COMPOSITIONS AND METHODS OF USING THE SAME FOR TREATMENT OF NEURODEGENERATIVE AND MITOCHONDRIAL DISEASE

The present disclosure is directed to N—(3-substituted-chroman-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine compounds, methods of making N—(3-substituted-chroman-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine compounds, and methods of treating disorders associated with PINK kinase activity including, but not limited to, neurodegenerative diseases, mitochondrial diseases, fibrosis, and/or cardiomyopathy using these compounds. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

BACKGROUND

Maintenance of mitochondrial function is essential for the health and survival of numerous cell types, including cardiomyoctes, hepatocytes, renal cells and neurons. Aberrant mitochondrial quality control has been demonstrated to be an important factor in the development of neurodegenerative diseases, kidney disease, and cardiomyopathy (Schapira, A. H. Mitochondrial disease. Lancet 379, 1825-1834, (2012) and Chen, Y. and Dom, G. PINK1-Phosphorylated Mitofusin-2 Is a Parkin Receptor for Culling Damaged Mitochondria. Science 340, 471-475, (2013)). The mitochondrial kinase PTEN Induced Kinase 1 (PINK1) plays an important role in the mitochondrial quality control processes by responding to damage at the level of individual mitochondria. The PINK1 pathway has also been linked to the induction of mitochondrial biogenesis and, critically, to the reduction of mitochondrially-induced apoptosis. See e.g., Narendra, D. P. et al. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol 8, e1000298 (2010), Wang, X., (2011). et al. PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility. Cell 147, 893-906, (2011), and Shin, J. H. et al. PARIS (ZNF746) repression of PGC-1alpha contributes to neurodegeneration in Parkinson's disease. Cell 144, 689-702, (2011).

Parkinson's Disease (PD) is one of the most common neurodegenerative disorders; however, no disease modifying therapies are currently approved to treat PD. Both environmental and genetic factors lead to progressive apoptosis of dopaminergic neurons, lowered dopamine levels, and, ultimately, PD. PINK1 kinase activity appears essential to mediate its neuroprotective activity. The regulation of mitochondrial movement, distribution, and clearance is a key part of neuronal oxidative stress response. Disruptions to these regulatory pathways have been shown to contribute to chronic neurodegenerative disease. See Schapira and Chen cited above.

Cardiomyopathy refers to a disease of cardiac muscle tissue, and it is estimated that cardiomyopathy accounts for 5-10% of the 5-6 million patients already diagnosed with heart failure in the United States. Based on etiology and pathophysiology, the World Health Organization created a classification of cardiomyopathy types which includes dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and unclassified cardiomyopathy. See e.g., Richardson P, et al. Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of cardiomyopathies. Circulation 1996; 93:841. PINK1 kinase activity appears to mediate its' cardio-protective activity. The regulation of mitochondrial movement, distribution, and clearance is a part of cardiac cell oxidative stress response. Disruptions to these regulatory pathways have been shown to contribute to cardiomyopathy. See Schapira and Chen cited above. Wang, X., (2011) et al. PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility Cell 147, 893-906, (2011) and Richardson P, et al. Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of cardiomyopathies. Circulation 1996; 93:841. Koh, H. & Chung, J.

Leigh syndrome is a severe neurological disorder caused by mutation of mitochondrial genes. Behavioral symptoms of LS patients can include (with a wide variety of clinical presentation) developmental retardation, hypotonia, ataxia, spasticity, dystonia, weakness, optic atrophy, defects in eye or eyelid movement, hearing impairment, breathing abnormalities, dysarthria, swallowing difficulties, failure to thrive, and gastrointestinal problems. Several cases of adult-onset LS have also been reported recently. See e.g., Longo, D, et al. Harrison's Internal Medicine. 18thed. (online), Ch. 238 (2011), Wang and Richardson cited above, and Samaranch, L. et al. PINK1-linked parkinsonism is associated with Lewy body pathology. Brain 133, 1128-1142, (2010) and Merrick, K. A. et al. Switching Cdk2 on or off with small molecules to reveal requirements in human cell proliferation. Mol Cell 42, 624-636, (2011). The cause of death in most LS cases is unclear, and the lack of a genetic model to study the disease progression and cause of death has impeded the development of adequate treatment. Prognosis for LS (and most diseases resulting from mitochondrial dysfunction) is very poor; there is no cure and treatment is often ineffective. In vivo imaging techniques such as MRI reveal bilateral hyperintense lesions in the basal ganglia, thalamus, substantia nigra, brainstem, cerebellar white matter and cortex, cerebral white matter, or spinal cord of LS patients. See e.g., Longo cited above and Shin, J. H. et al. PARIS (ZNF746) repression of PGC-1alpha contributes to neurodegeneration in Parkinson's disease.

Parkinson's Disease (PD) is one of the most common neurodegenerative disorder; however, no disease modifying therapies are currently approved to treat PD. Both environmental and genetic factors lead to progressive apoptosis of dopaminergic neurons, lowered dopamine levels, and, ultimately, PD. PINK1 kinase activity appears to mediate its' neuroprotective activity. The regulation of mitochondrial movement, distribution, and clearance is a key part of neuronal oxidative stress response. Disruptions to these regulatory pathways have been shown to contribute to chronic neurodegenerative disease. See Schapira and Chen cited above.

Despite the widespread prevalence of disorders associated with PINK1 pathway, compounds capable of selectively targeting this pathway and, thus, treating disorders associated with this pathway have remained elusive.

SUMMARY

In accordance with the purpose(s) of the disclosure, as embodied and broadly described herein, the disclosure, in some embodiments, relates to adenine compounds useful in the treatment of disorders associated with PINK1 kinase activity such as, for example, a neurodegenerative disease, a mitochondrial disease, fibrosis, and/or cardiomyopathy.

Thus, provided herein are compounds having a structure represented by a formula:

Also provided herein are compounds having a structure represented by a formula:

Also provided are compounds selected from:

or a pharmaceutically acceptable salt thereof.

Without wishing to be bound by theory, an advantage of the presently described compounds is that they possess improved potency and reduced toxicity. For example, the disclosed compounds can exhibit an EC50of less than about 0.03 NM with a highest dolerated dose of more than about 1,000 times that amount. See, e.g., Table 2 compound no. EP-0042538.

Also provided are methods for making a disclosed compound.

Also provided are pharmaceutical compositions comprising a therapeutically effective amount of a disclosed compound and a pharmaceutically acceptable carrier.

Also provided are methods of modulating PINK1 kinase activity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of at least one disclosed compound.

Also disclosed are methods of modulating PINK1 kinase activity in at least one cell, the method comprising contacting the cell with an effective amount of at least one disclosed compound.

Also provided are methods for treating a disorder in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of at least one disclosed compound, wherein the disorder is a neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, or a reperfusion injury.

Also provided are kits comprising a disclosed compound and one or more selected from: (a) at least one agent known for the treatment of one or more disorders selected from neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury; (b) instructions for administering the compound in connection with treating one or more disorders selected from a neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury; and (c) instructions for treating one or more disorders selected from a neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury.

Still other objects and advantages of the present disclosure will become readily apparent by those skilled in the art from the following detailed description, wherein it is shown and described only the preferred embodiments, simply by way of illustration of the best mode. As will be realized, the disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, without departing from the disclosure. Accordingly, the description is to be regarded as illustrative in nature and not as restrictive.

Additional advantages of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the disclosure. The advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure, as claimed.

DETAILED DESCRIPTION

The present invention can be understood more readily by reference to the following detailed description of the disclosure and the Examples included therein.

While embodiments of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each embodiment of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or embodiment set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of embodiments described in the specification.

Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.

As used herein, the terms “a” or “an” means that “at least one” or “one or more” unless the context clearly indicates otherwise. The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in various embodiments, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

The term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, “either,” “one of,” “only one of,” or “exactly one of.”

As used herein, the terms “comprising” (and any form of comprising, such as “comprise,” “comprises,” and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

As used herein, the term “about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical limitation is used, unless indicated otherwise by the context, “about” means the numerical value can vary by ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% and remain within the scope of the disclosed embodiments.

As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. In some embodiments of the disclosed methods, the subject has been diagnosed with a need for treatment of a disorder associated with PINK1 kinase activity such as, for example, a neurodegenerative disease, a mitochondrial disease, fibrosis, and/or cardiomyopathy, prior to the administering step. As used herein, the phrase “identified to be in need of treatment for a disorder,” or the like, refers to selection of a subject based upon need for treatment of the disorder. It is contemplated that the identification can, in some embodiments, be performed by a person different from the person making the diagnosis. It is also contemplated, in further embodiments, that the administration can be performed by one who subsequently performed the administration.

The term “contacting” as used herein refers to bringing a disclosed compound and a cell, target receptor, or other biological entity together in such a manner that the compound can affect the activity of the target (e.g., receptor, cell, etc.), either directly; i.e., by interacting with the target itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent.

As used herein, “IC50” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% inhibition of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. In some embodiments, an IC50can refer to the concentration of a substance that is required for 50% inhibition in vivo, as further defined elsewhere herein.

As used herein, “EC50” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is results in a half-maximal response (i.e., 50% of the maximum response) of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. In some embodiments, an EC50can refer to the concentration of a substance that is required to achieve 50% of the maximum response in vivo, as further defined elsewhere herein.

The compounds according to this disclosure may form prodrugs at hydroxyl or amino functionalities using alkoxy, amino acids, etc., groups as the prodrug forming moieties. For instance, the hydroxymethyl position may form mono-, di- or triphosphates and again these phosphates can form prodrugs. Preparations of such prodrug derivatives are discussed in various literature sources (examples are: Alexander et al., J. Med. Chem. 1988, 31, 318; Aligas-Martin et al., PCT WO 2000/041531, p. 30). The nitrogen function converted in preparing these derivatives is one (or more) of the nitrogen atoms of a compound of the disclosure.

“Derivatives” of the compounds disclosed herein are pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, solvates and combinations thereof. The “combinations” mentioned in this context are refer to derivatives falling within at least two of the groups: pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, and solvates. Examples of radio-actively labeled forms include compounds labeled with tritium, phosphorous-32, iodine-129, carbon-11, fluorine-18, and the like.

The term “leaving group” refers to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons. Examples of suitable leaving groups include sulfonate esters, including triflate, mesylate, tosylate, brosylate, and halides.

The terms “halo” and “halogen” as used herein refer to an atom selected from fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), and iodine (iodo, —I).

The term “alkyl,” as used herein, refers to a monovalent saturated, straight- or branched-chain hydrocarbon radical, having unless otherwise specified, 1-6 carbon atoms. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, n-pentyl, tert-pentyl, neopentyl, sec-pentyl, 3-pentyl, sec-isopentyl, hexyl, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, 2,3-dimentybutane and the like. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms. The term alkyl group can also be a C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, and the like up to and including a C1-C24 alkyl.

Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. Alternatively, the term “monohaloalkyl” specifically refers to an alkyl group that is substituted with a single halide, e.g. fluorine, chlorine, bromine, or iodine. The term “polyhaloalkyl” specifically refers to an alkyl group that is independently substituted with two or more halides, i.e. each halide substituent need not be the same halide as another halide substituent, nor do the multiple instances of a halide substituent need to be on the same carbon. The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “aminoalkyl” specifically refers to an alkyl group that is substituted with one or more amino groups. The term “hydroxyalkyl” specifically refers to an alkyl group that is substituted with one or more hydroxy groups. When “alkyl” is used in one instance and a specific term such as “hydroxyalkyl” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “hydroxyalkyl” and the like.

The term “heteroalkyl,” as used herein, refers to an alkyl group containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined above for alkyl groups.

The term “haloalkyl” includes mono, poly, and perhaloalkyl groups where the halogens are independently selected from fluorine, chlorine, bromine, and iodine.

“Alkoxy” is an alkyl group which is attached to another moiety via an oxygen linker (—O(alkyl)). Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy.

“Haloalkoxy” is a haloalkyl group which is attached to another moiety via an oxygen atom such as, e.g., but are not limited to —OCHCF2or —OCF3.

The term “9- to 10-membered carbocyclyl” means a 9- or 10-membered monocyclic, bicyclic (e.g., a bridged or spiro bicyclic ring), polycyclic (e.g., tricyclic), or fused hydrocarbon ring system that is saturated or partially unsaturated. The term “9- to 10-membered carbocyclyl” also includes saturated or partially unsaturated hydrocarbon rings that are fused to one or more aromatic or practically saturated hydrocarbon rings (e.g., dihydroindenyl and tetrahydronaphthalenyl). Bridged bicyclic cycloalkyl groups include, without limitation, bicyclo[4.3.1]decanyl and the like. Spiro bicyclic cycloalkyl groups include, e.g., spiro[3.6]decanyl, spiro[4.5]decanyl, spiro [4.4]nonyl and the like. Fused cycloalkyl rings include, e.g., decahydronaphthalenyl, dihydroindenyl, decahydroazulenyl, octahydroazulenyl, tetrahydronaphthalenyl, and the like. It will be understood that when specified, optional substituents on a carbocyclyl (e.g., in the case of an optionally substituted cycloalkyl) may be present on any substitutable position and, include, e.g., the position at which the carbocyclyl group is attached.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. In various aspects, the cycloalkyl group and heterocycloalkyl group can be monocyclic, bicyclic (e.g., bridged such as, for example, bicyclo[4.3.1]decanyl or spiro such as, for example, spiro[3.6]decanyl, spiro[4.5]decanyl, spiro [4.4]nonyl), polycyclic (e.g., tricyclic), or a fused hydrocarbon ring system that is saturated or partially unsaturated (e.g., decahydronaphthalenyl, dihydroindenyl, decahydroazulenyl, octahydroazulenyl, tetrahydronaphthalenyl).

The term “heterocycle” or “heterocyclyl” as used herein can be used interchangeably and refer to single and multi-cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Thus, the term is inclusive of, but not limited to, “heterocycloalkyl,” “heteroaryl,” “bicyclic heterocycle,” and “polycyclic heterocycle.” The heterocycle can be monocyclic, bicyclic (e.g., spiro or bridged), polycyclic, or a fused system that is saturated or partially saturated. Heterocycle includes pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole, thiadiazole, including, 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, triazole, including, 1,2,3-triazole, 1,3,4-triazole, tetrazole, including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridazine, pyrazine, triazine, including 1,2,4-triazine and 1,3,5-triazine, tetrazine, including 1,2,4,5-tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, dioxane, and the like. The term heterocyclyl group can also be a C2 heterocyclyl, C2-C3 heterocyclyl, C2-C4 heterocyclyl, C2-C5 heterocyclyl, C2-C6 heterocyclyl, C2-C7 heterocyclyl, C2-C8 heterocyclyl, C2-C9 heterocyclyl, C2-C10 heterocyclyl, C2-C11 heterocyclyl, and the like up to and including a C2-C18 heterocyclyl. For example, a C2 heterocyclyl comprises a group which has two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, dihydrodiazetyl, oxiranyl, thiiranyl, and the like. Alternatively, for example, a C5 heterocyclyl comprises a group which has five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, pyridinyl, and the like. It is understood that a heterocyclyl group may be bound either through a heteroatom in the ring, where chemically possible, or one of carbons comprising the heterocyclyl ring.

The term “bicyclic heterocycle” or “bicyclic heterocyclyl” as used herein refers to a ring system in which at least one of the ring members is other than carbon. Bicyclic heterocyclyl encompasses ring systems wherein an aromatic ring is fused with another aromatic ring, or wherein an aromatic ring is fused with a non-aromatic ring. Bicyclic heterocyclyl encompasses ring systems wherein a benzene ring is fused to a 5- or a 6-membered ring containing 1, 2, or 3 ring heteroatoms or wherein a pyridine ring is fused to a 5- or a 6-membered ring containing 1, 2, or 3 ring heteroatoms. Bicyclic heterocyclic groups include, but are not limited to, indolyl, indazolyl, pyrazolo[1,5-a]pyridinyl, benzofuranyl, quinolinyl, quinoxalinyl, 1,3-benzodioxolyl, 2,3-dihydro-1,4-benzodioxinyl, 3,4-dihydro-2H-chromenyl, 1H-pyrazolo[4,3-c]pyridin-3-yl; 1H-pyrrolo[3,2-b]pyridin-3-yl; and 1H-pyrazolo[3,2-b]pyridin-3-yl.

The term “heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturated or fully saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi- and tricyclic ring systems. The heterocycloalkyl ring-systems include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized and a nitrogen heteroatom optionally can be substituted. Representative heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

The term “9-membered fused heterocyclyl” means a 9-membered saturated or partially unsaturated fused monocyclic heterocyclic ring comprising at least one oxygen heteroatom and optionally two to four additional heteroatoms independently selected from N, O, and S. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical,” are used interchangeably herein. A heterocyclyl ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Examples of fused saturated or partially unsaturated heterocyclic radicals compristing at least one oxygen atom include, without limitation, dihydrobenzofuranyl, dihydrofuropyridinyl, octahydrobenzofuranyl, and the like. Where specified as being optionally substituted, substituents on a heterocyclyl (e.g., in the case of an optionally substituted heterocyclyl) may be present on any substitutable position and include, e.g., the position at which the heterocyclyl group is attached.

The term “aromatic group” as used herein refers to a ring structure having cyclic clouds of delocalized π electrons above and below the plane of the molecule, where the π clouds contain (4n+2) π electrons. A further discussion of aromaticity is found in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter 13, entitled “Aromaticity,” pages 477-497, incorporated herein by reference. The term “aromatic group” is inclusive of both aryl and heteroaryl groups.

The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, —NH2, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” In addition, the aryl group can be a single ring structure or comprise multiple ring structures that are either fused ring structures or attached via one or more bridging groups such as a carbon-carbon bond. For example, biaryl can be two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

The terms “amine” or “amino” as used herein are represented by the formula —NA1A2, where A1and A2can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. A specific example of amino is —NH2.

The term “ester” as used herein is represented by the formula —OC(O)A1or —C(O)OA1, where A1can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “polyester” as used herein is represented by the formula -(A1O(O)C-A2-C(O)O)a— or -(A1O(O)C-A2-OC(O))a—, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer from 1 to 500. “Polyester” is as the term used to describe a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two hydroxyl groups.

In some embodiments, a structure of a compound can be represented by a formula:

which is understood to be equivalent to a formula:

wherein n is typically an integer. That is, R″ is understood to represent five independent substituents, Rn(a), Rn(b), Rn(c), Rn(d), Rn(e). In each such case, each of the five R″ can be hydrogen or a recited substituent. By “independent substituents,” it is meant that each R substituent can be independently defined. For example, if in one instance Rn(a) is halogen, then Rn(b)is not necessarily halogen in that instance.

In some yet further embodiments, a structure of a compound can be represented by a formula:

wherein Ryrepresents, for example, 0-2 independent substituents selected from A1, A2, and A3, which is understood to be equivalent to the groups of formulae:wherein Ryrepresents 0 independent substituents

Again, by “independent substituents,” it is meant that each R substituent can be independently defined. For example, if in one instance Ry1is A1, then Ry2is not necessarily A1in that instance.

In some further embodiments, a structure of a compound can be represented by a formula,

wherein, for example, Q comprises three substituents independently selected from hydrogen and A, which is understood to be equivalent to a formula:

Again, by “independent substituents,” it is meant that each Q substituent is independently defined as hydrogen or A, which is understood to be equivalent to the groups of formulae:

wherein Q comprises three substituents independently selected from H and A

wherein Q comprises three substituents independently selected from H and A

In some embodiment, the disclosed compounds exists as geometric isomers. “Geometric isomer” refers to isomers that differ in the orientation of substituent atoms in relationship to a cycloalkyl ring, i.e., cis or trans isomers. When a disclosed compound is named or depicted by structure without indicating a particular cis or trans geometric isomer form, it is to be understood that the name or structure encompasses one geometric isomer free of other geometric isomers, mixtures of geometric isomers, or mixtures enriched in one geometric isomer relative to its corresponding geometric isomer. When a particular geometric isomer is depicted, i.e., cis or trans, the depicted isomer is at least about 60%, 70%, 80%, 90%, 99%, or 99.9% by weight pure relative to the other geometric isomer.

The compounds described herein may be present in the form of pharmaceutically acceptable salts. For use in medicines, the salts of the compounds described herein refer to non-toxic “pharmaceutically acceptable salts.” Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic/anionic or basic/cationic salts. Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include e.g., salts of inorganic acids (such as hydrochloric acid, hydrobromic, phosphoric, nitric, and sulfuric acids) and of organic acids (such as, acetic acid, benzenesulfonic, benzoic, methanesulfonic, and p-toluenesulfonic acids). Examples of pharmaceutically acceptable base addition salts include e.g., sodium, potassium, calcium, ammonium, organic amino, or magnesium salt.

As used herein, the phrase “pharmaceutically acceptable” means those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with tissues of humans and animals. In some embodiments, “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

Disease, disorder, and condition are used interchangeably herein.

As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed, i.e., therapeutic treatment. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of exposure to a particular organism, or other susceptibility factors), i.e., prophylactic treatment. Treatment may also be continued after symptoms have resolved, for example to delay their recurrence.

As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. The term “preventing” refers to preventing a disease, disorder, or condition from occurring in a human or an animal that may be predisposed to the disease, disorder and/or condition, but has not yet been diagnosed as having it; and/or inhibiting the disease, disorder, or condition, i.e., arresting its development.

As used herein, the term “salt” refers to acid or base salts of the compounds used in the methods of the present disclosure. Illustrative examples of acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.

The terms “subject” and “patient” may be used interchangeably, and means a mammal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). Typically, the subject is a human in need of treatment.

The term “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease (e.g., a protein associated disease, a symptom associated with a cardiomyopathy, neurodegenerative disease, or symptom associated with Parkinson's disease) means that the disease (e.g., cardiomyopathy, neurodegenerative disease or Parkinson's disease) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function. For example, a symptom of a disease or condition associated with a reduction in the level of PINK1 activity may be a symptom that results (entirely or partially) from a reduction in the level of PINK1 activity (e.g., loss of function mutation or gene deletion or modulation of PINK1 signal transduction pathway). As used herein, what is described as being associated with a disease, if a causative agent, could be a target for treatment of the disease. For example, a disease associated with PINK1, may be treated with an agent (e.g., compound as described herein) effective for increasing the level of activity of PINK1. In some embodiments, the compositions and compounds disclosed herein are useful to treat cancers associated with Pink1 kinase activity. “Cancer associated with PINK1 kinase activity” are those cancers derived from a cell or plurality of cells that comprise a mutation or mutations that confer impaired or dysfunctional PINK1 kinase activity, such dysfunctional PINK1 kinase activity resulting in an impaired or dysregulated growth cycle of the cell or cells. In some embodiments, cancer associated with PINK1 kinase activity is treated by one or a plurality of the pharmaceutical compositions comprising a therapeutic effective amount of an active the compounds or derivatives, salts or analogs disclosed herein.

“Control” or “control experiment” is used in accordance with its plain ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In some instances, the control is used as a standard of comparison in evaluating experimental effects.

“Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g., chemical compounds including biomolecules, or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated, however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be a compound as described herein and a protein or enzyme (e.g., PINK1). In some embodiments contacting includes allowing a compound described herein to interact with a protein or enzyme that is involved in a signaling pathway.

As defined herein, the term “inhibition,” “inhibit,” “inhibiting,” and the like in reference to a protein-inhibitor (e.g., antagonist) interaction means negatively affecting (e.g., decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor. In some embodiments inhibition refers to reduction of a disease or symptoms of disease. In some embodiments, inhibition refers to a reduction in the activity of a signal transduction pathway or signaling pathway. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein.

As defined herein, the term “activation,” “activate,” “activating,” and the like in reference to a protein-activator (e.g., agonist) interaction means positively affecting (e.g., increasing) the activity or function of the protein (e.g., PINK1) relative to the activity or function of the protein in the absence of the activator (e.g., compound described herein). In some embodiments, activation refers to an increase in the activity of a signal transduction pathway or signaling pathway (e.g., PINK1 pathway). Thus, activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein decreased in a disease (e.g., reduction of the level of PINK1 activity or protein associated with a cardiomyopathy or a neurodegenerative disease such as Parkinson's disease). Activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein (e.g., PINK1) that may modulate the level of another protein or increase cell survival (e.g., increase in PINK1 activity may increase cell survival in cells that may or may not have a reduction in PINK1 activity relative to a non-disease control).

The term “modulator” refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule. In some embodiments, the modulator is a modulator of PINK1. In some embodiments, the modulator is a modulator of PINK1 and is a compound that reduces the severity of one or more symptoms of a disease associated with PINK1 (e.g., reduction of the level of PINK1 activity or protein associated with a cardiomyopathy, neurodegenerative disease such as Parkinson's disease). In some embodiments, a modulator is a compound that reduces the severity of one or more symptoms of a cardiomyopathy or neurodegenerative disease that is not caused or characterized by PINK1 (e.g., loss of PINK1 function) but may benefit from modulation of PINK1 activity (e.g., increase in level of PINK1 or PINK1 activity).

“Disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with a compound, pharmaceutical composition, or method provided herein. In some embodiments, the disease is a disease related to (e.g., characterized by) a reduction in the level of PINK1. In some embodiments, the disease is a disease characterized by loss of dopamine-producing cells (e.g., Parkinson's disease). In some embodiments, the disease is a disease characterized by neurodegeneration. In some embodiments, the disease is a disease characterized by neural cell death. In some embodiments, the disease is a disease characterized by a reduction in the level of PINK1 activity. In some embodiments, the disease is Parkinson's disease. In some embodiments, the disease is a neurodegenerative disease. In some embodiments, the disease is a cardiomyopathy.

As used herein, the term “cardiomyopathy” refers to a disease condition that adversely affects cardiac cell tissue leading to a measurable deterioration in myocardial function (e.g., systolic function, diastolic function). Dilated cardiomyopathy is characterized by ventricular chamber enlargement with systolic dysfunction and no hypertrophy. Hypertrophic cardiomyopathy, is a genetic disease transmitted as an autosomal dominant trait. Hypertrophic cardiomyopathy is morphologically characterized by a hypertrophied and non-dialated left ventricle. Restrictive cardiomyopathy is characterized by nondialated nonhypertrophied morphology with diminished ventricular volume leading to poor ventricular filling. Arrhythmogenic right ventricular cardiomyopathy is an inheritable heart disease characterized by myocardial electric instability. Unclassified cardiomyopathy is a category for cardiomyopathies that do not match the features of any one of the other types. Unclassified cardiomyopathies may have features of multiple types or, for example, have the features of fibroelastosis, noncompacted myocardium, or systolic dysfunction with minimal dilatation.

The term “signaling pathway” as used herein refers to a series of interactions between cellular and optionally extra-cellular components (e.g., proteins, nucleic acids, small molecules, ions, lipids) that conveys a change in one component to one or more other components, which in turn may convey a change to additional components, which is optionally propagated to other signaling pathway components.

The compound of the disclosure can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compound individually or in combination (more than one compound or agent). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). The compositions of the present disclosure can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. The compositions of the present disclosure may additionally include components to provide sustained release and/or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present disclosure can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In some embodiments, the formulations of the compositions of the present disclosure can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present disclosure into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989). The compositions of the present disclosure can also be delivered as nanoparticles.

Pharmaceutical compositions provided by the present disclosure include compositions wherein the active ingredient (e.g., compounds described herein, including embodiments or examples) is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. When administered in methods to treat a disease, such compositions will contain an amount of active ingredient effective to achieve the desired result, e.g., modulating the activity of a target molecule (e.g., PINK1), and/or reducing, eliminating, or slowing the progression of disease symptoms (e.g., symptoms of cardiomyopathy or a neurodegeneration such as symptoms of Parkinson's disease). Determination of a therapeutically effective amount of a compound of the disclosure is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein.

The dosage and frequency (single or multiple doses) administered to a mammal can vary depending upon a variety of factors, for example, whether the mammal suffers from another disease, and its route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated (e.g., symptoms of cardiomyopathy or neurodegeneration such as Parkinson's disease and severity of such symptoms), kind of concurrent treatment, complications from the disease being treated or other health-related problems. Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of Applicants' disclosure. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the ability of those skilled in the art.

Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.

Utilizing the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be planned that does not cause substantial toxicity and yet is effective to treat the clinical symptoms demonstrated by the particular patient. This planning should involve the careful choice of active compound by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred mode of administration and the toxicity profile of the selected agent.

The compounds described herein can be used in combination with one another, with other active agents known to be useful in treating a disease associated neurodegeneration (e.g., Parkinson's disease such as levodopa, dopamine agonists (e.g., bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, lisuride), MAO-B inhibitors (e.g., selegiline or rasagiline), amantadine, anticholinergics, antipsychotics (e.g., clozapine), cholinesterase inhibitors, modafinil, or non-steroidal anti-inflammatory drugs), or with adjunctive agents that may not be effective alone, but may contribute to the efficacy of the active agent.

The compounds described herein can be used in combination with one another, with other active agents known to be useful in treating a cardiomyopathy such as Angiotensin Converting Enzyme Inhibitors (e.g., Enalipril, Lisinopril), Angiotensin Receptor Blockers (e.g., Losartan, Valsartan), Beta Blockers (e.g., Lopressor, Toprol-XL), Digoxin, or Diuretics (e.g., Lasixdisease associated neurodegeneration (e.g., Parkinson's disease such as levodopa, dopamine agonists (e.g., bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, lisuride), MAO-B inhibitors (e.g., selegiline or rasagiline), amantadine, anticholinergics, antipsychotics (e.g., clozapine), cholinesterase inhibitors, modafinil, or non-steroidal anti-inflammatory drugs), or with adjunctive agents that may not be effective alone, but may contribute to the efficacy of the active agent.

In some embodiments, co-administration includes administering one active agent within about 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of a second active agent. Co-administration includes administering two active agents simultaneously, approximately simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order. In some embodiments, co-administration can be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition including both active agents. In other embodiments, the active agents can be formulated separately. In some embodiments, the active and/or adjunctive agents may be linked or conjugated to one another. In some embodiments, the compounds described herein may be combined with treatments for neurodegeneration such as surgery. In some embodiments, the compounds described herein may be combined with treatments for cardiomyopathy such as surgery.

“PINK1” is used according to its common, ordinary meaning and refers to proteins of the same or similar names and functional fragments and homologs thereof. The term includes and recombinant or naturally occurring form of PINK1 (e.g., “PTEN induced putative kinase 1”; Entrez Gene 65018, OMIM 608309, UniProtKB Q9BXM7, and/or RefSeq (protein) NP_115785.1). The term includes PINK1 and variants thereof that maintain PINK1 activity (e.g., within at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% activity as compared to PINK1).

The term “neo-substrate” refers to a composition that is structurally similar to a composition that is a substrate for a protein or enzyme during the normal functioning of the protein or enzyme, but that is structurally distinct from the normal substrate of the protein or enzyme. In some embodiments, the composition comprises a neo-substrate. In some embodiments, the neo-substrate is a better substrate for the protein or enzyme than the normal substrate (e.g., the reaction kinetics are better (e.g., faster), binding is stronger, turnover rate is higher, reaction is more productive, equilibrium favors product formation). In some embodiments, the neo-substrate is a derivative of adenine, adenosine, AMP, ADP, or ATP. In some embodiments, the neo-substrate is a substrate for PINK1. In some embodiments, the neo-substrate is an N6 substituted adenine, adenosine, AMP, ADP, or ATP.

The term “derivative” as applied to a phosphate containing, monophosphate, diphosphate, or triphosphate group or moiety refers to a chemical modification of such group wherein the modification may include the addition, removal, or substitution of one or more atoms of the phosphate containing, monophosphate, diphosphate, or triphosphate group or moiety. In some embodiments, such a derivative is a prodrug of the phosphate containing, monophosphate, diphosphate, or triphosphate group or moiety, which is converted to the phosphate containing, monophosphate, diphosphate, or triphosphate group or moiety from the derivative following administration to a subject, patient, cell, biological sample, or following contact with a subject, patient, cell, biological sample, or protein (e.g., enzyme). In an embodiment, a triphosphate derivative is a gamma-thio triphosphate. In an embodiment, a derivative is a phosphoramidate. In some embodiments, the derivative of a phosphate containing, monophosphate, diphosphate, or triphosphate group or moiety is as described in Murakami et al. J. Med Chem., 2011, 54, 5902; Sofia et al., J. Med Chem. 2010, 53, 7202; Lam et al. ACC, 2010, 54, 3187; Chang et al., ACS Med Chem Lett., 2011, 2, 130; Furman et al., Antiviral Res., 2011, 91, 120; Vernachio et al., ACC, 2011, 55, 1843; Zhou et al, AAC, 2011, 44, 76; Reddy et al., BMCL, 2010, 20, 7376; Lam et al., J. Virol., 2011, 85, 12334; Sofia et al., J. Med. Chem., 2012, 55, 2481, Hecker et al., J. Med. Chem., 2008, 51, 2328; or Rautio et al., Nature Rev. Drug. Discov., 2008, 7, 255, all of which are incorporated herein by reference in their entirety for all purposes.

The term “mitochondrial dysfunction” is used in accordance with its ordinary meaning and refers to aberrant activity of function of the mitochondria, including for example aberrant respiratory chain activity, reactive oxygen species levels, calcium homeostasis, programmed cell death mediated by the mitochondria, mitochondrial fusion, mitochondrial fission, mitophagy, lipid concentrations in the mitochondrial membrane, and/or mitochondrial permeability transition.

As used herein, the term “mitochondrial disease” refers to a disease, disorder, or condition in which the function of a subject's mitochondria becomes impaired or dysfunctional. Examples of mitochondrial diseases that may be treated with a compound or method described herein include Alzheimer's disease, amyotrophic lateral sclerosis, Asperger's Disorder, Autistic Disorder, bipolar disorder, cancer, cardiomyopathy, Charcot Marie Tooth disease (CMT, including various subtypes such as CMT type 2b and 2b), Childhood Disintegrative Disorder (CDD), diabetes, diabetic nephropathy, epilepsy, Friedreich's Ataxia (FA), Hereditary motor and sensory neuropathy (HMSN), Huntington's Disease, Keams-Sayre Syndrome (KSS), Leber's Hereditary Optic Neuropathy (LHON, also referred to as Leber's Disease, Leber's Optic Atrophy (LOA), or Leber's Optic Neuropathy (LON)), Leigh Disease or Leigh Syndrome, macular degeneration, Mitochondrial Myopathy, Lactacidosis, and Stroke (MELAS), mitochondrial neurogastrointestinal encephalomyophathy (MNGIE), motor neuron diseases, Myoclonic Epilepsy With Ragged Red Fibers (MERRF), Neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP), Parkinson's disease, Peroneal muscular atrophy (PMA), Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), renal tubular acidosis, Rett's Disorder, Schizophrenia, and types of stroke.

The term “oxidative stress” is used in accordance with its ordinary meaning and refers to aberrant levels of reactive oxygen species.

As used herein, the term “animal” includes, but is not limited to, humans and non-human vertebrates such as wild, domestic, and farm animals.

As used herein, the term “antagonize” or “antagonizing” means reducing or completely eliminating an effect, such as an activity of GPR109a.

As used herein, the phrase “anti-receptor effective amount” of a compound can be measured by the anti-receptor effectiveness of the compound. In some embodiments, an anti-receptor effective amount inhibits an activity of the receptor by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or by at least 95%. In some embodiments, an “anti-receptor effective amount” is also a “therapeutically effective amount” whereby the compound reduces or eliminates at least one effect of GPR109a. In some embodiments, the effect is the B-arrestin effect. In some embodiments, the effect is the G-protein mediated effect.

As used herein, the term “carrier” means a diluent, adjuvant, or excipient with which a compound is administered. Pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used.

As used herein, the terms “comprising” (and any form of comprising, such as “comprise,” “comprises,” and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

As used herein, the term “contacting” means bringing together of two elements in an in vitro system or an in vivo system. For example, “contacting” a compound disclosed herein with an individual or patient or cell includes the administration of the compound to an individual or patient, such as a human, as well as, for example, introducing a compound into a sample containing a cellular or purified preparation containing the compounds or pharmaceutical compositions disclosed herein.

As used herein, the phrase “inhibiting activity,” such as enzymatic or receptor activity means reducing by any measurable amount the activity of PINK1.

As used herein, the phrase “in need thereof” means that the animal or mammal has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the animal or mammal can be in need thereof. In some embodiments, the animal or mammal is in an environment or will be traveling to an environment in which a particular disease, disorder, or condition is prevalent.

As used herein, the phrase “integer from X to Y” means any integer that includes the endpoints. For example, the phrase “integer from 1 to 5” means 1, 2, 3, 4, or 5.

As used herein, the term “isolated” means that the compounds described herein are separated from other components of either (a) a natural source, such as a plant or cell, or (b) a synthetic organic chemical reaction mixture, such as by conventional techniques.

As used herein, the term “mammal” means a rodent (i.e., a mouse, a rat, or a guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or a human. In some embodiments, the mammal is a human.

As used herein, the term “prodrug” means a derivative of a known direct acting drug, which derivative has enhanced delivery characteristics and therapeutic value as compared to the drug, and is transformed into the active drug by an enzymatic or chemical process. The compounds described herein also include derivatives referred to as prodrugs, which can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds. Examples of prodrugs include compounds of the disclosure as described herein that contain one or more molecular moieties appended to a hydroxyl, amino, sulfhydryl, or carboxyl group of the compound, and that when administered to a patient, cleaves in vivo to form the free hydroxyl, amino, sulfhydryl, or carboxyl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds of the disclosure. Preparation and use of prodrugs is discussed in T. Higuchi et al., “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference in their entireties.

As used herein, the term “purified” means that when isolated, the isolate contains at least 90%, at least 95%, at least 98%, or at least 99% of a compound described herein by weight of the isolate.

As used herein, the phrase “solubilizing agent” means agents that result in formation of a micellar solution or a true solution of the drug.

As used herein, the term “solution/suspension” means a liquid composition wherein a first portion of the active agent is present in solution and a second portion of the active agent is present in particulate form, in suspension in a liquid matrix.

As used herein, the phrase “substantially isolated” means a compound that is at least partially or substantially separated from the environment in which it is formed or detected.

As used herein, the phrase “therapeutically effective amount” means the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician. The therapeutic effect is dependent upon the disorder being treated or the biological effect desired. As such, the therapeutic effect can be a decrease in the severity of symptoms associated with the disorder and/or inhibition (partial or complete) of progression of the disorder, or improved treatment, healing, prevention or elimination of a disorder, or side-effects. The amount needed to elicit the therapeutic response can be determined based on the age, health, size and sex of the subject. Optimal amounts can also be determined based on monitoring of the subject's response to treatment.

It is further appreciated that certain features described herein, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

It should be noted that any embodiment of the disclosure can optionally exclude one or more embodiment for purposes of claiming the subject matter.

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 disclosure. 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 disclosure, or salt thereof. Methods for isolating compounds and their salts are routine in the art.

In various embodiments, the disclosure relates to compounds useful in treating disorders associated with PINK1 kinase activity such as, for example, neurodegenerative diseases, mitochondrial diseases, fibrosis, and/or cardiomyopathy.

In various embodiments, the compounds are useful in treating a disorder associated with PINK1 kinase activity in a mammal. In a further embodiment, the compounds are useful in treating PINK1 kinase activity in a human.

It is contemplated that each disclosed derivative can be optionally further substituted. It is also contemplated that any one or more derivative can be optionally omitted from the disclosure. It is understood that a disclosed compound can be provided by the disclosed methods. It is also understood that the disclosed compounds can be employed in the disclosed methods of using.

In some embodiments, provided are compounds having a structure represented by a formula:

In some embodiments, provided are compounds having a structure represented by a formula:

In some embodiments, the compound is selected from:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound has a structure represented by a formula:

In some embodiments, the compound has a structure represented by a formula:

In some embodiments, the compound has a structure represented by a formula:

In some embodiments, the compound has a structure represented by a formula:

In some embodiments, the compound has a structure represented by a formula:

wherein n is 1, 2, 3, or 4. In a further embodiment, n is 3. In a still further embodiment, each of R1a, R1b, and R1dis hydrogen. In yet a further embodiment, R1cis halogen. In an even further embodiment, R1cis —F.

In some embodiments, the compound has a structure represented by a formula:

wherein n is 1, 2, 3, or 4. In a further embodiment, n is 3.

In some embodiments, the compound has a structure represented by a formula selected from:

In some embodiments, the compound has a structure represented by a formula selected from:

In some embodiments, the compound has a structure represented by a formula selected from:

wherein n is 1, 2, 3, or 4. In a further embodiment, n is 3.

In some embodiments, the compound is selected from:

In some embodiments, the compound is selected from:

In some embodiments, the compound is selected from:

In some embodiments, the compound is selected from:

In some embodiments, the compound is selected from:

In some embodiments, the compound is selected from:

In some embodiments, the compound is selected from:

In some embodiments, the compound is selected from:

In some embodiments, m is 0 or 1. In a further embodiment, m is 0. In a still further embodiment, m is 1.

Specific examples of compounds are provided in the EXAMPLES section and are included herein. Pharmaceutically acceptable salts as well as the neutral forms of these compounds are also included.

In some embodiments, each of Q1and Q2is independently N or CH. In further embodiments, each of Q1and Q2is CH. In still further embodiments, each of Q1and Q2is N. In yet a further embodiment, Q1is N and Q2is CH. In an even further embodiment, Q1is CH and Q2is N.

In some embodiments, Q1is CH or N. In a further embodiment, Q1is N. In a still further embodiment, Q1is CH.

In some embodiments, Q2is CH or N. In a further embodiment, Q2is CH. In a still further embodiment, Q2is NH.

c. Z Groups

In some embodiments, Z is CR11aR11b, NR12, or O. In further embodiments, Z is CR11aR11bor NR12. In still further embodiments, Z is NR12or O.

In some embodiments, Z is CR11aR11bor O. In further embodiments, Z is CR11aR11b. In still further embodiments, Z is CH2. In yet further embodiments, Z is O.

In some embodiments, Z is NR12.

In further embodiments, each of R1a, R1b, R1c, and R1dis hydrogen.

In various embodiments, each of R1a, R1b, R1c, and R1dis independently selected from hydrogen and C1-C4 alkyl. In further embodiments, each of R1a, R1b, R1c, and R1dis independently selected from hydrogen, methyl, ethyl, n-propyl, and isopropyl. In still further embodiments, each of R1a, R1b, R1c, and R1dis independently selected from hydrogen, methyl, and ethyl. In yet further embodiments, each of R1a, R1b, R1c, and R1dis independently selected from hydrogen and methyl.

In various embodiments, each of R1a, R1b, R1c, and R1dis independently selected from hydrogen and halogen. In further embodiments, each of R1a, R1b, R1c, and R1dis independently selected from hydrogen, —F, —Cl, and —Br. In still further embodiments, each of R1a, R1b, R1c, and R1dis independently selected from hydrogen, —F, and —C1. In yet further embodiments, each of R1a, R1b, R1c, and R1dis independently selected from hydrogen and —Cl. In still further embodiments, each of R1a, R1b, R1c, and R1dis independently selected from hydrogen and —F.

In some embodiments, R2is selected from C5-C8 cyanoalkyl and —(C1-C8 alkyl)-O—(C1-C4 alkyl). In further embodiments, R2is selected from C5-C6 cyanoalkyl and —(C1-C4 alkyl)-O—(C1-C4 alkyl). In still further embodiments, R2is selected from —CH2CH2C(CH3)2CN, —CH2CH2OCH2CH3, —CH2CH2OCH3, and —CH2OCH3. In yet further embodiments, R2is selected from —CH2CH2C(CH3)2CN, —CH2CH2OCH2CH3, and —CH2CH2OCH3. In even further embodiments, R2is selected from —CH2CH2C(CH3)2CN and —CH2CH2OCH3.

In some embodiments, R2is selected from —O—(C1-C8 alkyl)-R13, —O—(C1-C8 haloalkyl)-R13, and —O—(C1-C8 alkyl substituted with a C3-C5 cycloalkyl)-R13. In further embodiments, R2is selected from —O—(C1-C4 alkyl)-R13, —O—(C1-C4 haloalkyl)-R13, and —O—(C1-C4 alkyl substituted with a C3-C5 cycloalkyl)-R13. In still further embodiments, R2is selected from —OCH2C(CH3)2R13, —OCH2CF2R13, and a structure:

In some embodiments, R3is a 3- to 6-membered cycloalkyl. In further embodiments, R3is a 3- to 5-membered cycloalkyl. In still further embodiments, R3is a 3- to 4-membered cycloalkyl. In yet further embodiments, R3is a 3-membered cycloalkyl. In an even further embodiment, R3is a 4-membered cycloalkyl.

In further embodiments, R3is hydrogen or (C1-C4)alkyl. In still further embodiments, R3is hydrogen, methyl, ethyl, n-propyl, or isopropyl. In yet further embodiments, R3is hydrogen, methyl, or ethyl. In an even further embodiment, R3is hydrogen or ethyl. In still further embodiments, R3is hydrogen or methyl.

In further embodiments, R3is (C1-C4)alkyl. In still further embodiments, R3is methyl, ethyl, n-propyl, or isopropyl. In yet further embodiments, R3is methyl or ethyl. In an even further embodiment, R3is ethyl. In still further embodiments, R3is methyl.

In further embodiments, R3is hydrogen or halogen. In still further embodiments, R3is hydrogen, —F, —Cl, or —Br. In yet further embodiments, R3is hydrogen, —F, or —C1. In an even further embodiment, R3is hydrogen or —F. In still further embodiments, R3is hydrogen or —C1.

In further embodiments, R3is hydrogen or 3- to 6-membered cycloalkyl. In still further embodiments, R3is hydrogen, cyclopropyl, cyclobutyl, or cyclopentyl. In yet further embodiments, R3is hydrogen, cyclopropyl, or cyclobutyl. In an even further embodiment, R3is hydrogen or cyclopropyl. In some embodiments, R3is not a methyl, ethyl or butyl. In some embodiments, R3is not an acyclic alkyl chain comprising from about 1 to about 5 substituted or unsubstituted carbons.

In further embodiments, R3is a 3-membered cycloalkyl or —CF3. In still further embodiments, R3is a 3-membered cycloalkyl. In yet further embodiments, R3is —CF3.

In some embodiments, R4is selected from hydrogen and C1-C4 alkyl. In further embodiments, R4is selected from hydrogen, methyl, ethyl, n-propyl, and isopropyl. In still further embodiments, R4is selected from hydrogen, methyl, and ethyl. In yet further embodiments, R4is selected from hydrogen and ethyl. In an even further embodiment, R4is selected from hydrogen and methyl.

In some embodiments, R4is C1-C4 alkyl. In further embodiments, R4is selected from methyl, ethyl, n-propyl, and isopropyl. In still further embodiments, R4is selected from methyl and ethyl. In yet further embodiments, R4is ethyl. In an even further embodiment, R4is methyl.

In some embodiments, each of R5aand R5b, when present, is hydrogen, or R5aand R5b, when present, are covalently bonded, and, together with the intermediate atoms, comprise a C3-C6 cycloalkyl.

In some embodiments, each of R5aand R5b, when present, is hydrogen.

In some embodiments, R5aand R5b, when present, are covalently bonded, and, together with the intermediate atoms, comprise a C3-C6 cycloalkyl. In further embodiments, R5aand R5b, when present, are covalently bonded, and, together with the intermediate atoms, comprise a C3-C5 cycloalkyl. In still further embodiments, R5aand R5b, when present, are covalently bonded, and, together with the intermediate atoms, comprise a C3-C4 cycloalkyl. In yet further embodiments, R5aand R5b, when present, are covalently bonded, and, together with the intermediate atoms, comprise a C5-C6 cycloalkyl. In even further embodiments, R5aand R5b, when present, are covalently bonded, and, together with the intermediate atoms, comprise a C4-C5 cycloalkyl.

In some embodiments, R5aand R5b, when present, are covalently bonded, and, together with the intermediate atoms, comprise a cyclopropyl. In further embodiments, R5aand R5b, when present, are covalently bonded, and, together with the intermediate atoms, comprise a cyclobutyl. In still further embodiments, R5aand R5b, when present, are covalently bonded, and, together with the intermediate atoms, comprise a cyclopentyl. In yet further embodiments, R5aand R5b, when present, are covalently bonded, and, together with the intermediate atoms, comprise a cyclohexyl.

In some embodiments, each of R11aand R11b, when present, is independently selected from hydrogen, halogen, —OH, and C1-C4 alkoxy, or wherein each of R11aand R11b, when present, together comprise ═O.

In some embodiments, each of R11aand R11b, when present, is independently selected from hydrogen, halogen, —OH, and C1-C4 alkoxy. In further embodiments, each of R11aand R11b, when present, is independently selected from hydrogen, —F, —Cl, —Br, —OH, methoxy, ethoxy, n-propoxy, and isopropoxy. In still further embodiments, each of R11aand R11bwhen present, is independently selected from hydrogen, —F, —Cl, —OH, methoxy, and ethoxy. In yet further embodiments, each of R11aand R11bwhen present, is independently selected from hydrogen, —F, —OH, and methoxy.

In some embodiments, each of R11aand R11b, when present, is independently selected from hydrogen, —OH, and C1-C4 alkoxy. In further embodiments, each of R11aand R11bwhen present, is independently selected from hydrogen, —OH, methoxy, ethoxy, n-propoxy, and isopropoxy. In still further embodiments, each of R11aand R11b, when present, is independently selected from hydrogen, —OH, methoxy, and ethoxy. In yet further embodiments, each of R11aand R11b, when present, is independently selected from hydrogen, —OH, and methoxy.

In some embodiments, each of R11aand R11b, when present, is independently selected from hydrogen and C1-C4 alkoxy. In further embodiments, each of R11aand R11b, when present, is independently selected from hydrogen, methoxy, ethoxy, n-propoxy, and isopropoxy. In still further embodiments, each of R11aand R11b, when present, is independently selected from hydrogen, methoxy, and ethoxy. In yet further embodiments, each of R11aand R11bwhen present, is independently selected from hydrogen and methoxy.

In some embodiments, each of R11aand R11b, when present, is independently selected from hydrogen and —OH. In further embodiments, each of R11aand R11b, when present, is —OH. In still further embodiments, each of R11aand R11b, when present, is hydrogen.

In some embodiments, each of R11aand R11b, when present, is independently selected from hydrogen and halogen. In further embodiments, each of R11aand R11bwhen present, is independently selected from hydrogen, —F, —Cl, and —Br. In still further embodiments, each of R11aand R11b, when present, is independently selected from hydrogen, —F, and —Cl. In yet further embodiments, each of R11aand R11b, when present, is independently selected from hydrogen and —F.

In some embodiments, each of R1aand R11, when present, together comprise ═O.

In some embodiments, R12, when present, is hydrogen or C1-C4 alkyl. In further embodiments, R12, when present, is hydrogen, methyl, ethyl, n-propyl, or isopropyl. In still further embodiments, R12, when present, is hydrogen, methyl, or ethyl. In yet further embodiments, R12, when present, is hydrogen or methyl.

In some embodiments, R12, when present, is C1-C4 alkyl. In further embodiments, R12, when present, is methyl, ethyl, n-propyl, or isopropyl. In still further embodiments, R12, when present, is methyl or ethyl. In yet further embodiments, R12, when present, is methyl.

In some embodiments, R12, when present, is hydrogen.

In some embodiments, R13, when present, is selected from —CN, —OH, and —NH2. In further embodiments, R13, when present, is selected from —CN and —OH. In still further embodiments, R13, when present, is selected from —OH and —NH2. In yet further embodiments, R13, when present, is selected from —CN and —NH2. In even further embodiments, R13, when present, is —CN. In still further embodiments, R13, when present, is —OH. In yet further embodiments, R13, when present, is —NH2.

In some embodiments, R13, when present, is C1-C4 haloalkyl. In further embodiments, R13, when present, is selected from —CH2F, —CH2CH2F, —CH(CH3)CH2F, —CH2CH2CH2F, —CH2Cl, —CH2CH2Cl, —CH(CH3)CH2Cl, and —CH2CH2CH2C1. In still further embodiments, R13, when present, is selected from —CH2F, —CH2CH2F, —CH2Cl, and —CH2CH2C1. In yet further embodiments, R13, when present, is selected from —CH2F and —CH2Cl.

In some embodiments, R13, when present, is C1-C4 alkoxy. In further embodiments, R13, when present, is selected from methoxy, ethoxy, n-propoxy, and isopropoxy. In still further embodiments, R13, when present, is selected from methoxy and ethoxy. In yet further embodiments, R13, when present, is methoxy. In even further embodiments, R13, when present, is ethoxy.

2. Example Compounds

In some embodiments, a compound can be present as one or more of the following structures:

or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound can be present as one or more of the following structures:

or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound can be present as one or more of the following structures:

or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound can be present as one or more of the following structures:

or a pharmaceutically acceptable salt thereof.

3. Prophetic Compound Examples

The following compound examples are prophetic, and can be prepared using the synthesis methods described herein above and other general methods as needed as would be known to one skilled in the art. It is anticipated that the prophetic compounds would be active as modulators of PINK1 kinase activity, and such activity can be determined using the assay methods described herein below.

In one aspect, a compound is selected from:

In one aspect, a compound is selected from:

In one aspect, a compound is selected from:

In one aspect, a compound is selected from:

It is contemplated that one or more compounds can optionally be omitted from the disclosed invention.

It is understood that the disclosed compounds can be used in connection with the disclosed methods, compositions, kits, and uses.

It is understood that pharmaceutical acceptable derivatives of the disclosed compounds can be used also in connection with the disclosed methods, compositions, kits, and uses. The pharmaceutical acceptable derivatives of the compounds can include any suitable derivative, such as pharmaceutically acceptable salts as discussed below, isomers, radiolabeled analogs, tautomers, and the like.

Also provided herein are pharmaceutical compositions comprising a disclosed compound, or pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier. Thus, in various embodiments, disclosed are pharmaceutical compositions comprising a therapeutically effective amount at least one disclosed compound and a pharmaceutically acceptable carrier. In a further embodiment, a pharmaceutical composition can be provided comprising a therapeutically effective amount of at least one disclosed compound. In a still further embodiment, a pharmaceutical composition can be provided comprising a prophylactically effective amount of at least one disclosed compound. In yet a further embodiment, the disclosure relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound, wherein the compound is present in an effective amount.

Thus, in various embodiments, provided herein are pharmaceutical compositions comprising a therapeutically effective amount of a compound having a structure represented by a formula:

In various embodiments, provided herein are pharmaceutical compositions comprising a therapeutically effective amount of a compound having a structure represented by a formula:

In various embodiments, provided herein are pharmaceutical compositions comprising a therapeutically effective amount of a compound selected from:

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable salts of the compounds are conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Exemplary acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Example base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethylammonium hydroxide. Chemical modification of a pharmaceutical compound into a salt is a known technique to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457.

The pharmaceutical compositions comprise the compounds in a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. The compounds can be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

In further embodiments, the pharmaceutical composition is administered to a mammal. In still further embodiments, the mammal is a human. In even further embodiments, the human is a patient.

In further embodiments, the pharmaceutical composition is administered following identification of the mammal in need of treatment of a disorder associated with PINK1 kinase activity. In still further embodiments, the mammal has been diagnosed with a need for treatment of a disorder associated with PINK1 kinase activity prior to the administering step.

The choice of carrier will be determined in part by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, rectal, and vaginal administration are merely exemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granule; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water, cyclodextrin, dimethyl sulfoxide and alcohols, for example, ethanol, benzyl alcohol, propylene glycol, glycerin, and the polyethylene alcohols including polyethylene glycol, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, the addition to the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.

The compounds of the present disclosure alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl-1, 3-dioxolane-4-methanol, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcelluslose, or emulsifying agents and other pharmaceutical adjuvants.

Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example. dimethyldialkylammonium halides, and alkylpyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl β-aminopropionates, and 2-alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.

The parenteral formulations typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

Pharmaceutically acceptable excipients are also well-known to those who are skilled in the art. The choice of excipient will be determined in part by the particular compound, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present disclosure. The following methods and excipients are merely exemplary and are in no way limiting. The pharmaceutically acceptable excipients preferably do not interfere with the action of the active ingredients and do not cause adverse side-effects. Suitable carriers and excipients include solvents such as water, alcohol, and propylene glycol, solid absorbants and diluents, surface active agents, suspending agent, tableting binders, lubricants, flavors, and coloring agents.

The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. SeePharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia, PA, Banker and Chalmers, Eds., 238-250 (1982) andASHP Handbook on Injectable Drugs, Toissel, 4thed., 622-630 (1986).

Formulations suitable for topical administration include lozenges comprising the active ingredient in a flavor, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier; as well as creams, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.

Additionally, formulations suitable for rectal administration may be presented as suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

One skilled in the art will appreciate that suitable methods of exogenously administering a compound of the present disclosure to an animal are available, and, although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective reaction than another route.

As regards these applications, the present method includes the administration to an animal, particularly a mammal, and more particularly a human, of a therapeutically effective amount of the compound effective in the treatment (e.g., prophylactic or therapeutic) of a disorder associated with PINK1 kinase activity. The method also includes the administration of a therapeutically effect amount of the compound for the treatment of patient having a predisposition for being afflicted with a disorder associated with PINK1 kinase activity. The dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the animal over a reasonable timeframe. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition of the animal, the body weight of the animal, as well as the severity and stage of the disorder.

The total amount of the compound of the present disclosure administered in a typical treatment is preferably from about 1 mg/kg to about 100 mg/kg of body weight for mice, and from about 10 mg/kg to about 50 mg/kg of body weight, and from about 20 mg/kg to about 40 mg/kg of body weight for humans per daily dose. This total amount is typically, but not necessarily, administered as a series of smaller doses over a period of about one time per day to about three times per day for about 24 months, and over a period of twice per day for about 12 months. In some embodiments, the compound of the present disclosure is administered in a dose range of from about 10 mg/kg to about 40 mg/kg of body weight of the subject. In some embodiments, the compound of the present disclosure is administered in a dose range of from about 10 mg/kg to about 50 mg/kg of body weight of the subject. In some embodiments, the compound of the present disclosure is administered in a dose range of from about 10 mg/kg to about 35 mg/kg of body weight of the subject. In some embodiments, the compound of the present disclosure is administered in a dose range of from about 10 mg/kg to about 30 mg/kg of body weight of the subject. In some embodiments, the compound of the present disclosure is administered in a dose range of from about 10 mg/kg to about 25 mg/kg of body weight of the subject.

The size of the dose also will be determined by the route, timing and frequency of administration as well as the existence, nature and extent of any adverse side effects that might accompany the administration of the compound and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states, in particular chronic conditions or disease states, may require prolonged treatment involving multiple administrations.

In certain embodiments, a composition described herein is formulated for administration to a patient in need of such composition. Compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.

A compound described herein can be administered alone or can be coadministered with an additional therapeutic agent. Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). Additional therapeutic agents include, but are not limited to, other active agents known to be useful in treating a disease associated neurodegeneration (e.g., Parkinson's disease such as levodopa), dopamine agonists (e.g., bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, lisuride), MAO-B inhibitors (e.g., selegiline or rasagiline), amantadine, anticholinergics, antipsychotics (e.g., clozapine), cholinesterase inhibitors, modafinil, or non-steroidal anti-inflammatory drugs), Angiotensin Converting Enzyme Inhibitors (e.g., Enalipril, Lisinopril), Angiotensin Receptor Blockers (e.g., Losartan, Valsartan), Beta Blockers (e.g., Lopressor, Toprol-XL), Digoxin, or Diuretics.

Suitable compositions include, but are not limited to, oral non-absorbed compositions. Suitable compositions also include, but are not limited to saline, water, cyclodextrin solutions, and buffered solutions of pH from about 3 to about 9.

In some embodiments, the formulation can be lyophilized to a solid and reconstituted with, for example, water prior to use.

When administered to a mammal (e.g., to an animal for veterinary use or to a human for clinical use) the compounds can be administered in isolated form.

When administered to a human, the compounds can be sterile. Water is a suitable carrier when the compound of Formula I is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

The compositions described herein can take the form of a solution, suspension, emulsion, tablet, pill, pellet, capsule, capsule containing a liquid, powder, sustained-release formulation, suppository, aerosol, spray, or any other form suitable for use. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. R. Gennaro (Editor) Mack Publishing Co.

In some embodiments, the compounds are formulated in accordance with routine procedures as a pharmaceutical composition adapted for administration to humans. Typically, compounds are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration may optionally include a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the compound is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The pharmaceutical compositions can be in unit dosage form. In such form, the composition can be divided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms.

In some embodiments, a composition of the present disclosure is in the form of a liquid wherein the active agent is present in solution, in suspension, as an emulsion, or as a solution/suspension. In some embodiments, the liquid composition is in the form of a gel. In other embodiments, the liquid composition is aqueous. In other embodiments, the composition is in the form of an ointment.

In some embodiments, the composition is in the form of a solid article. For example, in some embodiments, the ophthalmic composition is a solid article that can be inserted in a suitable location in the eye, such as between the eye and eyelid or in the conjunctival sac, where it releases the active agent as described, for example, U.S. Pat. Nos. 3,863,633; 3,867,519; 3,868,445; 3,960,150; 3,963,025; 4,186,184; 4,303,637; 5,443,505; and 5,869,079. Release from such an article is usually to the cornea, either via the lacrimal fluid that bathes the surface of the cornea, or directly to the cornea itself, with which the solid article is generally in intimate contact. Solid articles suitable for implantation in the eye in such fashion are generally composed primarily of polymers and can be bioerodible or non-bioerodible. Bioerodible polymers that can be used in the preparation of ocular implants carrying one or more of the compounds described herein in accordance with the present disclosure include, but are not limited to, aliphatic polyesters such as polymers and copolymers of poly(glycolide), poly(lactide), poly(epsilon-caprolactone), poly-(hydroxybutyrate) and poly(hydroxyvalerate), polyamino acids, polyorthoesters, polyanhydrides, aliphatic polycarbonates and polyether lactones. Suitable non-bioerodible polymers include silicone elastomers.

In some embodiments, the compound or pharmaceutical composition comprising the compounds discosed herein, or the pharmaceutically acceptable salts herein, are neo-substrates of PINK1. In some embodiments, the neo-substrate is not kinetin. In some embodiments, the neo-substrate is not kinetin riboside. In some embodiments, the neo-substrate is not kinetin riboside 5′ monophosphate. In some embodiments, the neo-substrate is not kinetin riboside 5′ diphosphate. In some embodiments, the neo-substrate is not kinetin riboside 5′ triphosphate. In some embodiments, the neo-substrate is not a derivative (e.g., prodrug) of kinetin, kinetin riboside, kinetin riboside 5′ monophosphate, kinetin riboside 5′ diphosphate, or kinetin riboside 5′ triphosphate. In some embodiments, the neo-substrate is not N6-(delta 2-Isopentenyl)-adenine. In some embodiments, the neo-substrate is not N6-(delta 2-Isopentenyl)-adenosine, N6-(delta 2-Isopentenyl)-adenosine 5′ monophosphate, N6-(delta 2-Isopentenyl)-adenosine 5′ diphosphate, N6-(delta 2-Isopentenyl)-adenosine 5′ triphosphate, or a derivative (e.g., prodrug) thereof. In some embodiments, the neo-substrate is not a cytokinin. In some embodiments, the neo-substrate is not a cytokinin riboside, cytokinin riboside 5′ monophosphate, cytokinin riboside 5′ diphosphate, cytokinin riboside 5′ triphosphate, or a derivative (e.g., prodrug) thereof.

It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be employed in the disclosed methods of using.

D. METHODS OF MAKING THE COMPOUNDS

In various embodiments, the disclosures relates to methods of making compounds useful to treat a disorder associated with PINK1 kinase activity. Thus, in some embodiments, disclosed are methods of making a disclosed compound.

Compounds according to the present disclosure can, for example, be prepared by the several methods outlined below. A practitioner skilled in the art will understand the appropriate use of protecting groups [see: Greene and Wuts, Protective Groups in Organic Synthesis] and the preparation of known compounds found in the literature using the standard methods of organic synthesis. There may come from time to time the need to rearrange the order of the recommended synthetic steps, however this will be apparent to the judgment of a chemist skilled in the art of organic synthesis. The following examples are provided so that the disclosure might be more fully understood, are illustrative only, and should not be construed as limiting.

In some embodiments, the disclosed compounds comprise the products of the synthetic methods described herein. In further embodiments, the disclosed compounds comprise a compound produced by a synthetic method described herein. In still further embodiments, the disclosure comprises a pharmaceutical composition comprising a therapeutically effective amount of the product of the disclosed methods and a pharmaceutically acceptable carrier. In still further embodiments, the disclosure comprises a method for manufacturing a medicament comprising combining at least one compound of any of disclosed compounds or at least one product of the disclosed methods with a pharmaceutically acceptable carrier or diluent.

1. Route I

In some embodiments, the disclosed compounds can be prepared as shown below.

Compounds are represented in generic form, wherein o is 0, 1, 2, 3, 4, 5, 6, or 7, PG is an alcohol protecting group, X is a halogen, R′ is —CN or C1-C4 alkoxy, and PG′ is an amine protecting group, and with other substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In some embodiments, compounds of type 1.9, and similar compounds, can be prepared according to reaction Scheme 1B above. Thus, compounds of type 1.2 can be prepared by reduction of an appropriate ketone, e.g., 1.1 as shown above. Appropriate ketones are commercially available or prepared by methods known to one skilled in the art. The reaction is carried out in the presence of an appropriate reducing agent, e.g., sodium borohydride. Compounds of type 1.3 can be prepared by protection of an appropriate alcohol, e.g., 1.2 as shown above, followed by reduction of the ester. The protection is carried out in the presence of an appropriate alcohol protecting agent, e.g., tert-butyldimethylsilyl chloride (TMSCl), and an appropriate base, e.g., imidazole, in an appropriate solvent, e.g., dimethylformamide (DMF). The reduction is carried out in the presence of an appropriate reducing agent, e.g., lithium aluminum hydride, in an appropriate solvent, e.g., tetrahydrofuran, at an appropriate temperature, e.g., 0° C. Compounds of type 1.4 can be prepared by activation and displacement of an appropriate alcohol, e.g., 1.3 as shown above. The reaction is carried out in the presence of an appropriate activating agent, e.g., mesyl chloride, and an appropriate base, e.g., triethylamine (TEA), in an appropriate solvent, e.g., dichloromethane (DCM), followed by addition of an appropriate halide agent, e.g., sodium iodide, in an appropriate solvent, e.g., acetone, at an appropriate temperature, e.g., 50° C. Compounds of type 1.5 can be prepared by substitution of an appropriate alkyl halide, e.g., 1.4 as shown above, followed by a deprotection reaction. The substitution is carried out in the presence of a cyanoalkyl analog, e.g., isopropylcyanide, and an appropriate base, e.g., lithium diisopropylamide, in an appropriate solvent, e.g., tetrahydrofuran, at an appropriate temperature, e.g., −78° C. The deprotection reaction is carried out in the presence of an appropriate deprotecting agent, e.g., tetrabutylammonium fluoride. Compounds of type 1.6 can be prepared by electrophilic addition of an appropriate cyanide analog, e.g., methylcyanide, and an appropriate alcohol, e.g., 1.5 as shown above. The electrophilic addition is carried out in the presence of appropriate acid, e.g., sulfuric acid, at an appropriate temperature, e.g., 40° C. Compounds of type 1.7 can be prepared by protection of an appropriate amide, e.g., 1.6 as shown above. The protection is carried out in the presence of an appropriate amine protecting agent, e.g., di-tert-butyl dicarbonates, and an appropriate base, e.g., 4-dimethylamino pyridine, in an appropriate solvent, e.g., tetrahydrofuran, at an appropriate temperature, e.g., 50° C. Compounds of type 1.8 can be prepared by reduction of an appropriate amide, e.g., 1.7 as shown above. The reduction is carried out in the presence of an appropriate base, e.g., potassium carbonate, in an appropriate solvent, e.g., methanol. Compounds of type 1.9 can be prepared by deprotection of an appropriate amine, e.g., 1.8 as shown above. The deprotection is carried out in the presence of an appropriate acid, e.g., trifluoracetic acid, in an appropriate solvent, e.g., dichloromethane. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, and 1.18), can be substituted in the reaction to provide compounds similar to Formula 1.19.

2. Route II

In some embodiments, the disclosed compounds can be prepared as shown below.

Compounds are represented in generic form, wherein X and X′ are independently halogen and R is —(C1-C8 alkyl)-R13, —(C1-C8 haloalkyl)-R13, or —(C1-C8 alkyl substituted with a C3-C5 cycloalkyl)-R13, and with other substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In some embodiments, compounds of type 2.10, and similar compounds, can be prepared according to reaction Scheme 2B above. Thus, compounds of type 2.2 can be prepared by epoxidation and subsequent ring-opening of an appropriate haloalcohol, e.g., 2.1 as shown above. Appropriate haloalcohols are commercially available or prepared by methods known to one skilled in the art. The epoxidation reaction is carried out in the presence of an appropriate base, e.g., aqueous potassium hydroxide, in an appropriate solvent, e.g., tetrahydrofuran. The resultant epoxide is then reacted with an appropriate imine, e.g., diphenylmethanimine, at an appropriate temperature, e.g., 95° C., for an appropriate period of time, e.g., 6 hours. Compounds of type 2.3 can be prepared by alkylation of an appropriate alcohol, e.g., 2.2 as shown above. The alkylation is carried out in the presence of an appropriate alkyl halide, e.g., 2.3 as shown above, and an appropriate base, e.g., sodium hydride, in an appropriate solvent, e.g., dimethylformamide. Compounds of type 2.5 can be prepared by reduction of an appropriate imine, e.g., 2.4 as shown above. The reduction is carried out in the presence of an appropriate acid, e.g., hydrochloric acid, in an appropriate solvent, e.g., dichloromethane. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 2.6, 2.7, 2.8, and 2.9), can be substituted in the reaction to provide compounds similar to Formula 2.10.

3. Route III

In some embodiments, the disclosed compounds can be prepared as shown below.

Compounds are represented in generic form, wherein X is halogen, and with other substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In some embodiments, compounds of type 3.3, and similar compounds, can be prepared according to reaction Scheme 3B above. Thus, compounds of type 3.2 can be prepared by reducing an appropriate ketone, e.g., 3.1, followed by condensation, formation of the bromohydrin, and coupling to a carboxylic acid. Appropriate ketones are commercially available or prepared by methods known to one skilled in the art. The reduction is carried out in the presence of an appropriate reducing agent, e.g., sodium borohydride, followed by addition of an appropriate acid, e.g., toluene-4-sulfonic acid, in an appropriate solvent, e.g., toluene, at an appropriate temperature, e.g., 120° C., thereby forming an alkene. The alkene is then reacted with an appropriate bromine source, e.g., N-bromosuccinimide, in an aqueous solvent system, e.g., tetrahydrofuran in water, at an appropriate temperature, e.g., 0° C., to form a bromohydrin, which is then coupled with an appropriate carboxylic acid, e.g., (2R)-2-phenyl-propanoic acid, in the presence of an appropriate activating agent, e.g., N,N′-dicyclohexylcarbodiimide, and an appropriate base, e.g., 4-dimethylaminopyridine, in an appropriate solvent, e.g., dichloromethane. Compounds of type 3.3 can be prepared by epoxidation and subsequent ring-opening. The epoxidation is carried out in the presence of an appropriate base, e.g., sodium methoxide, in an appropriate solvent, e.g., diethyl ether. The resultant epoxide is then reacted with an appropriate imine, e.g., diphenylmethanimine, at an appropriate temperature, e.g., 95° C. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 3.4 and 3.5), can be substituted in the reaction to provide compounds similar to Formula 3.6.

4. Route IV

In some embodiments, the disclosed compounds can be prepared as shown below.

Compounds are represented in generic form, wherein o is 1, 2, 3, 4, 5, or 6, X is halogen, and each occurrence of R is C1-C8 alkyl, and with other substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In some embodiments, compounds of type 4.5, and similar compounds, can be prepared according to reaction Scheme 4B above. Thus, compounds of type 4.3 can be prepared by displacement of an appropriate alkyl halide, e.g., 4.2 as shown above, with an appropriate alcohol, e.g., 4.1 as shown above. Appropriate alkyl halides and appropriate alcohols are commercially available or prepared by methods known to one skilled in the art. The displacement reaction is carried out in the presence of an appropriate base, e.g., sodium hyride, in an appropriate solvent, e.g., tetrahydrofuran, at an appropriate temperature, e.g., 0° C. Compounds of type 4.5 can be prepared by a Grignard reaction between an appropriate ester, and an appropriate Grignard reagent, e.g., 4.4 as shown above. Appropriate Grignard reagents are commercially available or prepared by methods known to one skilled in the art. The Grignard reaction is carried out in the presence of an appropriate solvent, e.g., tetrahydrofuran, at an appropriate temperature, e.g., −78° C. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 4.6, 4.7, 4.8, and 4.9), can be substituted in the reaction to provide compounds similar to Formula 4.10.

5. Route V

In some embodiments, the disclosed compounds can be prepared as shown below.

Compounds are represented in generic form, wherein R is C1-C4 alkyl and R′ is C1-C8 alkyl, and with other substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In some embodiments, compounds of type 5.7, and similar compounds, can be prepared according to reaction Scheme 5B above. Thus, compounds of type 5.2 can be prepared by condensation and silylation of an appropriate ketone, e.g., 5.2 as shown above. Appropriate ketones are commercially available or prepared by methods known to one skilled in the art. The reaction is carried out in the presence of an appropriate agent to generate a silyl enol ether, e.g., trimethylsilyl trifluoromethanesulfonate, and an appropriate base, e.g., triethylamine, in an appropriate solvent, e.g., dichloromethane, at an appropriate temperature, e.g., 0° C. Compounds of type 5.4 can be prepared by addition with an appropriate alkene, e.g., 5.3 as shown above. Appropriate alkenes are commercially available or prepared by methods known to one skilled in the art. The addition is carried out in the presence of an appropriate Lewis acid, e.g., titanium chloride, in an appropriate solvent, e.g., dichloromethane, at an appropriate temperature, e.g., 30° C. to −78° C. Compounds of type 5.5 can be prepared by reduction/oxidation of an appropriate ester. The reduction is carried out in the presence of an appropriate reducing agent, e.g., lithium aluminum hydride, in an appropriate solvent, e.g., tetrahydrofuran, at an appropriate temperature, e.g., 0° C., followed by selective oxidation of the secondary alcohol using an appropriate oxidizing agent, e.g., manganese dioxide, in an appropriate solvent, e.g., dichloromethane. Compounds of type 5.6 can be prepared alkylation of an appropriate alcohol, e.g., 5.5. The alkylation is carried out in the presence of an appropriate alkyl halide, e.g., methyl iodide, and an appropriate base, e.g., silver oxide, in an appropriate solvent, e.g., acetonitrile. Compounds of type 5.7 can be prepared by reductive amination of an appropriate ketone, e.g., 5.6 as shown above. The reductive amination is carried out in the presence of an appropriate amine, e.g., ammonium acetate, and an appropriate reducing agent, e.g., sodium cyanoborohydride, at an appropriate temperature, e.g., 80° C. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 5.8, 5.9, 5.10, 5.11, 5.12, and 5.13), can be substituted in the reaction to provide compounds similar to Formula 5.14.

6. Route VI

In some embodiments, the disclosed compounds can be prepared as shown below.

Compounds are represented in generic form, wherein X is halogen, and with other substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In some embodiments, compounds of type 6.3, and similar compounds, can be prepared according to reaction Scheme 6B above. Thus, compounds of type 6.3 can be prepared by arylation of an appropriate amine, e.g., 6.1 as shown above. The arylation is carried out in the presence of an appropriate halide, e.g., 6.2 as shown above, and an appropriate base, e.g., N,N-diisopropylethyl amine (DIEA), in an appropriate solvent, e.g., butanol (BuOH). Appropriate halides are commercially available or prepared by methods known to those skilled in the art. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 6.4 and 6.5), can be substituted in the reaction to provide compounds similar to Formula 6.6.

Compounds and compositions described herein are generally useful for modulating the activity of PINK1. In some embodiments, the compounds and compositions described herein inhibit the activity of PINK1.

E. Methods of Using the Compounds

The compounds and pharmaceutical compositions of the disclosure are useful in treating or controlling disorders associated with PINK1 kinase activity. To treat or control the disorder, the compounds and pharmaceutical compositions comprising the compounds are administered to a subject in need thereof, such as a vertebrate, e.g., a mammal, a fish, a bird, a reptile, or an amphibian. The subject can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. The subject is preferably a mammal, such as a human. Prior to administering the compounds or compositions, the subject can be diagnosed with a need for treatment of the disorder associated with PINK1 kinase activity.

The compounds or compositions can be administered to the subject according to any method. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. A preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. A preparation can also be administered prophylactically; that is, administered for prevention of a disease or condition.

The therapeutically effective amount or dosage of the compound can vary within wide limits. Such a dosage is adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg or more, a daily dosage of about 10 mg to about 10,000 mg, preferably from about 200 mg to about 1,000 mg, should be appropriate, although the upper limit may be exceeded. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, as a continuous infusion. Single dose compositions can contain such amounts or submultiples thereof of the compound or composition to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

In various embodiments, the use relates to a treatment of a disorder associated with PINK1 kinase activity in a mammalian subject. In some embodiments, the use is characterized in that the mammal is a human. In some embodiments, the use is characterized in that the disorder associated with PINK1 kinase activity is a neurodegenerative disease, a mitochondrial disease, fibrosis, and/or cardiomyopathy. In some embodiments, the disclosure relates to a method of treating a subject in need thereof, wherein the subject has been diagnosed or is suspected as having a disorder associated with PINK1 kinase activity. In some embodiments, the disclosure also relates to a method of treating a disease associated with PINK1 kinase activity in a subject in need thereof comprising administering to the subject a composition comprising a therapeutically effective amount of a compound disclosed herein, or the derivatives, pharmaceutically acceptable salts, or analogs thereof. In some embodiments, the disorder associated with PINK1 kinase activity is cancer associated with PINK1 kinase activity. In some embodiments, the disorder associated with PINK1 kinase activity is kidney disease, neurodegenerative disease, fibrosis or cardiomyopathy.

The disclosure also relates to a method of modulating PINK1 kinase activity in a cell or subject in need thereof comprising administering to the subject (or exposing the cell to) a composition comprising a therapeutically effective amount of a compound disclosed herein, or the derivatives, pharmaceutically acceptable salts, or analogs thereof. In some embodiments, the method further comprises allowing a time period sufficient for the PINK1 kinase activity to be activated or increased. In some embodiments, the time period sufficient to activate or increase PINK1 kinase activity is from about 5 to about 120 minutes after administration. In some embodiments, In some embodiments, the disclosure relates to a method of activating or increasing PINK1 kinase activity in a subject in need thereof without induction of cytochrome p450 relative to the cytochrome p450 of a subject who was not administered the disclosed compounds, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of the compounds, derivative, salts or analogs thereof; and a pharmaceutically acceptable carrier. In some embodiments, the CYP induction is less than about three times the CYP induction in a cell or in the subject at a concentration of about 10 micromolar of compound concentration. In some embodiments, the CYP inhibitions (e.g., the inhibition of CYPs 1A2, 2B6, 3A4) of the disclosed compounds are less than about 50% at a concentration of compound of about 5 micromolar (ie, IC50as between the compound and the >5 micromolar). In some embodiments, the time-dependent CYP inhibitions (e.g., the inhibition of CYPs following 30 minutes of compound incubation in a cell) of the disclosed compounds are less than about 50% at a concentration of compound at 3 μM compound concentration (i.e., IC50>3 μM). In some embodiments, the IC50of the compound or a metabolite thereof on CYP enzymes is greater than about 5, 4, or 3 mircomolar.

1. Treatment Methods

The compounds disclosed herein are useful for treating or controlling disorders associated with PINK1 kinase activity. Thus, provided is a method comprising administering a therapeutically effective amount of a composition comprising a disclosed compound to a subject.

Accordingly, in some embodiments, the present disclosure provides methods of treating or preventing a neurodegenerative disease (e.g., Parkinson's disease, Leigh's disease) in a subject comprising administering to the subject one or more compounds, or a pharmaceutically acceptable salt thereof, of any one of the compounds described herein or a pharmaceutical composition comprising one or more of the compounds described herein, or pharmaceutically acceptable salt thereof. In some embodiments, the treating of the neurodegenerative disease comprises ameliorating symptoms by stimulating PINK1 or a mutated PINK1.

In some embodiments, the present disclosure provides methods of treating or preventing a mitochondrial disease in a subject comprising administering to the subject one or more compounds, or a pharmaceutically acceptable salt thereof, of any one of the compounds described herein or a pharmaceutical composition comprising one or more of the compounds described herein, or pharmaceutically acceptable salt thereof. In some embodiments, the treating of the mitochondrial disease comprises ameliorating symptoms by stimulating PINK1 or a mutated PINK1.

In some embodiments, a method of treating one or more of the following mitochondrial diseases in a subject is provided: LHON, MELAS, and Charcot Marie Tooth.

In some embodiments, the present disclosure provides methods of treating or preventing cardiomyopathy in a subject comprising administering to the subject one or more compounds, or a pharmaceutically acceptable salt thereof, of any one of the compounds described herein or a pharmaceutical composition comprising one or more of the compounds described herein, or pharmaceutically acceptable salt thereof. In some embodiments, the treating of the cardiomyopathy comprises ameliorating symptoms by stimulating PINK1 or a mutated PINK1.

In some embodiments, the present disclosure provides methods of treating or preventing fibrosis in a subject comprising administering to the subject one or more compounds, or a pharmaceutically acceptable salt thereof, of any one of the compounds described herein or a pharmaceutical composition comprising one or more of the compounds described herein, or pharmaceutically acceptable salt thereof. In some embodiments, the treating of the fibrosis comprises ameliorating symptoms by stimulating PINK1 or a mutated PINK1.

In some embodiments, the present disclosure provides methods of treating or preventing a kidney disease in a subject comprising administering to the subject one or more compounds, or a pharmaceutically acceptable salt thereof, of any one of the compounds described herein or a pharmaceutical composition comprising one or more of the compounds described herein, or pharmaceutically acceptable salt thereof. In some embodiments, the treating of the kidney disease comprises ameliorating symptoms by stimulating PINK1 or a mutated PINK1.

In some embodiments, the present disclosure provides methods of treating or preventing a fibrotic disorder in a subject comprising administering to the subject one or more compounds, or a pharmaceutically acceptable salt thereof, of any one of the compounds described herein or a pharmaceutical composition comprising one or more of the compounds described herein, or pharmaceutically acceptable salt thereof. In some embodiments, the treating of the fibrotic disorder comprises ameliorating symptoms by stimulating PINK1 or a mutated PINK1.

In some embodiments, the present disclosure provides methods of treating or preventing a reperfusion injury in a subject comprising administering to the subject one or more compounds, or a pharmaceutically acceptable salt thereof, of any one of the compounds described herein or a pharmaceutical composition comprising one or more of the compounds described herein, or pharmaceutically acceptable salt thereof. In some embodiments, the treating of the reperfusion injury comprises ameliorating symptoms by stimulating PINK1 or a mutated PINK1.

In some embodiments, the method comprises administering to a subject one or more compounds described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds described herein, or pharmaceutically acceptable salt thereof. In some embodiments, the method comprises administering to a subject a compound or pharmaceutically acceptable salt thereof that acts as a PINK1 substrate with one or more compounds described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one or more compounds described herein, or pharmaceutically acceptable salt thereof. In some embodiments, the subject is a subject in need thereof.

a. Treating a Disorder Associated with PINK1 Activity

In some embodiments, compounds and compositions described herein are useful in treating a disorder associated with PINK1 function. Thus, provided herein are methods of treating a disorder associated with PINK1 function, comprising administering to a subject in need thereof, a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, or a composition comprising a disclosed compound or pharmaceutically acceptable salt thereof. Disorders treatable by the present compounds and compositions include, e.g., a neurodegenerative disease, a mitochondrial disease, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury.

In some embodiments, the disclosure relates to any of the above disclosed methods disclosed herein, wherein the administrating step comprises administering a pharmaceutical composition comprising: (i) a pharmaceutically effective amount of any of the disclosed compounds; and (ii) a pharmaceutically acceptable carrier.

Thus, in various embodiments, disclosed are methods of treating a disorder in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a disclosed compound, wherein the disorder is a neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, or a reperfusion injury.

In various embodiments, disclosed are methods of treating a disorder in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a compound having a structure represented by a formula:

In various embodiments, the compound has a structure represented by a formula:

In various embodiments, disclosed are methods of treating a disorder in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a compound selected from:

or a pharmaceutically acceptable salt thereof, wherein the disorder is a neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, or a reperfusion injury.

Cardiomyopathy refers to a disease condition that adversely affects cardiac cell tissue leading to a measurable deterioration in myocardial function (e.g., systolic function, diastolic function). Dilated cardiomyopathy is characterized by ventricular chamber enlargement with systolic dysfunction and no hypertrophy. Hypertrophic cardiomyopathy, is a genetic disease transmitted as an autosomal dominant trait. Hypertrophic cardiomyopathy is morphologically characterized by a hypertrophied and non-dialated left ventricle. Restrictive cardiomyopathy is characterized by nondialated nonhypertrophied morphology with diminished ventricular volume leading to poor ventricular filling. Arrhythmogenic right ventricular cardiomyopathy is an inheritable heart disease characterized by myocardial electric instability. Unclassified cardiomyopathy is a category for cardiomyopathies that do not match the features of any one of the other types. Unclassified cardiomyopathies may have features of multiple types or, for example, have the features of fibroelastosis, noncompacted myocardium, or systolic dysfunction with minimal dilatation.

Examples of kidney diseases that may be treated with a compound or composition described herein include chronic kidney disease (e.g., autosomal dominant polycystic kidney disease, diabetic nephropathy, hypertension-induced renal injury, crescentic glomerulonephritis, membranous nephropathy, membranous nephropathy, IgA nephropathy, amyloid A amyloidosis, secondary nephrotic syndrome) or acute kidney injury (AKI).

Examples of fibrotic disorders that may be treated with a compound or composition described herein include pulmonary fibrosis, liver fibrosis, heart fibrosis, mediastinal fibrosis, retroperitoneal cavity fibrosis, bone marrow fibrosis, skin fibrosis, scleroderma, pancreatic fibrillation, prostatic hyperplasia caused by fibrillation, and renal fibrosis.

Examples of reperfusion injuries that may be treated with a compound or composition described herein include reperfusion injuries induced by a mitochondrial disease (e.g., myocardial ischemia or stroke caused by Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes (MELAS)) and reperfusion injuries that are not induced by a mitochondrial disease (e.g., transplantation reperfusion, hepatic ischemia reperfusion, renal ischemia reperfusion, cerebral ischemia reperfusion).

In certain some embodiments, the compounds and compositions described herein can be used to treat Parkinson's disease by decreasing the production of Lewy bodies, decreasing the accumulation of alpha-synuclein, decreasing cell death, decreasing loss of dopamine-generating cells, decreasing loss of cells in the substantia nigra, decreasing loss of dopamine production, decreasing a symptom of Parkinson's disease, decreasing loss of motor function, decreasing shaking or slowing an increase in shaking (tremor), decreasing rigidity or an increase in rigidity, decreasing slowness (bradykinesia) of movement or a slowing of movement, decreasing sensory symptoms, decreasing insomnia, decreasing sleepiness, increasing mental wellbeing, increasing mental function, slowing the decrease of mental function, decreasing dementia, delaying the onset of dementia, improving cognitive skills, decreasing the loss of cognitive skills, improving memory, decreasing the degradation of memory, or extending survival. In certain some embodiments, the compounds and compositions described herein can be used to treat cardiomyopathy by increasing cardiac performance, improving exercise tolerance, preventing heart failure, increasing blood oxygen content, or improving respiratory function.

In certain some embodiments, the disease treated by a disclosed compound or composition is one that is characterized by a reduction in the level of PINK1. In certain some embodiments, the disease is one characterized by loss of dopamine-producing cells (e.g., Parkinson's disease). In certain some embodiments, the disease is one characterized by neurodegeneration. In certain some embodiments, the disease is one characterized by neural cell death. In certain some embodiments, the disease is one characterized by a reduction in the level of PINK1 activity. In certain some embodiments, the disease is Parkinson's disease. In some embodiments, the disease is a neurodegenerative disease. In certain some embodiments, the disease is a cardiomyopathy.

In further embodiments, the neurodegenerative disorder is Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis.

In further embodiments, the subject has been diagnosed with a need for treatment of a disorder associated with PINK1 kinase activity prior to the administering step.

In further embodiments, the subject is a mammal. In still further embodiments, the mammal is a human.

In further embodiments, the method further comprises the step of identifying a subject in need of treatment of a disorder associated with PINK1 kinase activity.

In various embodiments, the method further comprises administering an effective amount of an agent associated with the treatment of a neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, or a reperfusion injury.

Thus, in various embodiments, the method further comprises administering an agent known for the treatment of a neurodegenerative disorder. Examples of agents known for the treatment of neurodegenerative disorders include, but are not limited to, cholinesterase inhibitor, an antidepressant, memantine, rilutek, radicava, levodopa, carbidopa, a dopamine agonist, a MAO-B inhibitor, a catechol-O-methyltransferase inhibitor, an anticholinergic, spinraza, tetrabenazine, an antipsychotic agent, levetiracetam, clonazepam, an antipsychotic agent, a mood-stabilizing agent, and amantadine.

In various embodiments, the method further comprises administering an agent known for the treatment of a mitochondrial disease. Examples of agents known for the treatment of mitochondrial diseases include, but are not limited to, vitamins and supplements such as coenzyme Q10, B complex vitamins (e.g., thiamine (B1) and riboflavin (B2)), alpha lipoic acid, L-carnitine (Carnitor), creatine, and L-arginine.

In various embodiments, the method further comprises administering an agent known for the treatment of fibrosis such as, for example, idiopathic pulmonary fibrosis (IPF), non-alcoholic fatty liver disease (NASH), liver fibrosis, heart fibrosis, mediastinal fibrosis, bone marrow fibrosis, retroperitoneal cavity fibrosis, and renal fibrosis. Examples of agents known for the treatment of fibrosis include, but are not limited to, pirfenidone, nintedanib, a prostaglandin such as latanoprost and bimaotoprost, a beta blocker such as timolol and betaxolol, an alpha-adrenergic agonist such as apraclonidine and brimonidine, a carbonic anhydrase inhibitor such as dorzolamide and brinzolamide, a moitic or cholinergic agent such as pilocarpine, a diuretic, an angiotenisin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker, an anti-inflammatory agent, and an anti-fibrotic agent.

In various embodiments, the method further comprises administering an agent known for the treatment of cardiomyopathy. Examples of agents known for the treatment of cardiomyopathy include, but are not limited to, ACE inhibitors, angiotensin II receptor blockers, beta blockers, calcium channel blockers, digoxin, and antiarrhythmics. In various embodiments, the agent known for the treatment of cardiomyopathy is a medical device such as, for example, an implantable cardioverter-defibrillator (ICD), a ventricular assist device (VAD), or a pacemaker.

In various embodiments, the method further comprises administering an agent associated with the treatment of a kidney disease or a fibrotic disorder. Examples of agents associated with the treatment of a kidney disease or a fibrotic disorder include, but are not limited to an angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, Ramipril, trandolapril), an angiotensin II receptor blockers (e.g., azilsartan, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan), nintedanib, pirfenidone, autotaxin inhibitors, and peroxisome proliferator-activated receptor (PPAR) modulators (e.g., ADGE, EPI-001, INT-131, K-0533, S26948).

In various embodiments, the method further comprises administering an agent associated with the treatment of a reperfusion injury. Examples of agents associated with the treatment of a reperfusion injury include, but are not limited to, hydrogen sulfide, cyclosporine, TR040303, superoxide dismutase, metformin, elamipretide, and cannabinoids.

In some embodiments, the compound and the agent are administered simultaneously.

In some embodiments, the compound and the agent are administered sequentially.

2. Methods of Modulating PINK1 Kinase Activity in a Subject

In some embodiments, disclosed are methods of modulating PINK1 kinase activity in a subject, the method comprising the step of administering to the mammal a therapeutically effective amount of at least one disclosed compound, or a pharmaceutically acceptable salt thereof.

Thus, in various embodiments, disclosed are methods of modulating PINK1 kinase activity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a compound having a structure represented by a formula:

In various embodiments, the compound has a structure represented by a formula:

In various embodiments, disclosed are methods of modulating PINK1 kinase activity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a compound selected from:

or a pharmaceutically acceptable salt thereof.

As used herein, “modulation” can refer to either inhibition or enhancement of a specific activity. For example, the modulation of PINK1 activity can refer to the inhibition and/or activation of PINK1 dependent activities, such as a decrease in Parkin recruitment. In some embodiments, the modulation refers to the inhibition or activation of Parkin recruitment. In some embodiments, the compounds described herein activate PINK1 activity by a factor from about 1% to about 50%. The activity of PINK1 can be measured by any method including but not limited to the methods described herein.

The compounds described herein are neo-substrates of PINK1. The ability of the compounds to stimulate or inhibit PINK1 activity may be measured using any assay known in the art used to detect Parkin recruitment or PINK1 phosphorylation, or the absence of such signaling/activity. “PINK1 activity” refers to the ability of PINK1 to phosphorylate any substrate. Such activity can be measured, e.g., in a cell(s), by expressing mutant PINK1, administering the compounds disclosed herein and measuring the degree to which cells expressing the mutant PINK1 were able to phosphorylate an enzymatically active substrate as compared to a cell(s) expressing wild-type PINK1.

PINK1 activity can be measured by changes in the time necessary to recruit 50% of a substrate (“R50”). In some embodiments, the compounds reduce a R50 by a factor of about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%. In some embodiments, the compounds reduce a R50by a factor from about 1% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 2% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 3% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 4% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 5% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 6% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 7% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 8% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 9% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 10% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 15% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 20% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 25% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 30% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 35% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 40% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 45% to about 50%. In some embodiments, the compounds reduce a R50by a factor from about 10% to about 40%. In some embodiments, the compounds reduce a R50by a factor from about 10% to about 30%. In some embodiments, the compounds reduce a R50by a factor from about 10% to about 20%.

Plasmids expressing PINK1 can be transfected into an isolated cell and expressed in an isolated cell, expressed in a membrane derived from a cell, expressed in tissue or in an animal. For example, neuronal cells, cells of the immune system, transformed cells, or membranes can be used to test the PINK1 activity described above. Modulation is tested using one of the in vitro or in vivo assays described herein. Other assays generally known can also be used to test the compounds. Signal transduction can also be examined in vitro with soluble or solid state reactions, using a chimeric molecule such as an extracellular domain of a receptor covalently linked to a heterologous signal transduction domain, or a heterologous extracellular domain covalently linked to the transmembrane and or cytoplasmic domain of a receptor. Furthermore, ligand-binding domains of the protein of interest can be used in vitro in soluble or solid state reactions to assay for ligand binding.

In some embodiments, a compound's effect on the modulation of PINK1 will be measured using cells expressing mutant and wild-type verisons of PINK1. PINK1 is generally known. In some embodiments, the enzymatic rescue is measured. Enzymatic rescue experiments are experiments in which cells expressing mutated forms of the PINK1 with reduced or deficient enzymatic activity are contacted with compounds of the present invention and are able to re-activate the mutated PINK1 enzymatic activity. PINK1 molecules are known. In some embodiments, the compounds of the present invention are able to enzymatically rescue human PINK1 (accession number NM_032409.3, which is incorporated by reference in its entirety) having the following amino acid sequence:

In some embodiment, the compounds of the present invention are able to enzymatically rescue mouse PINK1 (accession number XM_924521, which is incorporated by reference in its entirety) having the following amino acid sequence:

In some embodiments, the compounds of the present invention are able to enzymatically rescue rat PINK1 (accession number XM_216565, which is incorporated by reference in its entirety) having the following amino acid sequence:

In further embodiments, modulating is inhibiting. In still further embodiments, modulating is decreasing.

In further embodiments, the compound exhibits inhibition of PINK1 kinase activity with an IC50of less than about 30 μM. In still further embodiments, the compound exhibits inhibition of PINK1 kinase activity with an IC50of less than about 25 μM. In yet further embodiments, the compound exhibits inhibition of PINK1 kinase activity with an IC50of less than about 20 μM. In an even further embodiment, the compound exhibits inhibition of PINK1 kinase activity with an IC50of less than about 15 μM. In still further embodiments, the compound exhibits inhibition of PINK1 kinase activity with an IC50of less than about 10 μM. In yet further embodiments, the compound exhibits inhibition of PINK1 kinase activity with an IC50of less than about 5 μM. In an even further embodiment, the compound exhibits inhibition of PINK1 kinase activity with an IC50of less than about 1 μM. In still further embodiments, the compound exhibits inhibition of PINK1 kinase activity with an IC50of less than about 0.5 μM.

In further embodiments, the subject is a mammal. In still further embodiments, the subject is a human.

In further embodiments, the subject has been diagnosed with a need for treatment of an disorder associated with PINK1 kinase dysfunction prior to the administering step. In still further embodiments, the method further comprises the step of identifying a subject at risk of becoming infected with a disorder associated with PINK1 kinase dysfunction prior to treatment of the disorder.

3. Methods of Modulating PINK1 Kinase Activity in at Least One Cell

In some embodiments, disclosed are methods for modulating PINK1 kinase activity in at least one cell, the method comprising the step of contacting the at least one cell with an effective amount of at least one disclosed compound, or a pharmaceutically acceptable salt thereof.

Thus, in various embodiments, disclosed are methods of modulating PINK1 kinase activity in at least one cell, the method comprising contacting the cell with an effective amount of a compound having a structure represented by a formula:

In various embodiments, the compound has a structure represented by a formula:

In various embodiments, disclosed are methods of modulating PINK1 kinase activity in at least one cell, the method comprising contacting the cell with an effective amount of a compound selected from:

or a pharmaceutically acceptable salt thereof.

In further embodiments, the cell is mammalian. In still further embodiments, the cell is human. In yet further embodiments, the cell has been isolated from a mammal prior to the contacting step.

In further embodiments, modulating is inhibiting. In still further embodiments, modulating is decreasing.

In further embodiments, contacting is via administration to a mammal.

In further embodiments, the step of contacting is performed in vitro.

4. Use of Compounds

Also provided herein is the use of a compound described herein, or a pharmaceutically acceptable salt thereof, or a composition comprising a disclosed compound or pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating a disorder described herein. Also provided is a compound described herein, or a pharmaceutically acceptable salt thereof, or a composition comprising a disclosed compound or pharmaceutically acceptable salt thereof, for use in treating a disorder described herein.

Thus, in some embodiments, the disclosure relates to the use of a disclosed compound or a product of a disclosed method. In further embodiments, a use relates to the manufacture of a medicament for the treatment of a disorder associated with PINK1 kinase activity in a mammal.

Also provided are the uses of the disclosed compounds and products. In some embodiments, the disclosure relates to use of at least one disclosed compound; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof. In further embodiments, the compound used is a product of a disclosed method of making.

In further embodiments, the use relates to a process for preparing a pharmaceutical composition comprising a therapeutically effective amount of a disclosed compound or a product of a disclosed method of making, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, for use as a medicament.

In further embodiments, the use relates to a process for preparing a pharmaceutical composition comprising a therapeutically effective amount of a disclosed compound or a product of a disclosed method of making, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, wherein a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of the compound or the product of a disclosed method of making.

In various embodiments, the use relates to a treatment of a disorder associated with PINK1 kinase activity in a mammal. In some embodiments, the use is characterized in that the mammal is a human. In some embodiments, the use is characterized in that the disorder associated with PINK1 kinase activity is a neurodegenerative disease, a mitochondrial disease, fibrosis, and/or cardiomyopathy.

In further embodiments, the use relates to the manufacture of a medicament for the treatment of a disorder associated with PINK1 kinase activity in a mammal.

It is understood that the disclosed uses can be employed in connection with the disclosed compounds, products of disclosed methods of making, methods, compositions, and kits. In further embodiments, the disclosure relates to the use of a disclosed compound or a disclosed product in the manufacture of a medicament for the treatment of a disorder associated with PINK1 kinase activity in a mammal.

5. Manufacture of a Medicament

In some embodiments, the disclosure relates to a method for the manufacture of a medicament for treating a disorder associated with PINK1 kinase activity in a mammal, the method comprising combining a therapeutically effective amount of a disclosed compound or product of a disclosed method with a pharmaceutically acceptable carrier or diluent.

As regards these applications, the present method includes the administration to an animal, particularly a mammal, and more particularly a human, of a therapeutically effective amount of the compound effective in treatment of a disorder associated with PINK1 kinase activity. The dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the animal over a reasonable timeframe. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition of the animal and the body weight of the animal.

The total amount of the compound of the present disclosure administered in a typical treatment is preferably between about 1 mg/kg and about 100 mg/kg of body weight for mice, and between about 10 mg/kg and about 50 mg/kg of body weight, and more preferably between 20 mg/kg and about 40 mg/kg of body weight for humans per daily dose. This total amount is typically, but not necessarily, administered as a series of smaller doses over a period of about one time per day to about three times per day for about 24 months, and preferably over a period of twice per day for about 12 months.

The size of the dose also will be determined by the route, timing and frequency of administration as well as the existence, nature and extent of any adverse side effects that might accompany the administration of the compound and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states, in particular chronic conditions or disease states, may require prolonged treatment involving multiple administrations.

The additional medicament can be administered in co-therapy (including co-formulation) with the one or more of the compounds described herein.

In some embodiments, the response of the disease or disorder to the treatment is monitored and the treatment regimen is adjusted if necessary in light of such monitoring.

Frequency of administration is typically such that the dosing interval, for example, the period of time between one dose and the next, during waking hours is from about 2 to about 12 hours, from about 3 to about 8 hours, or from about 4 to about 6 hours. It will be understood by those of skill in the art that an appropriate dosing interval is dependent to some degree on the length of time for which the selected composition is capable of maintaining a concentration of the compound(s) in the subject and/or in the target tissue (e.g., above the EC50(the minimum concentration of the compound which modulates the receptor's activity by 90%). Ideally the concentration remains above the EC50for at least 100% of the dosing interval. Where this is not achievable it is desired that the concentration should remain above 5% of the EC50, above 10% of the EC50, above 25% of the EC50, or above 50% of the EC50for the dosing period.

Thus, in some embodiments, the disclosure relates to the manufacture of a medicament comprising combining a disclosed compound or a product of a disclosed method of making, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, with a pharmaceutically acceptable carrier or diluent.

In some embodiments, disclosed are kits comprising a disclosed compound and one or more selected from: (a) at least one agent known for the treatment of one or more disorders selected from neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury; (b) instructions for administering the compound in connection with treating one or more disorders selected from a neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury; and (c) instructions for treating one or more disorders selected from a neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury.

Thus, in various embodiments, disclosed are kits comprising a compound having a structure represented by a formula:

wherein m is 0 or 1; wherein each of Q1and Q2is independently N or CH; wherein Q3is CH2or NH; wherein Z is CR11aR11b, NR12, or O; wherein each of R11aand R11b, when present, is independently selected from hydrogen, halogen, —OH, and C1-C4 alkoxy, or wherein each of R11aand R11b, when present, together comprise ═O; wherein R12, when present, is hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl, or —(C1-C4 alkyl)(C3-C6 cycloalkyl); wherein each of R1a, R1b, R1c, and R1dis independently selected from hydrogen, halogen, —CN, —NH2, —OH, —NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; wherein R2is selected from hydrogen, C5-C8 cyanoalkyl, —O—(C1-C8 alkyl)-R13, —O—(C1-C8 haloalkyl)-R13, —O—(C1-C8 alkyl substituted with a C3-C5 cycloalkyl)-R13, and —(C1-C8 alkyl)-O—(C1-C4 alkyl); wherein R13, when present, is selected from —CN, —OH, —NH2, C1-C4 haloalkyl, C1-C4 alkoxy, and C1-C4 haloalkoxy; wherein R3is a 3- to 6-membered cycloalkyl, a C1-C6 haloalkyl, C1-C6 haloalkoxy, or C1-C6 halohydroxyalkyl; wherein R4is selected from hydrogen and C1-C4 alkyl; and wherein each of R5aand R5b, when present, is hydrogen, or wherein one of R5aand R5b, when present, is C1-C4 alkyl and one of R5aand R5b, when present, is —(C1-C8 alkyl)-O—(C1-C4 alkyl), or wherein R5aand R5b, when present, are covalently bonded, and, together with the intermediate atoms, comprise a C3-C6 cycloalkyl, provided that when m is 1 and each of R5aand R5bis hydrogen, then R2is C5-C8 cyanoalkyl, —O—(C1-C8 alkyl)-R13, —O—(C1-C8 haloalkyl)-R13, —O—(C1-C8 alkyl substituted with a C3-C5 cycloalkyl)-R13, or —(C1-C8 alkyl)-O—(C1-C4 alkyl), and provided that when m is 1 and R5aand R5btogether comprise a C3-C6 cycloalkyl, then R2is hydrogen, or a pharmaceutically acceptable salt thereof, and one or more selected from: (a) at least one agent known for the treatment of one or more disorders selected from neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury; (b) instructions for administering the compound in connection with treating one or more disorders selected from a neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury; and (c) instructions for treating one or more disorders selected from a neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury.

In various embodiments, the compound has a structure represented by a formula:

In various embodiments, disclosed are kits comprising a compound selected from:

or a pharmaceutically acceptable salt thereof, and one or more selected from: (a) at least one agent known for the treatment of one or more disorders selected from neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury; (b) instructions for administering the compound in connection with treating one or more disorders selected from a neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury; and (c) instructions for treating one or more disorders selected from a neurodegenerative disorder, a mitochondrial disorder, a fibrosis, cardiomyopathy, a kidney disease, a fibrotic disorder, and a reperfusion injury.

In further embodiments, the agent is known for the treatment of a neurodegenerative disorder. Examples of agents known for the treatment of neurodegenerative disorders include, but are not limited to, cholinesterase inhibitor, an antidepressant, memantine, rilutek, radicava, levodopa, carbidopa, a dopamine agonist, a MAO-B inhibitor, a catechol-O-methyltransferase inhibitor, an anticholinergic, spinraza, tetrabenazine, an antipsychotic agent, levetiracetam, clonazepam, an antipsychotic agent, a mood-stabilizing agent, and amantadine.

In further embodiments, the agent is known for the treatment of a mitochondrial disease. Examples of agents known for the treatment of mitochondrial diseases include, but are not limited to, vitamins and supplements such as coenzyme Q10, B complex vitamins (e.g., thiamine (B1) and riboflavin (B2)), alpha lipoic acid, L-carnitine (Carnitor), creatine, and L-arginine.

In further embodiments, the agent is known for the treatment of fibrosis such as, for example, idiopathic pulmonary fibrosis (IPF), non-alcoholic fatty liver disease (NASH), liver fibrosis, heart fibrosis, mediastinal fibrosis, bone marrow fibrosis, retroperitoneal cavity fibrosis, and renal fibrosis. Examples of agents known for the treatment of fibrosis include, but are not limited to, pirfenidone, nintedanib, a prostaglandin such as latanoprost and bimaotoprost, a beta blocker such as timolol and betaxolol, an alpha-adrenergic agonist such as apraclonidine and brimonidine, a carbonic anhydrase inhibitor such as dorzolamide and brinzolamide, a moitic or cholinergic agent such as pilocarpine, a diuretic, an angiotenisin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker, an anti-inflammatory agent, and an anti-fibrotic agent.

In further embodiments, the agent is known for the treatment of cardiomyopathy. Examples of agents known for the treatment of cardiomyopathy include, but are not limited to, ACE inhibitors, angiotensin II receptor blockers, beta blockers, calcium channel blockers, digoxin, and antiarrhythmics. In various embodiments, the agent known for the treatment of cardiomyopathy is a medical device such as, for example, an implantable cardioverter-defibrillator (ICD), a ventricular assist device (VAD), or a pacemaker.

In further embodiments, the agent is known for the treatment of a kidney disease or a fibrotic disorder. Examples of agents known for the treatment of a kidney disease or a fibrotic disorder include, but are not limited to an angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, Ramipril, trandolapril), an angiotensin II receptor blockers (e.g., azilsartan, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan), nintedanib, pirfenidone, autotaxin inhibitors, and peroxisome proliferator-activated receptor (PPAR) modulators (e.g., ADGE, EPI-001, INT-131, K-0533, S26948, ASP1128).

In further embodiments, the agent is known for the treatment of a reperfusion injury. Examples of agents known for the treatment of a reperfusion injury include, but are not limited to, hydrogen sulfide, cyclosporine, TR040303, superoxide dismutase, metformin, elamipretide, and cannabinoids.

In further embodiments, the at least one compound and the at least one agent are co-formulated. In further embodiments, the at least one compound and the at least one agent are co-packaged.

In further embodiments, the compound and the agent are administered sequentially. In still further embodiments, the compound and the agent are administered simultaneously.

The kits can also comprise compounds and/or products co-packaged, co-formulated, and/or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and/or product and another component for delivery to a patient.

It is understood that the disclosed kits can be prepared from the disclosed compounds, products, and pharmaceutical compositions. It is also understood that the disclosed kits can be employed in connection with the disclosed methods of using.

The foregoing description illustrates and describes the disclosure. Additionally, the disclosure shows and describes only the preferred embodiments but, as mentioned above, it is to be understood that it is capable to use in various other combinations, modifications, and environments and is capable of changes or modifications within the scope of the disclosure concepts as expressed herein, commensurate with the above teachings and/or the skill or knowledge of the relevant art. The embodiments described herein above are further intended to explain best modes known by applicant and to enable others skilled in the art to utilize the disclosure in such, or other, embodiments and with the various modifications required by the particular applications or uses thereof. Accordingly, the description is not intended to limit the disclosure to the form disclosed herein. Also, it is intended to the appended claims be construed to include alternative embodiments.

Representative examples of the disclosed compounds are illustrated in the following non-limiting methods, schemes, and examples.

1. General Experimental Method

All temperatures are in degrees Celsius (° C.) and are uncorrected. Reagent grade chemicals and anhydrous solvent were purchased from commercial sources and unless otherwise mentioned, were used without further purification. The names of the products were determined using the naming software included in Biovia electronic lab notebook. Silica gel chromatography was performed on Teledyne Isco instruments using pre-packaged disposable SiO2 stationary phase columns with eluent flow rate range of 15 to 200 m/min, UV detection (254 and 280 nm). Reverse phase preparative HPLC was carried out using C18 columns, UV detection (214 and 254 nm) eluting with gradients of MeCN in H2O (0.03% (NH4)2CO3/0.375% NH4OH, high pH) or MeCN in H2O (0.1% HCOOH, low pH). The analytical HPLC chromatograms were performed using an Agilent 1100 series instrument with DAD detector (190 nm to 300 nm). The mass spectra were recorded with a Waters Micromass ZQ detector at 130° C. The mass spectrometer was equipped with an electrospray ion source (ESI) operated in a positive ion mode and was set to scan between m/z 150-750 with a scan time of 0.3 s. Products and intermediates were analyzed by HPLC/MS on a Gemini-NX (5 m, 2.0×30 mm) using a high pH buffer gradient of 5% to 100% of MeCN in H2O (0.03% (NH4)2CO3/0.375% NH4OH) over 2.5 min at 1.8 m/min for a 3.5 min run (B05) and EVO C18 (5 m, 3.0×50 mm) using a low pH buffer gradient of 5% to 100% of MeCN in H2O (0.1% HCOOH) over 2.5 min at 2.2 m/min for a 3.5 min run (A05). The 1H NMR spectra were recorded on a Bruker UltraShield 500 MHz/54 mm instrument (BZH 43/500/70B, D221/54-3209). The chemical shifts are referenced to solvent peaks, which in 1H NMR appear at 7.26 ppm for CDCl3, 2.50 for DMSO-d6, and 3.31 ppm for CD3OD.

2. Analytical Methods

Analytical Methods were performed using Discovery L3 instruments as detailed in Table 1 below.

a. Preparation of ethyl 2-(4-hydroxychroman-3-yl)acetate

To a solution of ethyl 2-(4-oxochroman-3-yl)acetate (3.50 g, 14.94 mmol) in EtOH (45.0 mL) was added NaBH4(170 mg, 0.3 mmol) at −10° C. The mixture was stirred at −10° C. for 45 minutes. The reaction mixture was poured into water and extracted with EtOAc (100 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (0-20% EA in PE) to afford the title product (2.00 g, 56.7%) as a colorless oil. m/z: (ES+): [M-OH]+: 219.2; (A05) tR=1.66 min.

b. Preparation of ethyl 2-(4-((tert-butyldimethylsilyl)oxy)chroman-3-yl)acetate

To a solution of ethyl 2-(4-hydroxychroman-3-yl)acetate (2.00 g, 8.46 mmol) in DMF (30.0 mL) was added imidazole (1.70 g, 25.39 mmol) and stirred at room temperature for 5 minutes. TBSCl (1.90 g, 12.7 mmol) was slowly added to the mixture and stirred for another 40 minutes. The reaction mixture was poured into water then extracted with EtOAc (80.0 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (0-10% EA in PE) to afford product (2.50 g, 84.2%) as a colorless oil. m/z: (ES+): [M-OTBS]+: 219.2; (A05) tR=2.51 min.

c. Preparation of 2-(4-((tert-butyldimethylsilyl)oxy)chroman-3-yl)ethan-1-ol

To a solution of ethyl 2-(4-((tert-butyldimethylsilyl)oxy)chroman-3-yl)acetate (2.50 g, 7.13 mmol) in dry THF (30.0 mL) was added LiAlH4(540 mg, 14.26 mmol) at 0° C. The mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into water and extracted with EtOAc (80.0 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product (2.20 g, quant) as a colorless oil, which was directly used for the next step without further purification. m/z: (ES+): [M-OTBS]+: 177.2; (A05) tR=2.20 min.

To a solution of 2-(4-((tert-butyldimethylsilyl)oxy)chroman-3-yl)ethan-1-ol (2.20 g, crude) in dry DCM (25.0 mL) was added Et3N (2.10 g, 21.40 mmol) at 0° C. The solution was stirred at 0° C. for 5 minutes. MsCl (1.20 g, 10.70 mmol) was slowly added to the mixture and stirred at 0° C. for another 40 minutes. The reaction mixture was poured into water and extracted with EtOAc (80.0 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product (2.20 g) as a yellow oil, which was directly used for the next step without further purification. m/z: (ES+): [M-OTBS-OMs]+: 159.1; (A05) tR=2.25 min.

e. Preparation of tert-butyl((3-(2-iodoethyl)chroman-4-yl)oxy)dimethylsilane

To a solution of 2-(4-((tert-butyldimethylsilyl)oxy)chroman-3-yl)ethyl methanesulfonate (2.20 g) in acetone (20.0 mL) was added NaI (2.60 g, 17.83 mmol). The mixture was stirred at 50° C. overnight. The reaction mixture was poured into water and extracted with EtOAc (80.0 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (0-5% EA in PE) to afford product (2.10 g, 73.7%) as a light yellow oil. m/z: (ES+): [M-OTBS]+: 287.1; (A05) tR=2.77 min.

f. Preparation of 4-(4-((tert-butyldimethylsilyl)oxy)chroman-3-yl)-2,2-dimethylbutanenitrile

To a solution of isobutyronitrile (694 mg, 10.04 mmol) in dry THF (10.0 mL) was added LDA (2M in THF, 5.00 mL, 10.0 mmol) dropwise at −78° C. After 1 hour, a solution of tert-butyl((3-(2-iodoethyl)chroman-4-yl)oxy)dimethylsilane (2.10 g, 5.02 mmol) in THF (15.0 mL) was slowly added to the mixture and stirred at −78° C. for another 30 minutes. The reaction mixture was poured into NH4Cl (sat. aq., 100 mL) and extracted with EtOAc (70.0 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (0-10% EA in PE) to afford product (1.60 g, 88.6%) as a yellow solid. m/z: (ES+): [M-OTBS]+: 228.2; (A05) tR=2.45 min.

g. Preparation of 4-(4-hydroxychroman-3-yl)-2,2-dimethylbutanenitrile

To a solution of 4-(4-((tert-butyldimethylsilyl)oxy)chroman-3-yl)-2,2-dimethylbutanenitrile (1.60 g, 4.46 mmol) in THF (20.0 mL) was added TBAF (5.00 mL). The solution was stirred at room temperature for 40 minutes. The reaction mixture was poured into water and extracted with EtOAc (70.0 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product (1.40 g, quant) as a light yellow oil, which was directly used for the next step without further purification. m/z: (ES+): [M+H]+: 246.3; (A05) tR=1.69 min.

h. Preparation of N-(3-(3-cyano-3-methylbutyl)chroman-4-yl)acetamide

A solution of 4-(4-hydroxychroman-3-yl)-2,2-dimethylbutanenitrile (1.40 g, crude) in MeCN (12.0 mL) was added Con. H2SO4(4.00 mL). The solution was stirred at 40° C. for 2 hours. The reaction mixture was poured into NaHCO3(sat., aq., 100 mL) and extracted with EtOAc (60.0 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product (1.50 g) as a yellow oil, which was directly used for the next step without further purification. m/z: (ES+): [M+H]+: 287.3; (A05) tR=1.66 min.

To a solution of the crude product N-(3-(3-cyano-3-methylbutyl)chroman-4-yl)acetamide (1.50 g, crude) in THF (15.0 mL) was added DMAP (123 mg, 1.01 mmol) followed by (Boc)2O (3.30 g, 15.06 mmol). The mixture was heated to 50° C. and stirred for 16 hours. The reaction mixture was directly used for the next step without further purification. m/z: (ES+): [M-BOC]+: 287.3; (A05) tR=2.09 min.

To the previous reaction mixture was added MeOH (15.0 mL) followed by K2CO3(1.40 g, 10.04 mmol) at room temperature. The reaction mixture was stirred for 4 hours. The reaction mixture was poured into water and extracted with EtOAc (70.0 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (0-5% MeOH in DCM) to afford product (1.45 g, 94.6%) as a pale-yellow solid. m/z: (ES+): [M-tBu]+: 289.3; (A05) tR=2.03 min.

k. Preparation of 4-(4-aminochroman-3-yl)-2,2-dimethylbutanenitrile

To a solution of tert-butyl (3-(3-cyano-3-methylbutyl)chroman-4-yl)carbamate (1.50 g, 4.21 mmol) in DCM (15.0 mL) was added TFA (5.00 mL). The solution was stirred at room temperature for 2 hours. The reaction mixture was poured into water and extracted with EtOAc (70.0 mL×3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by prep-HPLC (ACN-H2O (0.05% NH4OH)) to afford product as two separated diastereomers.

a. Preparation of N-[(3R,4,S)-3-(2-methoxyethoxy)chroman-4-yl]-1,1-diphenyl-methanimine

b. Preparation of (3R,4,S)-3-(2-methoxyethoxy)chroman-4-amine

a. Preparation of (3R,4S)-4-(benzhydrylideneamino)chroman-3-ol

KOH (0.612 g, 0.0109 mol) was dissolved into H2O (5.00 mL) and the solution was added to a mixture of (3R,4R)-3-bromochroman-4-ol (1.00 g, 4.37 mmol) in THF (10.0 mL). The resulting solution was stirred for 2 h at 22° C. H2O (20.0 mL) was added and the layers were separated. The aqueous phase was extracted with DCM (3×20.0 mL). The organic layers were combined, dried (Na2SO4), filtered, and concentrated under reduced pressure to afford crude epoxide. The previous epoxide was mixed with diphenylmethanimine (1.42 g, 7.86 mmol) and heated to 95° C. for 6 h. The mixture was cooled to 20° C. and purified by silica gel chromatography (40 g cartridge) with hexane and EtOAc (0-20%) to provide the title compound (1.02 g, 67.4%) as a solid. m/z: (ES+): [M+H]+: 330.24; (A05) tR=2.48 min.

b. Preparation of N-[(3R,4S)-3-(3-methoxypropoxy)chroman-4-yl]-1,1-diphenyl-methanimine

c. Preparation of (3R,4S)-3-(3-methoxypropoxy)chroman-4-amine

a. Preparation of [(3R,4R)-3-bromochroman-4-yl](2R)-2-phenylpropanoate

NaBH4(11.2 g, 297 mmol) was added to a mixture of chroman-4-one (40.0 g, 270 mmol) in dry MeOH (500 mL) at 0° C. under air. The mixture was warmed to 22° C. and stirred for 1.5 h. 1 M HCl (200 mL) and brine (200 mL) were added to the mixture, and the aqueous phase was extracted with ethyl acetate (4×200 mL). The combined organic phases were washed with brine (300 mL), dried (MgSO4), filtered, and concentrated under reduced pressure to provide the chromanol intermediate, which was used immediately in the next step. m/z: (ES+): [M−H2O]+=133.21; LCMS (A05); tR=1.83 m.

NBS (41.6 g, 234 mmol) was added to a mixture of 2H-chromene (29.4 g, 222 mmol), in THF (200 mL), and H2O (100 mL) at 0° C. under nitrogen. The mixture warmed to 22° C., and was stirred for 45 min. The mixture was diluted with water (200 mL) and ethyl acetate (200 mL), and the aqueous phase extracted with ethyl acetate (200 mL). The combined organic phases were washed with brine (200 mL), dried (MgSO4), filtered, and concentrated under reduced pressure to provide 3-bromochroman-4-ol intermediate.

DMAP (534 mg, 4.37 mmol) was added to a mixture of rac-(trans)-3-bromochroman-4-ol (910 mg, 3.97 mmol), (2R)-2-phenylpropanoic acid (656 mg, 4.37 mmol) and DCC (902 mg, 4.37 mmol) in DCM (20.0 mL) at 22° C. under nitrogen. The mixture was stirred at 22° C. for 3 h, filtered over celite, and the filtrate was concentrated. The residue was purified by silica gel chromatography (40 g cartridge) with hexanes and diethyl ether (0-4%) to provide the separated diastereomers as oils (889 mg, 62%).

b. Preparation of (3R,4S)-4-(benzhydrylideneamino)chroman-3-ol

NaOMe (1.57 g, 29.1 mmol) was added to a solution of [(3R,4R)-3-bromochroman-4-yl](2R)-2-phenylpropanoate (3.00 g, 8.30 mmol) in dry Et2O (25.0 mL), and the mixture was stirred for 2 h at 22° C. H2O (30.0 mL) was added and the layers were separated. The aqueous phase was extracted with DCM (3×20.0 mL). The organic layers were combined, dried (Na2SO4), filtered, and concentrated under reduced pressure to afford crude epoxide.

The previous epoxide was mixed with diphenylmethanimine (3.01 g, 16.6 mmol) and heated to 95° C. for 6 h. The mixture was cooled to 20° C. and purified by silica gel chromatography (120 g cartridge) with hexane and EtOAc (0-20%) to provide the title compound (70% pure, 2.90 g, 74%) as a solid. m/z: (ES+): [M+H]+: 330.15; (A05); tR=2.55 m.

c. Preparation of N-[(3R,4S)-3-(2-methoxyethoxy)chroman-4-yl]-1,1-diphenyl-methanimine

To a solution of (3R,4S)-4-(benzhydrylideneamino)chroman-3-ol (1.00 g, 3.04 mmol) in THF (40.0 mL) in pressure tube at 0° C. was added NaH (60%, 128 mg, 3.34 mmol). The mixture was stirred at room temperature for 1 h then, 1-bromo-2-methoxy-ethane (422 mg, 3.04 mmol) was added. The tube was sealed and the reaction was stirred at 70° C. for 16 h and cooled down at room temperature. Water (40.0 mL) was added and the reaction was extracted with DCM (2×40.0 mL). The combined organics were washed with brine, dried (Na2SO4) and concentrated under reduced pressure. The residue was purified by silica gel chromatography (80 g cartridge) with EtOAc and Hexanes (0-20%) to afford the title product (95% pure, 105 mg, 8%) as an oil. m/z: (ES+): [M+H]+: 388.21; (A05); tR=2.69 m.

d. Preparation of (3R,4S)-3-(2-methoxyethoxy)chroman-4-amine

a. Preparation of (3R,4S)-4-aminochroman-3-ol

NaOMe (1.67 g, 30.9 mmol) was added to a solution of [(3R,4R)-3-bromochroman-4-yl] (2R)-2-phenylpropanoate (2.23 g, 6.17 mmol) in dry Et2O (20.0 mL), and the mixture was stirred for 16 h at 22° C. H2O (30.0 mL) was added and the layers were separated. The aqueous phase was extracted with DCM (3×30.0 mL). The organic layers were combined, dried (Na2SO4), filtered, and concentrated under reduced pressure to afford crude epoxide.

b. Preparation of (3R,4S)-4-[[6-cyclopropyl-7-(2-trimethylsilylethoxymethyl)pyrrolo[2,3-d]pyrimidin-4-yl]amino]chroman-3-ol

c. Preparation of 3-[(3S,4R)-4-[[6-cyclopropyl-7-(2-trimethylsilylethoxymethyl)pyrrolo[2,3-d]pyrimidin-4-yl]amino]chroman-3-yl]oxy-2,2-dimethyl-propanenitrile

To a solution of (3R,4S)-4-[[6-cyclopropyl-7-(2-trimethylsilylethoxymethyl)pyrrolo[2,3-d]pyrimidin-4-yl]amino]chroman-3-ol (280 mg, 0.619 mmol) in dry DMF (4.00 mL) was added NaH (71.1 mg, 1.86 mmol). The mixture was stirred at room temperature for 15 min before 3-bromo-2,2-dimethyl-propanenitrile (200 mg, 1.24 mmol) was added and the mixture was stirred at 80° C. for 16 h. Water (15.0 mL) was added and the reaction was extracted with EtOAc (3×15.0 mL). The combined organic layers were washed with brine (15.0 mL), dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (24 g cartridge) eluting with DCM and MeOH (0-5%) to afford the title compound (284 mg, 86%) as a solid. m/z: (ES+): [M+H]+: 534.35; (A05) tR=2.92 min.

a. Preparation of ethyl 2-[(3R,4S)-4-(benzhydrylideneamino)chroman-3-yl]oxyacetate

NaH (60% dispersion in mineral oil, 143 mg, 3.73 mmol) was added to a stirring solution of (3R,4S)-4-(benzhydrylideneamino)chroman-3-ol (615 mg, 1.87 mmol) in THF (8.39 mL) at 0° C. under nitrogen atmosphere. The mixture was warmed to room temperature, stirred for 30 min and cooled to 0° C. Ethyl 2-bromoacetate (248 μL, 2.24 mmol) was added and the mixture was warmed to room temperature and stirred for 16 h. The mixture was concentrated under reduced pressure and diluted with water (25.0 mL) and extracted with EtOAc (3×20.0 mL). The combined organic phases were washed with brine (30.0 mL), dried (Na2SO4), filtered and concentrated. The residue was purified by silica chromatography (40 g cartridge) eluting with hexanes and EtOAc (0-35%) to provide the title compound as an oil (319 mg, 41%). m/z: (ES+) [M+H]+=416.61; (A05); tR=2.71 m.

b. Preparation of 1-[(3R,4,S)-4-(benzhydrylideneamino)chroman-3-yl]oxy-2-methyl-propan-2-ol

MeMgBr (3.00 M in Et2O, 0.512 mL, 1.54 mmol) was added to a stirring solution of ethyl 2-[(3R,4S)-4-(benzhydrylideneamino)chroman-3-yl]oxyacetate (319 mg, 0.000768 mol) in THF (3.13 mL) at −78° C. under N2. The mixture was warmed to room temperature, stirred for 1 h, and then concentrated under reduced pressure. The residue was diluted with water (10.0 mL) and extracted with EtOAc (3×15.0 mL). The combined organics were washed with brine (15.0 mL), dried (Na2SO4), filtered, and concentrated. The residue was purified by silica chromatography (24 g cartridge) with hexanes and EtOAc (0-30%) to provide the title compound as an oil (275 mg, 89%). m/z: (ES+) [M+H]+=402.70; (A05); tR=2.59 m.

c. Preparation of 1-[(3R,4S)-4-amino-7-fluoro-chroman-3-yl]oxy-2-methyl-propan-2-ol

To a solution of 1-[(3R,4S)-4-(benzhydrylideneamino)chroman-3-yl]oxy-2-methyl-propan-2-ol (275 mg, 0.685 mmol) in DCM (2.87 mL) was added HCl (3 M aq, 0.685 mL). The residue was stirred at room temperature for 16 h. The solvent was removed at reduced pressure and 3N HCl (2.00 mL) was added. The acidic solution was extracted with ether (2.00 mL) and the aqueous layer was basified with 3N NaOH until pH 13. The basic aqueous layer was extracted with ethyl acetate (3×2.00 mL), the combined organic phases were washed with brine (2.00 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure to afford the title compound as an oil (121 mg, 74%). The product was used in the next step without further purification. m/z: (ES+) [M-NH2—OH]+=203.09; (A05); tR=1.63 m.

To a solution of (3R,4S)-4-[[6-cyclopropyl-7-(2-trimethylsilylethoxymethyl)pyrrolo[2,3-d]pyrimidin-4-yl]amino]chroman-3-ol (200 mg, 0.442 mmol) in dry DMF (3.00 mL) was added NaH (60% dispersion in mineral oil, 50.8 mg, 1.33 mmol). The mixture was stirred at room temperature for 15 min before (2-fluoro-2-methyl-propyl) trifluoromethanesulfonate (198 mg, 0.884 mmol) was added and the mixture was stirred at 70° C. for 2 h. Water (15.0 mL) was added and the reaction was extracted with EtOAc (3×10.0 mL). The combined organic layers were washed with brine (15.0 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (24 g cartridge) eluting with hexanes and EtOAc (0-70%) to afford the title compound (187 mg, 80%) as an oil. m/z: (ES+): [M+H]+: 529.75; (A05) tR=2.84 min.

a. Preparation of 2H-chromen-4-yloxy(trimethyl)silane

Chroman-4-one (20.0 g, 135 mmol) was dissolved in dry DCM (240 mL) under nitrogen and cooled down to 0° C. before TEA (20.7 mL, 148 mmol) was added followed by dropwise addition of TMSOTf (25.7 mL, 142 mmol). The mixture was stirred at room temperature for 1 h before water (200 mL) was added. The layers were separated, and the aqueous layer was extracted with DCM (3×200 mL). The organics were combined, washed with brine (200 mL), dried over (Na2SO4), filtered and concentrated under pressure to afford the title compound (80% pure, 35.2 g, 95%) as oil which was used in the next step without further purification. m/z: (ES+): [M+H]+: 221.04; (A05) tR=2.82 min.

b. Preparation of methyl 3-(4-oxochroman-3-yl)propanoate

TiCl4(1.00 mol/L in DCM, 153 mL, 153 mmol) was put under nitrogen and cooled down to −30° C. before a solution of methyl prop-2-enoate (11.6 mL, 128 mmol) in dry DCM (125 mL) was added dropwise. The mixture was cooled down to −78° C. 2H-chromen-4-yloxy(trimethyl)silane (80% pure, 35.2 g, 128 mmol) in dry DCM (125 mL) was added dropwise and the mixture was stirred for 2 h at −78° C., slowly warmed to room temperature and further stirred for 16 h. Water (200 mL) was added and the layers were separated. The aqueous layer was extracted with DCM (2×150 mL), combined, washed with brine (150 mL), dried (Na2SO4), filtered, concentrated under reduced pressure. The residue was purified by silica gel chromatography (330 g cartridge) with Hexane and EtOAc (0-40%) to afford the title product (90% pure, 3.08 g, 9%) as an oil. m/z: (ES+): [M-OMe]+: 203.3; (A05) tR=2.27 min.

c. Preparation of 3-(3-hydroxypropyl)chroman-4-ol

A solution of LiAlH4(2 M in THF, 13.2 mL, 26.4 mmol) was added dropwise to a solution of methyl 3-(4-oxochroman-3-yl)propanoate (90% pure, 3.08 g, 11.8 mmol) in dry THF (25.0 mL) at 0° C. The reaction was warmed at 22° C. and stirred for 1 h. The reaction was cooled down to 0° C. before water (20.0 mL) was added and the mixture was stirred at 22° C. for 1 h. The precipitate was removed by filtration through a pad of Celite. The filtrate was extracted with DCM (3×20.0 mL). The combined organic phases were washed with brine (30.0 mL), dried (MgSO4), filtered, and concentrated under reduced pressure to afford the title compound (90% pure, 1.70 g, 62%) as an oil. m/z: (ES+): [M−2OH]+=174.12; (A05); tR=1.75 and 1.81 m.

d. Preparation of 3-(3-hydroxypropyl)chroman-4-one

A solution of 3-(3-hydroxypropyl)chroman-4-ol (1.70 g, 7.35 mmol) in DCM (30.0 mL) was treated with MnO2 (3.19 g, 36.7 mmole). The suspension was stirred at room temperature for 72 h and the solids were removed by filtration on a pad of celite. The organic phase was concentrated under reduced pressure and the residue was purified by silica gel chromatography (80 g cartridge) with Hexane and EtOAc (0-100%) to afford the title Compound (708 mg, 47%) as an oil. m/z: (ES+): [M-OH]+=189.04; (A05); tR=1.97 m.

e. Preparation of 3-(3-methoxypropyl)chroman-4-one

3-(3-hydroxypropyl)chroman-4-one (240 mg, 1.16 mmol) was dissolved in dry MeCN (4.00 mL) followed by addition of Ag2O (809 mg, 3.49 mmol) and iodomethane (0.159 mL, 2.56 mmol). The mixture was stirred at room temperature for 24 h. Iodomethane (0.159 mL, 2.56 mmol) was added and the mixture was further stirred for 24 h. The mixture was filtered and the washed with MeCN (5×5.00 mL). The filtrate was concentrated under reduced to afford the title product (216 mg, 84%) as an oil which was used in the next step without further purification. m/z: (ES+): [M-OMe]+: 190.06; (A05) tR=2.23 min.

f. Preparation of 3-(3-methoxypropyl)chroman-4-amine

To a solution of 3-(3-methoxypropyl)chroman-4-one (216 mg, 0.981 mmol) in MeOH (6.00 mL) in microwave vial were added ammonium acetate (756 mg, 9.81 mmol) and NaBH3CN (616 mg, 9.81 mmol). The vial was sealed and heated at 80° C. for 16 h. The solvent was removed at reduced pressure and 3N HCl (5.00 mL) was added. The acidic solution was extracted with ether (15.0 mL) and the aqueous layer was basified with 3N NaOH until pH 13. The basic aqueous layer was extracted with ethyl acetate (2×15.0 mL), the combined organic phases were washed with brine (10.0 mL), dried (MgSO4), filtered, and concentrated under reduced pressure to afford the title compound as oil (90% pure, 158 mg, 66%) which was used in the next step without further purification. m/z: (ES+): [M+H]+: 222.17; (A05) tR=1.67 min

To a solution of (3R,4S)-4-(benzhydrylideneamino)chroman-3-ol (500 mg, 1.52 mmol) in dry DMF (10.0 mL) was added NaH (174 mg, 4.55 mmol). The mixture was stirred at room temperature for 15 min before 3-bromo-2,2-dimethyl-propanenitrile (492 mg, 3.04 mmol) was added and the mixture was stirred at 80° C. for 2 h. Water (15.0 mL) was added and the reaction was extracted with EtOAc (3×15.0 mL). The combined organic layers were washed with brine (15.0 mL), dried (MgSO4), filtered, and concentrated under reduced pressure to afford the crude intermediate.

The intermediate was dissolved in THF (10.0 mL) before 2M HCl (10.0 mL) was added and the mixture was stirred at room temperature for 16 h. The aqueous phase was washed with DCM (3×20.0 mL), basified with 3 M NaOH until pH 14 and extracted with CHCl3; iPrOH 3:1 (3×30.0 mL). The combined organic phases were washed with brine (20.0 mL), dried (MgSO4), filtered, and concentrated under reduced pressure to give the title compound (60% pure, 30.0 mg, 5%) as an oil. m/z: (ES+): [M+H]+: 247.17; (A05) tR=1.75 min.

28. Evaluation of PINK1 Kinase Activity

A list of compounds evaluated and their corresponding activity is shown in Tables 2 and 3 below.

Briefly, SKOV3 cells (ATCC HTB-77) were plated in DMEM (Corning 10-013-CV) medium containing 25 mM glucose or DMEM with no glucose (Gibco 11966-025) supplemented with 10 mM galactose at 9,000 cells/well in 96 well plates (Corning costar 3610). 24 hrs later, compounds, in DMSO, were added at various concentrations to a final 0.1% DMSO for all wells. Following 20 hours of incubation with compound, all wells had their growth medium replaced with DMEM containing 25 mM of glucose followed by the addition of Promega CellTiter-Glo® reagent as per manufacturer's instructions. After a 15 minute incubation cellular ATP levels were read out using a Promega GloMax® DiscoverMicroplate Reader.

The ratio of relative CellTiter-Glo values from cells grown in galactose to cells grown in glucose was determined (gal/glc ratio). The gal/glc ratio vs concentration is then graphed, and non-linear regression is used to find the highest concentration of MTK compound at which gal/glc>0.80 (the “highest mito-safe dose”, or IC20). The Mito-Safety Index in Table 2 is equal to the highest mito-safe dose divided by the mt-Keima EC50dose.

Primary hippocampal neurons were derived following standard protocols, and treated with sonicated PFFs according to the methods in Volpicelli-Daley et al (Methods Mol Biol2019). Following addition of PFFs or PBS on DIV7, Mitokinin compound or vehicle control was added on DIV9 and DIV12. At DIV14, cultures were harvested for biochemical analysis using either whole cell lysis buffer or fractionation buffer containing NP-40.

For whole cell lysates, cultures were collected in WC lysis buffer [100 mM Bicine pH 8.0, 0.27M Sucrose (Sigma S-1888, FW: 342.3), 1 mM EDTA, 1 mM EGTA, 5 mM Na4P2O7(ESB422, MW 446.06 g/mol), 100 mM Tris pH 7.5, 1% Triton X-100, 1× Halt™ Protease and Phosphatase Inhibitor Cocktail, with Benzonase (1:1000)]. Samples were incubated on ice for 30 min, sonicated, and then cleared by centrifugation at 18,000 g for 20 min at 4° C. Supernatant protein concentration was determined by BCA assay, and samples were analyzed by western blot using commercially available antibodies. Neuronal health markers included NfL, total caspase-3, cleaved caspase-3, pS65 Ub, LC3, TUJ1, and actin.

31. Inspection of Crystallization and Round Cells

Briefly, the Hela MKYP (mt-Keima/YFP-Parkin) cells were seeded at 10K cells/well. EP/MTK compounds were added at seeding (cells were still in suspension). The cells were incubated with the EP/MTK compounds for 16 hours, then 1 μM FCCP/oligomycin was added for 6 hours. Prior to harvesting, cells were scored by eye under 20× magnification for the presence or absence of crystalline or aggregated compound, or round cells.

Briefly, HeLa cells expressing mt-Keima and YFP-Parkin (HeLa MKYP) were plated at 10,000 cells/well in 96 well plates along with compounds at various concentrations. Following 16 hrs of incubation, cells were treated with 1 uM FCCP/oligomycin for 6 hours, then analyzed via FACS for the presence of mitochondria in lysosomes (as determined by an emissions spectrum shift from the pH-sensitive mt-Keima tag).

G. Prophetic Experimental Methods

1. In Vivo Pre-Formed Fibril Model

Mouse and human alpha-synuclein monomers will be sourced commercially, then pre-formed fibrils will be generated as per the detailed protocol provided by the MJFF. Pre-formed fibrils will be introduced via stereotactic injection into the striatum of wt (C57BL/6J, Jax #000664) and transgenic A53T mice (B6; C3-Tg(Prnp-SNCA*A53T)83Vle/J, Jax #004479). Cohorts of drug-treated (oral gavage) and untreated animals will then be allowed to age for up to 6 months, at which point they will be sacrificed and perfused. Brains will be removed and fixed, then sectioned for analysis. It is anticipated that untreated A53T animals will show significant spreading of aggregated alpha-synuclein pathology and pS129 staining, as well as some possible neurodegeneration; wt animals are also expected to show pS129 synuclein staining and synuclein aggregation, albeit less than that seen in the A53T background. Drug treated animals are expected to show significantly less pS129 staining and reduced synuclein spreading.

Briefly, HeLa MKYP cells will be plated at 1,300,000 cells/plate in 10 cm plates in 10 mL of medium containing compound at various concentrations. Following 16 hrs of incubation, cells will bere treated with 0.5 uM FCCP/oligomycin for 2 hours, then harvested. Mitochondria will then be isolated according to published protocols (Ordureau et al, 2014; https://doi.org/10.1016/j.molcel.2014.09.007). Equal amounts of samples will be loaded on 26 well gradient gels, and a western blot analysis will be performed using commercially available antibodies for various markers, including phospho serine 65 (pS65) ubiquitin, MFN2, PINK1, Parkin, and actin.

A. In-Life Procedures Cisplatin Challenge and Dosing Regimen

Mice will be provided at least one week of acclimation to the animal facility and group housed. Mice will be injected intraperitoneally with 1 mg/ml cisplatin solution (BluePoint Labs) or 10 ml/kg sterile-filtered saline using 29G insulin syringes. Mice will be weighed and administered vehicle or compound by oral gavage. Mice will be monitored for excessive weight loss and euthanized if moribund.

Compounds will be formulated at ten-fold the dosing concentration in NMP (N-methylpyrrolidone) followed by dilution with solutol-15 and water for a final vehicle concentration of 10% NMP/10% solutol-15/80% water.

c. Sacrifice and Tissue Collection and Storage

For tissue harvest, mice will be anesthetized using isofluorane. Cardiac puncture will be performed to withdraw blood for serum collection. Blood will be deposited into serum separator tubes and left undisturbed for 30 min to 1 hr at room temperature to allow clotting prior to serum separation by centrifugation for 2 min (10,000 g, room temperature). Collected serum will be transferred to Eppendorf tubes and frozen on dry ice. After cervical dislocation, left and right kidneys will be extracted and frozen until analysis.

d. Kidney Homogenate Preparation and Mitochondrial Isolation

Kidneys will be removed from −80° C. and minced on an ice block. Minced tissues were transferred to a dounce homogenizer and homogenized with 20× strokes of the “loose” pestle and 20× strokes of the “tight” pestle using 1 mL of cold mitochondrial isolation buffer (MIB, 50 mM Tris-HCl (pH 7.5), 70 mM sucrose, 210 mM sorbitol, 1 mM EDTA, 1 mM EGTA, 100 mM chloroacetamide, Halt™ Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100×) (PI), 10 μM PR619). Kidney homogenate will be transferred to a 1.5 ml Eppendorf tube and were centrifuged at 300×g for 5 min at 4° C. Approximately 800 μL of supernatant will be transferred to a new 1.5 ml microcentrifuge tube. The supernatant (cytosol+mitochondria) will be transferred to a new tube and centrifuged at 10,000 g for 20 min at 4° C. to pellet the mitochondrial fraction. After removing residual supernatant, mitochondria will be resuspended in lysis buffer (100 mM Bicine pH 8.0, 0.27M Sucrose, 1 mM EDTA, 1 mM EGTA, 5 mM Na4P2O7, 100 mM Tris pH 7.5, 1% Triton X-100), containing benzonase (1:1000), HALT protease/phosphatase inhibitors (1:100), and PR-619 de-ubiquitinase inhibitor (1:1000). e. BLOOD UREA NITROGEN (BUN) DETERMINATION

Serum will be thawed on ice and subsequently diluted 1:50 in MilliQ water. BUN levels in the serum sample will be analyzed using ThermoFisher's Urea Nitrogen (BUN) Colorimetric Detection Kit. Assay will be performed following manufacturer's published protocol.

Urine will be collected from scruffed mice (serial collection) or directly from bladder using insulin syringe during harvest (terminal collection). KIM-1 will be measured in mouse urine using R&D System's Mouse TIM-1/KIM-1/HAVCR DuoSet ELISA following manufacturer's published protocol.

g. Kidney RNA Extraction and Quantitative PCR

RNA will be isolated from kidney samples using Rneasy Mini kit (Qiagen) according to its product manual. RNA concentration will be measured using NanoDrop™ 2000/2000c Spectrophotometers (Thermo Scientific). 50 ng of RNA for each sample was used to generate cDNA. cDNA will be synthesized using High-Capacity RNA-to-cDNA™ Kit (Thermo Scientific) according to its product manual. Quantitative PCR will be performed using Power SYBR™ Green PCR Master Mix (Applied Biosystems) according to its product manual. The following primers will be used to analyze gene expression levels in the kidney:

All gene expression levels will be normalized to expression levels of beta-actin using ΔΔCt and expressed as fold change relative to cisplatin vehicle treated mice.

A small piece of frozen kidney tissue (˜12 mg) will be homogenized and DNA extracted using the Qiagen QIAamp DNA mini kit. mtDNA/nucDNA ratio will be determined using a qPCR protocol from the Aurwex lab (Quiros et al, 2017), using the following primers:

For pS65-Ub ELISA, capture monoclonal rabbit antibody anti-pS65-Ub will be diluted to 1 μg/ml in PBS and pipetted into 96 well half-area polystyrene plates (50 μL/well). Sealed plates will be shaken at 800 rpm for 5 minutes and incubated overnight at 4° C. on an even surface. The next day, blocking solution (5% BSA in TBST, sterile filtered) will be added to each well (100 μL/well) and shaken for 1 hr at 800 rpm at room temperature. Plates will either be used immediately or stored sealed at 4° C. for maximum one week. Samples will be diluted in lysis buffer to a concentration of 10 ug/ul and 50 μL will be loaded onto plates in duplicate after washing 5× with TBST using an automated plate washer (used for all subsequent wash steps). Standard protein recombinant pS65-Ub was diluted in lysis buffer+0.1% BSA and serial dilutions (4000 ng/ml-0 ng/ml) will be added in duplicate to the sample plate (50 μL/well). Plates will be shaken at 800 rpm at room temperature for 2 hr. After washing 5× with TBST, 50 μL of mouse anti-Ub detection antibody (1 ug/ml in 5% BSA in TBST) was added to the wells. Plates will be shaken at 800 rpm at room temperature for 1 hr, followed by washing 5× with TBST, and shaking at 800 rpm at room temperature for 45 minutes with goat anti-mouse peroxidase-conjugated IgG antibody (1:10,000 dilution in 5% BSA in TBST) (50 μL/well). For peroxidase reaction, 50 μL of TMB reagent (Pierce #34029) will be added to the wells after washing and wells will be monitored closely for reaction development. To stop the ELISA reaction, 50 μL 2N sulfuric acid will be added. Absorbance was measured at 450 nm using LifeTechnologies SpectraMax).

The total protein concentration of kidney mitopreps will be measured with the Thermo Scientific Pierce BCA Protein Assay Kit (Thermo Scientific), according to its product manual. These samples will be normalized with their respective lysis buffers. For SDS-PAGE, the samples will be prepared with 4× Laemmli Sample Buffer with the reducing agent 2 mercaptoethanol. For each lane of a 26 well gel (4-20% Criterion™ Tris-HCl Protein Gel, Bio-Rad Laboratories), 10 μg per sample will be loaded and analyzed by Western Blotting. Indicated bands will be quantified using ImageStudio Lite and normalized to beta actin band intensity.

4. In Vitro Data

Briefly, HeLa cells expressing a YFP-tagged Parkin will be treated with 1 mM of FCCP and Oligomycin followed by the specified dose of compound, then analyzed by longitudinal imaging.

5. PK Data

Briefly, mice (C57Bl/6, fed) will be dosed by oral gavage with compound in NMP/solutol vehicle. Plasma concentrations of compound will be determined by mass spectrometry in at least 3 mice per study.

Briefly, P0 to P2 mice will be sacrificed and their cortical tissue dissected and plated according to standard methods to obtain primary mixed cortical cultures. Cultures will be maintained for 14 days. On or around Day 15, MTK compound will be added and allowed to incubate for 24 hours. After incubation with compound, the cells will be challenged with 100 ng/ml LPS. 24 hours after challenge initiation, cellular media is collected for analysis of cytokine levels via ELISA. A commercial ELISA kit for IL-6, TNF-α, and IL1-β will be used.

The expression of a deletion mutant of dOTC yields Triton X-100 insoluble protein aggregates in the mitochondrial matrix. This misfolded protein expression is capable of recruiting PINK1/Parkin to mitochondria without depolarizing the inner mitochondrial membrane. Thus, without wishing to be bound by theory, it may represent a more physiological mechanism of PINK1 stabilization.

Here, HeLa cells stably expressing YFP parkin, containing doxycycline inducible expression of dOTC, are obtained. The cells are seeded at 20000 cells/well plus doxycycline (1 μg/mL) plus MTK on a 96-well plate. On Day 3, the cells are fixed and permeabilized and bound with OTC antibody. DAPI and cell mask are added. There is no wash off of dox. The results are imaged at 40×, non-confocal. 85-600 cells are analyzed per well. Each condition has 1-3 wells.

8. Effect of Compounds on Cisplatin-Mediated Kidney Fibrosis Model

Repeated low-level tissue damage can lead to fibrosis and chronic disease in the affected tissue. Cisplatin can cause lung and kidney fibrosis in humans (Guinee et al., Cancer 1993), and repeated low-dose cisplatin challenge in mice causes fibrosis in mice (Sharp et al,AJPNephrology,2016; Katagiri et al,Kidney International,2015). By reducing cisplatin-mediated mtDNA damage, though a PINK1-dependent mechanism identical for evidence provided above, MTK compounds 35985 and 40180 will be shown to be protective for kidney fibrosis.

Sharp et al. describe a protocol by which mice (FVB strain) are injected weekly with 7 mg/kg cisplatin by intraperitoneal injection. N=12-15 mice per group will be injected with saline or 7 mg/kg cisplatin weekly for four weeks, and dosed with either vehicle or MTK compounds by oral gavage at doses of about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, or about 50 mg/kg, either once a day or twice a day. Then, blood urea nitrogen or creatinine (urine), and kidney-injury marker-1 (KIM-1) will be assessed to evaluate kidney function and injury, respectively. Furthermore, quantitative PCR (qPCR) will be used to measure the expression of inflammatory markers such TNFalpha, IL-1beta, and IL-6. TGFbeta and fibronectin will be measured using western blot or commercially available ELISA kit as the principle readout for fibrosis, and count the number of infiltrating reactive immune cells in kidney sections by immunofluorescence or immunohistochemistry as a secondary measure of kidney fibrosis. Without wishing to be bound by theory, it is expected that administration of a disclosed compound will reduce fibrosis by 50% or more at therapeutic doses.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the disclosure. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.