method comprised of administering a therapeutic amount of the compound of claim 1 to a subject.

3. The method of claim 2, wherein the ailment is an age-related disease or disorder.

4. The method of claim 3, wherein the age-related disease or disorder is selected from Alzheimer’s disease, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis, hair graying, sarcopenia, adiposity, neurogenesis, fibrosis and glaucoma.PATENT SR3-005WO5. The method of claim 2, wherein the ailment is a mitophagy-related disease or disorder.

6. The method of claim 5, wherein the mitophagy-related disease or disorder is a neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's, ALS), a cardiovascular disease, a cancer, kidney disease, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), Kearns-sayre syndrome (KSS), Leigh syndrome or leber hereditary optic neuropathy (LHON).

7. The method of claim 2, wherein the ailment is premature aging or a senescence-associated disease or disorder.

8. A method of slowing the aging process or reducing signs of aging, the method comprising administering a therapeutic amount of the compound of claim 1.

9. A method of inducing or promoting mitophagy in a cell, the method comprising administering a therapeutic amount of the compound of claim 1.

10. The method of claim 9, wherein the cell is a mammalian cell.

11. The method of claim 9, wherein the cell is a human cell.

12. A method of treating an ailment, the method comprised of administering a therapeutic amount of the compound of Formula (II) or an enantiomer thereof:PATENT SR3-005WO13. The method of claim 12, wherein the ailment is an age-related disease or disorder.

14. The method of claim 13, wherein the age-related disease or disorder is selected from Alzheimer’s disease, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis, hair graying, sarcopenia, adiposity, neurogenesis, fibrosis and glaucoma.

15. The method of claim 12, wherein the ailment is a mitophagy-related disease or disorder.

16. The method of claim 15, wherein the mitophagy-related disease or disorder is a neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's, ALS), a cardiovascular disease, a cancer, kidney disease, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), Kearns-sayre syndrome (KSS), Leigh syndrome or leber hereditary optic neuropathy (LHON).

17. The method of claim 12, wherein the ailment is premature aging or a senescence-associated disease or disorder.

18. A method of slowing the aging process or reducing signs of aging, the method comprising administering a therapeutic amount of the compound of claim 12.PATENT SR3-005WO19. A method of inducing or promoting mitophagy in a cell, the method comprising administering a therapeutic amount of the compound of claim 12.

20. The method of claim 19, wherein the cell is a mammalian cell.

21. The method of claim 19, wherein the cell is a human cell.

22. A method of treating an ailment, the method comprised of administering a therapeutic amount of an analog of Formula II to a subject, wherein the analog is selected from Formula A, Formula B, Formula C, Formula D, Formula E, Formula F, Formula G, Formula H, Formula I, Formula J, Formula K, Formula L and Formula M:PATENT SR3-005WOPATENT SR3-005WOor a pharmaceutically acceptable salt.

23. The method of claim 22, wherein the ailment is an age-related disease or disorder.

24. The method of claim 23, wherein the age-related disease or disorder is selected from Alzheimer’s disease, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis, hair graying, sarcopenia, adiposity, neurogenesis, fibrosis and glaucoma.

25. The method of claim 22, wherein the ailment is a mitophagy-related disease or disorder.

26. The method of claim 22, wherein the ailment is a mitochondrial disorder associated with pathogenic mitochondrial DNA (mtDNA) mutation.

27. The method of claim 25, wherein the mitophagy-related disease or disorder is a neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's, ALS), a cardiovascular disease, a cancer, kidney disease, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), Kearns-sayre syndrome (KSS), Leigh syndrome or leber hereditary optic neuropathy (LHON).

28. The method of claim 22, wherein the ailment is premature aging or a senescence-associated disease or disorder.PATENT SR3-005WO29. The method of claim 22, wherein the analog of Formula II inhibits ATPase inhibitory factor 1 (ATPIF1).

30. The method of claim 22, wherein the analog of Formula II increases selective mitophagy of dysfunctional mitochondria.

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