Adenosine derivatives for use in the treatment of neurodegenerative disorders and cancer

Abstract

The present invention provides compounds of general formula (I): (general formula I) (wherein R 1 and R 2 (wherein is as defined herein). The present invention also relates to pharmaceutical compositions and combination therapies comprising the compounds of formula (I) for treating these conditions, and to methods of treating such diseases and conditions using compounds of general formula (I).

Description

[Technical Field]

[0001] The present invention relates to therapeutic agents for use in the treatment of neurodegenerative diseases or conditions and / or cancers characterized by high levels of ubiquitin Ser65 phosphorylation, and to methods of treating such diseases or conditions in which a therapeutic agent or composition or combination therapy according to the invention is administered to a subject having, or suspected of having, a neurodegenerative disease or condition and / or cancer characterized by high levels of ubiquitin Ser65 phosphorylation. [Background technology]

[0002] Parkinson's disease belongs to a group of conditions called motor system disorders that result from the loss of dopamine-producing brain cells. Parkinson's disease (PD) is the second leading cause of neurodegeneration in humans, and to date, there is no treatment that can slow or halt its clinical progression.

[0003] Mitochondria play a crucial role in the pathogenesis of PD and other neurodegenerative diseases and cardiomyopathies. In this context, mitochondrial serine / threonine PTEN-induced kinase 1 (PINK1) has emerged as a central player in mitochondrial quality control. In healthy mitochondria, PINK1 is constitutively recruited to the mitochondrial membrane, where it undergoes N-terminal cleavage by proteases and subsequent proteasomal degradation in the cytosol (Figure 1a). However, in damaged mitochondria, after depolarization of the inner mitochondrial membrane, PINK1 is stabilized in its full-length form on the outer mitochondrial membrane (OMM). Accumulation of PINK1 leads to trans-autophosphorylation and subsequent activation. Active PINK1 then phosphorylates the E3 ubiquitin ligase Parkin at serine 65 and also phosphorylates ubiquitin at serine 65 (Ub pSer65). This ultimately leads to the ubiquitination of various proteins on the OMM, leading to mitochondrial degradation by the autophagic machinery, a process called mitophagy.

[0004] Therefore, activating PINK1 and inducing mitophagy has been generally proposed as a potential new therapeutic target for Parkinson's disease. Specifically, it has been emphasized that PINK1 kinase activity is essential to prevent the onset of neurodegeneration exemplified by its loss-of-function mutations that lead to a form of early-onset Parkinson's disease (PD). This observation led to the discovery of N 6 This led to the discovery that the substituted adenine, kinetin (1, Figure 1b), potentiates PINK1 activation in cells exposed to the depolarizing agent carbonyl cyanide m-chlorophenylhydrazine (CCCP), a drug used to stimulate mitochondrial damage. It was noted that kinetin activation of PINK1 results from its bioconversion to the active metabolite kinetin riboside triphosphate (3, Figure 1b), which acts as an ATP neosubstrate for PINK1. With this observation in mind and as a result of interest in developing nucleoside analog therapeutics, we subsequently demonstrated that a nucleoside derivative of kinetin, termed kinetin riboside 2, exhibits more potent CCCP-independent activation of PINK1 in cells compared to its nucleobase derivative, kinetin, as evidenced by parkin Ser65 phosphorylation.

[0005] Thus, phosphorylation of ubiquitin by the mitochondrial protein kinase PINK1 upon mitochondrial depolarization is typically considered a key step in mitochondrial repair and recycling by autophagy, and activators of PINK1 are therefore considered promising therapeutic agents in the treatment of many forms of PD.

[0006] However, recent evidence in postmortem analyses of brain tissue from PD and dementia patients without a discernible PINK1 genetic causative factor, such as dementia with Lewy bodies and idiopathic forms of PD (i.e., age-related and sporadic PD patients), has shown that the opposite is also true, with high levels of Ub Ser65 phosphorylation. 9、10Therefore, these subsets of PD patients and dementia patients exhibit high levels of phospho-ubiquitin (ubiquitin Ser65 phosphorylation) in the brain. This implies that conventional activators of PINK1, and the resulting increase in ubiquitin phosphorylation, are ineffective in such patients, in whom elevated Ub Ser65 phosphorylation is evident. Furthermore, although the role of mitophagy in tumorigenesis has not yet been fully elucidated, a significant body of evidence indicates that dysregulation of mitophagy is often associated with many types of cancer and is accompanied by the accumulation of dysfunctional mitochondria, which contributes to tumorigenesis.

[0007] The present inventors herein demonstrate that N 6 Substituted adenosines, e.g., N 6 -(2-furanylmethyl)adenosine (known as kinetin riboside) and N 6 We disclose that N-benzyladenosine unexpectedly inhibits ubiquitin phosphorylation induced by the established mitochondrial depolarizing agents, CCCP and niclosamide. These nucleoside analogs inhibited niclosamide- and CCCP-induced ubiquitin phosphorylation but did not prevent mitochondrial membrane depolarization. Notably, treatment of cells with these nucleoside analogs alone induced low levels of mitophagy and did not cause mitochondrial fragmentation. Together, this study identifies N-benzyladenosine as a novel inhibitor of PINK1-mediated ubiquitin phosphorylation. 6 We present a substituted adenosine, highlighting its potential utility in the unexpected treatment of patients with age-related and sporadic PD and dementia with Lewy bodies, who have high levels of phosphorylated ubiquitin and are refractory to conventional activators of PINK1 activity, and / or in the treatment of cancer. Our findings suggest that these compounds and their analogs may hold promise as therapeutic agents for halting the progression of neuronal death in idiopathic Parkinson's disease and related disorders and / or halting the progression of tumorigenesis due to the accumulation of dysfunctional mitochondria. Summary of the Invention

[0008] The invention, in its various aspects, is as set out in the appended claims.

[0009] According to a first aspect, the present invention provides compounds of general formula (I), including all tautomers thereof, for use in the treatment of neurodegenerative diseases or conditions and / or cancers characterized by high levels of ubiquitin Ser65 phosphorylation: [ka] General formula (I) (In the formula, R 1 OH, halo, nitro, C 1~4 Alkyl, C 1~4 Haloalkyl, -O(C 1~4 alkyl), -O(C 1~4 haloalkyl), NH2, NH(C 1~4 alkyl) and N(C 1~4 C may be substituted with one or more substituents selected from alkyl)2 1~10 Alkyl, C 3~10 Cycloalkyl, C 6~10 is an aryl, heterocycloalkyl, or heteroaryl group; R 2 is the furanose moiety of general formula (II): [ka] General formula (II) (In the formula, X is O, NH, S or CH; R 3 H, OH, halo, nitro, C 1~4 Alkyl, C 1~4 Haloalkyl, -O(C 1~4 alkyl), -O(C 1~4 haloalkyl), NH2, NH(C 1~4 alkyl) and N(C 1~4 alkyl)2 or a mono-, di- or tri-phosphate derivative of general formula (VIII)11 are independently selected from OH or aryloxy, amino acid ester, or pivaloyloxymethyl masking groups. [ka] General formula (VIII) and; R 4 and R 5 H, OH, halo, nitro, C 1~4 Alkyl, C 1~4 Haloalkyl, -O(C 1~4 alkyl), -O(C 1~4 haloalkyl), NH2, NH(C 1~4 alkyl) and N(C 1~4 alkyl) independently selected from is) or a pharmaceutically or veterinarily acceptable salt or hydrate thereof.

[0010] Compounds of general formula (I) have been shown to inhibit PINK1-mediated ubiquitin phosphorylation and are therefore useful in treating disorders and conditions associated with high levels of phosphorylated ubiquitin, for which conventional activators of PINK1 activity are ineffective.

[0011] The compounds of general formula (I) are particularly useful for the treatment of idiopathic (i.e., age-related and / or sporadic) Parkinson's disease, dementia with Lewy bodies, and / or cancer. More particularly, the compounds of general formula (I) are useful for the treatment of idiopathic Parkinson's disease and / or dementia with Lewy bodies.

[0012] In the present invention, "C 1~10 The term "alkyl" refers to a straight or branched chain saturated hydrocarbon group having from 1 to 10 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, t-butyl, and n-hexyl. As will be readily understood, other alkyl groups are as defined above but have a different number of carbon atoms. For example, "C 1~4An "alkyl" has 1 to 4 carbon atoms.

[0013] "C 3~10 The term "cycloalkyl", in the context of the present specification, refers to a cyclic saturated hydrocarbon group containing from 3 to 10 carbon atoms and containing a single ring or multiple condensed rings.

[0014] "C 6~10 The term "aryl" refers to a ring system having aromatic character, having 6 to 10 ring carbon atoms, and containing a single ring or multiple fused rings. If an aryl group contains two fused rings, both rings need not be fully aromatic. Examples of aromatic moieties are phenyl and naphthyl.

[0015] In the context of the present specification, the term "heterocycloalkyl" refers to a saturated ring system having 3 to 10 ring atoms (unless otherwise specified), at least one, and optionally two or three, of which are heteroatoms selected from N, O and S, and containing a single ring or multiple fused rings.

[0016] In the context of this specification, the term "heteroaryl" refers to a ring system having 6 to 10 ring atoms (unless otherwise specified), at least one, and optionally two or three, of which are heteroatoms selected from N, O, and S, and containing a single ring or multiple fused rings, and having aromaticity. When a heteroaryl group contains more than one ring, not all rings need to be fully aromatic. Examples of heteroaryl groups include pyridine, pyrimidine, indole, pyrrole, imidazole, triazole, tetrazole, oxazole, thiazole, benzofuran, benzimidazole, and indoline.

[0017] As used herein, "halo" refers to fluoro, chloro, bromo or iodo, and more suitably to chloro or bromo.

[0018] In this specification, "C 1~4 "Haloalkyl" refers to a C alkyl group substituted with one or more halo atoms, up to oversubstitution.1~4 Refers to an alkyl group. Examples include chloromethyl, trifluoromethyl, 2-chloroethyl, 1-bromoethyl, etc.

[0019] The term "aryloxy" is used in the context of the present specification to refer to a masking group of general formula (IX-A) 12 is C 5~25 aryl or 5- to 25-membered heteroaryl group, either of which may be substituted with one or more functional groups selected from hydroxy, thiol, thioether, alkoxy, and amino: [ka] General formula (IX-A) The term "amino acid ester" in the context of this specification refers to a masking group of general formula (IX-B) 13 is H or a saturated or unsaturated hydrocarbon optionally substituted with one or more functional groups selected from thiol, thioether, alkoxy and amino, preferably C 1~4 is an alkyl chain; R 14 is a saturated or unsaturated hydrocarbon, preferably C, optionally substituted with one or more functional groups selected from hydroxy, thiol, thioether, alkoxy and amino; 1~4 alkyl chain or C6 aryl group): [ka] General formula (IX-B) The term "pivaloyloxymethyl" in the context of the present specification refers to a masking group of general formula (IX-C) 15 and R 16 each of which is independently optionally substituted with hydrogen, halo, or one or more substituents selected from hydroxy, thiol, thioether, alkoxy, and amino; 1~10 is an alkyl group or R 15 and R 16 is R 15 and R16 together with the carbon to which it is bonded, C 3~4 forming a cycloalkyl group or a 3- or 4-membered heterocycloalkyl group: [ka] General formula (IX-C) Salts of compounds of general formula (I) are suitably pharmaceutically or veterinarily acceptable salts. 1 ~R 5 Depending on the nature of the bases, these may be base addition salts such as sodium, potassium, calcium, aluminum, zinc, magnesium, and other metal salts summarized in Paulekuhn et al., (2007) J. Med. Chem. 50:6665-6672, and / or known to those skilled in the art, as well as choline, diethanolamine, ethanolamine, ethyldiamine, meglumine, and other well-known base addition salts. Alternatively, when the compound of general formula (I) contains an amino group, it may be quaternized to form a salt with a counterion such as a halide, hydroxide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetate, fumarate, citrate, tartarate, oxalate, succinate, mandelate, methanesulfonate, and p-toluenesulfonate.

[0020] In the compound of general formula (I), R 1 is preferably OH and C 1~4 substituted C, optionally substituted with one or more substituents selected from alkyl; 1~6 Alkyl, C 3~6 It is cycloalkyl or C6 aryl.

[0021] More preferably, R 1 is a group of general formula (III-A), (III-B), (III-C), (III-D), (III-E) or (III-F): [ka] General formula (III-A); [ka] General formula (III-B); [ka] General formula (III-C); [ka] General formula (III-D); [ka] General formula (III-E); [ka] General formula (III-F) (In the formula, n is 0, 1, 2 or 3; m is 0, 1, 2 or 3; Z is O, NH, S or CH2, preferably CH2; R 6 is OH or O(C 1~4 alkyl) is.

[0022] Most preferably, R 1 teeth, [ka] is selected from.

[0023] As noted above, compounds of general formula (I) comprise a furanose moiety of general formula (II). In a preferred embodiment, X is O and / or R 4 and R 5 are independently selected from H and OH, and / or R 3 is H, OH or a mono-, di- or tri-phosphate derivative of general formula (VIII).

[0024] In some compounds of the present invention, R 3 , R 4 and R 5Each of is OH. As will be readily understood by one of skill in the art, such compounds are adenosine ribonucleoside analogs.

[0025] In other suitable compounds, R 3 and R 4 are both hydroxyl and R 5 is H. As will be readily appreciated by one of skill in the art, such compounds are adenosine deoxyribonucleoside analogs.

[0026] Exemplary compounds of general formula (I) include kinetin riboside, a known compound having the structure of formula (IV): [ka] Formula (IV) Further exemplary compounds of general formula (I) include the following novel N 6 Substituted adenosine analogs include: [ka] In particularly preferred embodiments, the compound of general formula (I) is selected from the following structures: [ka] As mentioned above, the known compound kinetin riboside is a compound of general formula (I) as defined above. Thus, according to a second aspect, the present invention provides a compound of general formula (I), including all its tautomers: [ka] General formula (I) (In the formula, R 1 OH, halo, nitro, C 1~4 Alkyl, C 1~4 Haloalkyl, -O(C 1~4 alkyl), -O(C 1~4 haloalkyl), NH2, NH(C 1~4 alkyl) and N(C 1~4C may be substituted with one or more substituents selected from alkyl)2 1~10 Alkyl, C 3~10 Cycloalkyl, C 6~10 is an aryl, heterocycloalkyl, or heteroaryl group; R 2 is the furanose moiety of general formula (II): [ka] General formula (II) (In the formula, X is O, NH, S or CH; R 3 H, OH, halo, nitro, C 1~4 Alkyl, C 1~4 Haloalkyl, -O(C 1~4 alkyl), -O(C 1~4 haloalkyl), NH2, NH(C 1~4 alkyl) and N(C 1~4 alkyl)2 or a mono-, di- or tri-phosphate derivative of general formula (VIII) 11 are independently selected from OH or aryloxy, amino acid ester, or pivaloyloxymethyl masking groups. [ka] General formula (VIII) and; R 4 and R 5 H, OH, halo, nitro, C 1~4 Alkyl, C 1~4 Haloalkyl, -O(C 1~4 alkyl), -O(C 1~4 haloalkyl), NH2, NH(C 1~4 alkyl) and N(C 1~4 alkyl) independently selected from is) or a pharmaceutically or veterinarily acceptable salt or hydrate thereof, with the proviso that the compound is not kinetin riboside.

[0027] R 1 , R 2 , R 3 , R 4 , R 5 and preferred features for X are as described above for the first aspect of the invention.

[0028] Compounds of general formula (I) can be prepared by the methods disclosed below.

[0029] In particular, N in general formula (I) 6 Substituted adenosine analogs can be synthesized by bimolecular nucleophilic substitution (S) via coupling of 6-halopurine nucleoside derivatives with amine nucleophiles. N2 ) reaction.

[0030] In such reactions, compounds of general formula (I) can be prepared in a one-step process by reacting a 6-halopurine derivative of general formula (V) with a nucleophilic compound of general formula (VI): [ka] (V) [ka] wherein R7 is halo, preferably chloro, and R1 and R2 are as defined for general formula (I). In a preferred process, this nucleophilic substitution reaction is carried out in the presence of a tertiary amine, preferably triethylamine ("TEA"), and / or at elevated temperatures (i.e., 30°C or above).

[0031] Alternatively, N in general formula (I) 6 Substituted adenosine analogs can be prepared by reacting a nucleoside derivative of hypoxanthine with an amine nucleophile in the presence of a peptide coupling reagent.

[0032] In such a reaction, compounds of general formula (I) can be prepared in a one-step process by reacting a hypoxanthine derivative of general formula (VII) with a nucleophilic compound of general formula (VI) in the presence of benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate ("PyBOP"): [ka] (VII) [ka] (VI) (In the formula, R 1 and R 2 is as defined for general formula (I) In a preferred process, the peptide coupling reaction is carried out in the presence of a tertiary amine, preferably N,N-diisopropylethylamine ("DIPEA").

[0033] These processes for preparing compounds of general formula (I) represent a third aspect of the present invention.

[0034] Many compounds of general formulas (V), (VI), and (VII) are well known and readily available. Other compounds of general formulas (V), (VI), and (VII) can be readily synthesized by one skilled in the art using standard methods.

[0035] According to a fourth aspect, the present invention provides the use of kinetin riboside or a compound according to the second aspect of the invention in the preparation of a medicament for treating cancer or a disorder or condition associated with high levels of phosphorylated ubiquitin.

[0036] According to a fifth aspect, the present invention extends to a method of treating cancer or a disorder or condition associated with high levels of phosphorylated ubiquitin, comprising administering to a patient in need of such treatment an effective amount of kinetin riboside or a compound according to the second aspect of the invention.

[0037] In preferred embodiments of the fourth and fifth aspects, the disorder or condition is selected from idiopathic (i.e., age-related and / or sporadic) Parkinson's disease, dementia with Lewy bodies, and / or cancer. More preferably, the disorder or condition is selected from idiopathic Parkinson's disease and / or dementia with Lewy bodies.

[0038] It will be appreciated that kinetin riboside and / or the compound of the second aspect of the invention will typically be administered as part of a pharmaceutical composition. Accordingly, in a sixth aspect of the invention, there is provided a pharmaceutical composition comprising kinetin riboside or a compound according to the second aspect of the invention and a pharmaceutically or veterinarily acceptable excipient or carrier.

[0039] Suitable pharmaceutical excipients are well known to those skilled in the art. The pharmaceutical compositions may be formulated for administration by any suitable route, for example, oral, rectal, nasal, bronchial (inhalation), topical (including ophthalmic, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, and may be prepared by any method well known in the art of pharmacy.

[0040] The compositions can be prepared by bringing into association kinetin riboside or a compound of the second aspect of the present invention with a carrier. In general, the formulations are prepared by uniformly and intimately bringing the compound into association with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0041] Formulations for oral administration in the present invention may be presented as discrete units such as capsules, sachets or tablets, each containing a predetermined amount of compound; as a powder or granules; as a solution or suspension of compound in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion; or as a bolus, etc.

[0042] For compositions for oral administration (e.g., tablets and capsules), the term "acceptable carrier" includes common excipients, such as binders, e.g., syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose, and starch; fillers and carriers, e.g., corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid; and lubricants, e.g., magnesium stearate, sodium stearate, and other metal stearates, glycerol stearate, stearic acid, silicone fluid, talc wax, oils, and colloidal silica. Flavoring agents, such as peppermint, oil of wintergreen, and cherry flavoring, can also be used. It may be desirable to add coloring agents to make the dosage form easily identifiable. Tablets can also be coated by methods well known in the art.

[0043] Tablets can be made by compression or molding, optionally with one or more accessory ingredients.Compressed tablets can be prepared by compressing a free-flowing compound such as powder or granules, which may be mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersant, using a suitable machine.Molded tablets can be made by molding a mixture of powdered compounds moistened with an inert liquid diluent using a suitable machine.Tablets can be coated or scored, and can be formulated to provide delayed or controlled release of active agents.

[0044] Other formulations suitable for oral administration include lozenges comprising the active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.

[0045] Parenteral formulations are generally sterile.

[0046] For topical application to the skin, the compositions may be made up into creams, ointments, jellies, solutions or suspensions, etc. Cream or ointment formulations which may be used for drugs are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.

[0047] In a preferred embodiment of this aspect of the invention, the composition is formulated for oral delivery.

[0048] The exact amount of the therapeutically effective composition defined herein and the best route of administration of such compounds can be easily determined by those skilled in the art.Of course, such amount depends on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size and weight, the duration of treatment, the nature of concomitant therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of medical professionals.These factors are well known to those skilled in the art and can be addressed only by routine experimentation.It is generally preferred to use the maximum dose of each component or their combination, i.e., the highest safe dose according to sound medical judgment.However, it will be understood by those skilled in the art that a patient may insist on a lower dose or a tolerable dose for medical reasons, psychological reasons, or virtually any other reason.

[0049] The dose of compound or composition according to the present invention that is administered to the subject can be selected according to different parameters, particularly according to the administration mode used and the condition of the subject.Other factors include the desired treatment duration.If the response of the subject is insufficient with the initial dose that is applied, a higher dose (or a substantially higher dose by a different, more local delivery route) can be used until the patient's tolerance allows it.

[0050] In some cases, the compounds of general formula (I) may be used in combination with further therapeutic agents, particularly those used in the treatment of cancer or neurodegenerative diseases or conditions characterized by high levels of ubiquitin Ser65 phosphorylation.

[0051] Thus, in a seventh aspect of the present invention, there is provided a combination therapy comprising a compound of general formula (I) and a further therapeutic agent used in the treatment of cancer or a neurodegenerative disease or condition characterised by high levels of ubiquitin Ser65 phosphorylation, for simultaneous, separate or sequential use in the treatment of cancer or a neurodegenerative disease or condition characterised by high levels of ubiquitin Ser65 phosphorylation.

[0052] When the compound of general formula (I) and the further therapeutic agent are used simultaneously, they may be provided in a pharmaceutical composition, and therefore the present invention also provides a pharmaceutical composition for use in treating a neurodegenerative disease or condition characterized by high levels of ubiquitin Ser65 phosphorylation, comprising a compound of general formula (I), a further therapeutic agent used in the treatment of a neurodegenerative disease or condition, and a pharmaceutically acceptable excipient or carrier.

[0053] In some cases, the neurodegenerative disease or condition is idiopathic (i.e., age-related or sporadic) Parkinson's disease, and the additional therapeutic agent is a drug for treating Parkinson's disease. Examples of therapeutic agents used to treat Parkinson's disease include levodopa, ropinirole, rotigotine, pramipexole and amantadine, and folic acid or deoxynucleosides and their monophosphates.

[0054] Throughout this description and the claims, the terms "comprise" and "contain," as well as variations of these words, such as "comprising" and "comprises," mean "including but not limited to" and do not exclude other moieties, additives, ingredients, integers, or steps. Throughout this description and the claims, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood as contemplating plural as well as singular unless the context requires otherwise.

[0055] All references cited herein, including any patents or patent applications, are hereby incorporated by reference. No admission is made that the references constitute prior art. Further, no admission is made that any of the prior art constitutes part of the general knowledge in the art.

[0056] Preferred features of each aspect of the invention may be as described in relation to any of the other aspects.

[0057] Other features of the present invention will become apparent from the following examples. Generally speaking, the present invention extends to any novel or novel combination of features disclosed in this specification (including the accompanying claims and drawings). Accordingly, it should be understood that any feature, integer, property, compound, or chemical moiety described in connection with a particular aspect, embodiment, or example of the present invention can also be applied to any other aspect, embodiment, or example described herein, unless inconsistent.

[0058] Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

[0059] The invention will now be described, by way of example only, with reference to the following examples and the following figures. [Brief explanation of the drawings]

[0060] [Figure 1A] Kinetin riboside suppresses niclosamide- and CCCP-induced Ub Ser65 phosphorylation. (A) Schematic of PINK1 / Parkin signaling in healthy and damaged mitochondria. (B) Chemical structure of kinetin and its metabolism to generate kinetin riboside triphosphate, a PINK1 ATP-neosubstrate. (C) Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 hours, then lysed or treated with 10 μM CCCP for 3 hours. Cell lysates were probed for Ub Ser65 phosphorylation, OPA1, and GAPDH. UU: untreated and untransfected; UT: untreated and transfected. (D) YFP-Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 hours, then lysed or treated with 10 μM niclosamide or CCCP for 1 hour. Cell lysates were probed for Ub Ser65 phosphorylation, LC3B-I / -II, and α-tubulin. NT: untreated cells. (E) As in (d), but samples were examined for Ub Ser65 phosphorylation, YFP-parkin expression, and TOM20 using immunofluorescence. Scale bar = 40 μm. [Figure 1B]Kinetin riboside suppresses niclosamide- and CCCP-induced Ub Ser65 phosphorylation. (A) Schematic of PINK1 / Parkin signaling in healthy and damaged mitochondria. (B) Chemical structure of kinetin and its metabolism to generate kinetin riboside triphosphate, a PINK1 ATP-neosubstrate. (C) Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 hours, then lysed or treated with 10 μM CCCP for 3 hours. Cell lysates were probed for Ub Ser65 phosphorylation, OPA1, and GAPDH. UU: untreated and untransfected; UT: untreated and transfected. (D) YFP-Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 hours, then lysed or treated with 10 μM niclosamide or CCCP for 1 hour. Cell lysates were probed for Ub Ser65 phosphorylation, LC3B-I / -II, and α-tubulin. NT: untreated cells. (E) As in (d), but samples were examined for Ub Ser65 phosphorylation, YFP-parkin expression, and TOM20 using immunofluorescence. Scale bar = 40 μm. [Figure 1C]Kinetin riboside suppresses niclosamide- and CCCP-induced Ub Ser65 phosphorylation. (A) Schematic of PINK1 / Parkin signaling in healthy and damaged mitochondria. (B) Chemical structure of kinetin and its metabolism to generate kinetin riboside triphosphate, a PINK1 ATP-neosubstrate. (C) Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 hours, then lysed or treated with 10 μM CCCP for 3 hours. Cell lysates were probed for Ub Ser65 phosphorylation, OPA1, and GAPDH. UU: untreated and untransfected; UT: untreated and transfected. (D) YFP-Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 hours, then lysed or treated with 10 μM niclosamide or CCCP for 1 hour. Cell lysates were probed for Ub Ser65 phosphorylation, LC3B-I / -II, and α-tubulin. NT: untreated cells. (E) As in (d), but samples were examined for Ub Ser65 phosphorylation, YFP-parkin expression, and TOM20 using immunofluorescence. Scale bar = 40 μm. [Figure 1D]Kinetin riboside suppresses niclosamide- and CCCP-induced Ub Ser65 phosphorylation. (A) Schematic of PINK1 / Parkin signaling in healthy and damaged mitochondria. (B) Chemical structure of kinetin and its metabolism to generate kinetin riboside triphosphate, a PINK1 ATP-neosubstrate. (C) Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 hours, then lysed or treated with 10 μM CCCP for 3 hours. Cell lysates were probed for Ub Ser65 phosphorylation, OPA1, and GAPDH. UU: untreated and untransfected; UT: untreated and transfected. (D) YFP-Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 hours, then lysed or treated with 10 μM niclosamide or CCCP for 1 hour. Cell lysates were probed for Ub Ser65 phosphorylation, LC3B-I / -II, and α-tubulin. NT: untreated cells. (E) As in (d), but samples were examined for Ub Ser65 phosphorylation, YFP-parkin expression, and TOM20 using immunofluorescence. Scale bar = 40 μm. [Figure 1E]Kinetin riboside suppresses niclosamide- and CCCP-induced Ub Ser65 phosphorylation. (A) Schematic of PINK1 / Parkin signaling in healthy and damaged mitochondria. (B) Chemical structure of kinetin and its metabolism to generate kinetin riboside triphosphate, a PINK1 ATP-neosubstrate. (C) Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 hours, then lysed or treated with 10 μM CCCP for 3 hours. Cell lysates were probed for Ub Ser65 phosphorylation, OPA1, and GAPDH. UU: untreated and untransfected; UT: untreated and transfected. (D) YFP-Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 hours, then lysed or treated with 10 μM niclosamide or CCCP for 1 hour. Cell lysates were probed for Ub Ser65 phosphorylation, LC3B-I / -II, and α-tubulin. NT: untreated cells. (E) As in (d), but samples were examined for Ub Ser65 phosphorylation, YFP-parkin expression, and TOM20 using immunofluorescence. Scale bar = 40 μm. [Figure 2A] Chemical synthesis of N6-substituted adenines and adenosines and their activation of PINK1 in cells. (A) Method A: Reagents and conditions: triethylamine, ethanol, heating, 16 hours; Method B: Reagents and conditions: PyBOP, DIPEA, acetonitrile / DMF, 3 days, rt. (B) Parkin-transfected HeLa cells were treated with 50 μM of 1, 2, 8a-8f, and 9a-9f for 1 hour. CCCP was used at 10 μM, and treatment was for 3 hours. Cells were then lysed and probed for anti-phospho-Ser65 parkin (pS65 parkin), total parkin, OPA1, and GAPDH. UU: untreated and non-transfected HeLa cells. UT: untreated and parkin-transfected HeLa cells. Data represent triplicates. [Figure 2B]Chemical synthesis of N6-substituted adenines and adenosines and their activation of PINK1 in cells. (A) Method A: Reagents and conditions: triethylamine, ethanol, heating, 16 hours; Method B: Reagents and conditions: PyBOP, DIPEA, acetonitrile / DMF, 3 days, rt. (B) Parkin-transfected HeLa cells were treated with 50 μM of 1, 2, 8a-8f, and 9a-9f for 1 hour. CCCP was used at 10 μM, and treatment was for 3 hours. Cells were then lysed and probed for anti-phospho-Ser65 parkin (pS65 parkin), total parkin, OPA1, and GAPDH. UU: untreated and non-transfected HeLa cells. UT: untreated and parkin-transfected HeLa cells. Data represent triplicates. [Figure 3A] N6-benzyladenosine induces mitophagy and inhibits Ub Ser65 phosphorylation in cells and astrocytes. (A) YFP-Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside or N6-benzyladenosine for 24 h and then lysed or treated with 10 μM CCCP for 1 h. Immunofluorescence data examining Ub Ser65 phosphorylation, YFP-Parkin expression, and TOM20. Scale bar = 40 μm. (B) Quantification of Ub pSer65 localization to mitochondria. (C) Astrocytes were treated with 50 μM kinetin (1), N6-methyladenosine (9a), and N6-benzyladenosine (9c), followed by 10 nM valinomycin for 5 h. Samples were examined for phospho-Ub. (D) Induction of mitophagy in PINK1 wild-type and PINK1 knockout MEFs after treatment with 5 μM CCCP, N6-benzyladenosine (9c), and kinetin riboside (KR) for xx hours (n=3). (E) Quantification of mitophagy-treated MEFs from d. [Figure 3B]N6-benzyladenosine induces mitophagy and inhibits Ub Ser65 phosphorylation in cells and astrocytes. (A) YFP-Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside or N6-benzyladenosine for 24 h and then lysed or treated with 10 μM CCCP for 1 h. Immunofluorescence data examining Ub Ser65 phosphorylation, YFP-Parkin expression, and TOM20. Scale bar = 40 μm. (B) Quantification of Ub pSer65 localization to mitochondria. (C) Astrocytes were treated with 50 μM kinetin (1), N6-methyladenosine (9a), and N6-benzyladenosine (9c), followed by 10 nM valinomycin for 5 h. Samples were examined for phospho-Ub. (D) Induction of mitophagy in PINK1 wild-type and PINK1 knockout MEFs after treatment with 5 μM CCCP, N6-benzyladenosine (9c), and kinetin riboside (KR) for xx hours (n=3). (E) Quantification of mitophagy-treated MEFs from d. [Figure 3C]N6-benzyladenosine induces mitophagy and inhibits Ub Ser65 phosphorylation in cells and astrocytes. (A) YFP-Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside or N6-benzyladenosine for 24 h and then lysed or treated with 10 μM CCCP for 1 h. Immunofluorescence data examining Ub Ser65 phosphorylation, YFP-Parkin expression, and TOM20. Scale bar = 40 μm. (B) Quantification of Ub pSer65 localization to mitochondria. (C) Astrocytes were treated with 50 μM kinetin (1), N6-methyladenosine (9a), and N6-benzyladenosine (9c), followed by 10 nM valinomycin for 5 h. Samples were examined for phospho-Ub. (D) Induction of mitophagy in PINK1 wild-type and PINK1 knockout MEFs after treatment with 5 μM CCCP, N6-benzyladenosine (9c), and kinetin riboside (KR) for xx hours (n=3). (E) Quantification of mitophagy-treated MEFs from d. [Figure 3D]N6-benzyladenosine induces mitophagy and inhibits Ub Ser65 phosphorylation in cells and astrocytes. (A) YFP-Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside or N6-benzyladenosine for 24 h and then lysed or treated with 10 μM CCCP for 1 h. Immunofluorescence data examining Ub Ser65 phosphorylation, YFP-Parkin expression, and TOM20. Scale bar = 40 μm. (B) Quantification of Ub pSer65 localization to mitochondria. (C) Astrocytes were treated with 50 μM kinetin (1), N6-methyladenosine (9a), and N6-benzyladenosine (9c), followed by 10 nM valinomycin for 5 h. Samples were examined for phospho-Ub. (D) Induction of mitophagy in PINK1 wild-type and PINK1 knockout MEFs after treatment with 5 μM CCCP, N6-benzyladenosine (9c), and kinetin riboside (KR) for xx hours (n=3). (E) Quantification of mitophagy-treated MEFs from d. [Figure 3E]N6-benzyladenosine induces mitophagy and inhibits Ub Ser65 phosphorylation in cells and astrocytes. (A) YFP-Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside or N6-benzyladenosine for 24 h and then lysed or treated with 10 μM CCCP for 1 h. Immunofluorescence data examining Ub Ser65 phosphorylation, YFP-Parkin expression, and TOM20. Scale bar = 40 μm. (B) Quantification of Ub pSer65 localization to mitochondria. (C) Astrocytes were treated with 50 μM kinetin (1), N6-methyladenosine (9a), and N6-benzyladenosine (9c), followed by 10 nM valinomycin for 5 h. Samples were examined for phospho-Ub. (D) Induction of mitophagy in PINK1 wild-type and PINK1 knockout MEFs after treatment with 5 μM CCCP, N6-benzyladenosine (9c), and kinetin riboside (KR) for xx hours (n=3). (E) Quantification of mitophagy-treated MEFs from d. [Figure 4] FIG. 1 shows that kinetin riboside and nucleosides 9a and 9c have no effect on mitochondrial fragmentation, parkin localization, or ubiquitin phosphorylation. [Figure 5A] Docking of kinetin riboside and nucleoside 9c into the human AlphaFold structure of human PINK1. (A) Docking of kinetin riboside into human PINK1. (B) 2D interaction between the dynamic riboside and human PINK1. (C) Docking of nucleoside 9c into human PINK1. (D) 2D interaction between nucleoside 9c and human PINK1. [Figure 5B]Docking of kinetin riboside and nucleoside 9c into the human AlphaFold structure of human PINK1. (A) Docking of kinetin riboside into human PINK1. (B) 2D interaction between the dynamic riboside and human PINK1. (C) Docking of nucleoside 9c into human PINK1. (D) 2D interaction between nucleoside 9c and human PINK1. [Figure 5C] Docking of kinetin riboside and nucleoside 9c into the human AlphaFold structure of human PINK1. (A) Docking of kinetin riboside into human PINK1. (B) 2D interaction between the dynamic riboside and human PINK1. (C) Docking of nucleoside 9c into human PINK1. (D) 2D interaction between nucleoside 9c and human PINK1. [Figure 5D] Docking of kinetin riboside and nucleoside 9c into the human AlphaFold structure of human PINK1. (A) Docking of kinetin riboside into human PINK1. (B) 2D interaction between the dynamic riboside and human PINK1. (C) Docking of nucleoside 9c into human PINK1. (D) 2D interaction between nucleoside 9c and human PINK1. [Figure 6A] FIG. 1 shows the effects of kinetin riboside, nucleoside 9c, and its triphosphate derivative on TcPINK1 in vitro using either ubiquitin (A) or parkin (B) as a substrate. [Figure 6B] FIG. 1 shows the effects of kinetin riboside, nucleoside 9c, and its triphosphate derivative on TcPINK1 in vitro using either ubiquitin (A) or parkin (B) as a substrate. [Figure 7]Protein sequence alignment of human PINK1 (hPINK1; UniProt ID Q9BXM7) and Tribolium castaneum PINK1 (TcPINK1; UniProt ID D6WMX4). Data were obtained using UniProt Align. Human PINK1 Cyc166 (equivalent to TcPINK1 T172) and Cys387 (equivalent to TcPINK1 Cys362) are identified by red rectangles. Data indicate that hPINK1 Cyc166 is not conserved, but hPINK1 Cyc387 is. [Figure 8A] (A) Superposition of full-length AlphaFold human PINK1 (hPINK1, shown in red; UniProt ID Q9BXM7) and full-length Tribolium castaneum PINK1 (TcPINK1, shown in blue; UniProt ID D6WMX4). (B) ATP-binding pockets of hPINK1 and (C) TcPINK1. [Figure 8B] (A) Superposition of full-length AlphaFold human PINK1 (hPINK1, shown in red; UniProt ID Q9BXM7) and full-length Tribolium castaneum PINK1 (TcPINK1, shown in blue; UniProt ID D6WMX4). (B) ATP-binding pockets of hPINK1 and (C) TcPINK1. [Figure 8C] (A) Superposition of full-length AlphaFold human PINK1 (hPINK1, shown in red; UniProt ID Q9BXM7) and full-length Tribolium castaneum PINK1 (TcPINK1, shown in blue; UniProt ID D6WMX4). (B) ATP-binding pockets of hPINK1 and (C) TcPINK1. DETAILED DESCRIPTION OF THE INVENTION

[0061] material and method All reagents and solvents were of general-purpose or analytical grade and were purchased from Sigma-Aldrich Ltd. (Merck), Fisher Scientific, Fluorochem, or Acros. H and C NMR data were recorded on a Bruker AVANCE DPX500 spectrometer operating at 202, 500, and 125 MHz, respectively. Chemical shifts (δ) are quoted in ppm, and J values ​​are quoted in Hz. The following abbreviations were used when reporting spectral data: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), td (triplet of doublet), and m (multiplet). All reactions were performed under a nitrogen atmosphere and monitored using analytical thin-layer chromatography on precoated silica plates (Kiesel Gel 60 F254, BDH). Compounds were visualized by irradiation under UV light (254 nm) or by the use of KMnO4 stain followed by heating. Flash column chromatography was performed using silica gel 60 (230-400 mesh) (Merck). HPLC was performed on a SHIMADZU Prominence-i quaternary low-pressure gradient pump equipped with a Prominence-i UV detector (190-700 nm). All solvents for HPLC were HPLC-grade and purchased from Fisher Scientific. HPLC data analysis was performed using the SHIMADZU Lab Solutions software package. The purity of the tested prodrugs was determined by HPLC; all were ≥95% pure, except where specified.

[0062] cell culture Both HeLa cells and Parkin-overexpressing HeLa cells were maintained in DMEM high glucose (Gibco) with 10% FBS (Sigma-Aldrich) at 37°C and 5% CO2. For experiments, cells were counted and seeded into various culture plates depending on the experiment. Cellometer Auto T4 cell counts were performed using Trypan Blue (Gibco). HeLa cells were incubated in T75 flasks (Corning) at 37°C and 5% CO2 until 70-80% confluency was achieved, at which point the cells were used for experiments. Alternatively, HeLa cells were seeded into well plates, and then transfected with 0.2 μg / ml Parkin cDNA using the PEI method once the seeded cells reached 60% confluency. After 6 hours, the medium was replaced. Cells were subcultured when they reached approximately 90% confluency.

[0063] Preparation of total protein lysates and protein concentration determination Cell cultures with 70-80% confluency were lysed as follows: Cells were washed with phosphate-buffered saline (PBS) (Sigma). 150 μl of lysis buffer was used for each well containing either: 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.27 M sucrose, 1 mM Na3VO4, 50 mM NaF, 5 mM Na pyrophosphate and fresh 1 mM benzamidine, 1% NP-40 and 0.1%; or · 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 10 mM Na β-glycerophosphate, 0.27 M sucrose, 1 mM Na3VO4, 50 mM NaF, 10 mM Na pyrophosphate with fresh 1 mM benzamidine, 1% Triton X-100, complete EDTA-free protease inhibitors, phosphatase inhibitor cocktail 3 and 100 μM 2-chloroacetamide.

[0064] The cells were dissociated, transferred to a microtube, and finally centrifuged at 12,000 rpm for 15 minutes at 4°C. Finally, the supernatant was transferred to a new microtube and stored at -20°C. Protein concentration was measured using the Bradford assay. Serial concentrations of bovine serum albumin (BSA) (Sigma-Aldrich) at 0.125, 0.25, 0.5, and 1 mg / ml were used as standards. Samples were boiled in SDS sample loading buffer at 90°C for 5 minutes.

[0065] antibody Anti-GAPDH (1:1,000 in 5% BSA / TBS-T, Cell Signaling), anti-PARKIN Phospho Ser65 (2 μg / ml, 5% milk / TBS-T, S210D, second bleed, University of Dundee), anti-PARKIN Phospho Ser65 (2 μg / ml, 5% milk / TBS-T, S210D, third bleed, University of Dundee), anti-PARKIN total (2 μg / ml, 5% milk / TBS-T, S966C, second bleed, University of Dundee), anti-PARKIN Phospho Ser65 (1:10,000 in 5% BSA / TBS-T, rabbit monoclonal, MJF foundation), non-phosphopeptide PARKIN Ser65 (2 mg / ml, 5% milk / TBS-T, University of Dundee), anti-PINK1 whole (2 µg / ml, 5% milk / TBS-T, S085D, third bleed, University of Dundee), anti-Bcl-xL whole (1:1,000, 5% BSA / TBS-T, Cell Signaling), anti-Bcl-xL Phospho Ser62 (1:1,000, 5% BSA / TBS-T, Invitrogen), anti-PINK1 Phospho Thr257 (2 µg / ml, 5% milk / TBS-T, S114D, third bleed, University of Dundee), non-phosphopeptide PINK1 Thr257 (2 mg / ml, 5% milk / TBS-T, University of Dundee), anti-OPA1 (1:1,000, 5% BSA / TBS-T, BD Biosciences). Anti-rabbit IgG HRP-conjugated (1:1,000 5% BSA / TBS-T, Cell Signaling), anti-sheep IgG HRP-conjugated (1:5,000 5% milk / TBS-T, Abcam), anti-mouse IgG HRP-conjugated (1:1,000 5% BSA / TBS-T, Cell Signaling).

[0066] Phospho-Ub immunoblotting Cells were pretreated with kinetin riboside for 23 hours, followed by treatment with 10 μM CCCP or niclosamide for 1 hour. Cells were then harvested using AP lysis buffer (50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1% IGEPAL, 20 mM MgCl2, 5 mM 2-mercaptoethanol, 10% glycerol (v / v), 1x protease inhibitor cocktail (Roche), 1x phosphatase inhibitor cocktail (Roche). Denatured proteins were loaded onto a Mini-PROTEAN TGX 4-12% precast gel, followed by protein separation. Proteins were then transferred to a methanol-activated PVDF membrane using a Trans-Blot Turbo System (Bio-Rad). The membrane was then blocked with 5% nonfat dry milk in TBS / 0.1% Tween (TBST) for 1 hour at room temperature. After blocking, the membrane was washed with TBST and 5% The membranes were incubated overnight at 4°C with agitation in primary antibodies diluted in BSA / TBST or 5% milk / TSBT. The next day, the membranes were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated antibodies for 1 hour at room temperature with agitation. After TBST washing, proteins were visualized using the Amersham™ ECL™ Prime kit (GE Biosciences) and imaged using a Bio-Rad ChemicDoc. Primary antibodies used for Western blotting were GAPDH (Abcam catalog no. ab9485), phospho-Ub Ser 65 (Sigma-Aldrich catalog no. ABS1513-I), and LC3B (Cell Signaling catalog no. 3868S). Secondary antibodies used for Western blotting were anti-rabbit HRP (Agilent Dako catalog no. P0399), and anti-mouse HRP (Agilent Dako catalog no. P0260).

[0067] Immunofluorescence Cells were seeded in PerkinElmer PhenoPlate 96-well plates and subsequently treated with compounds. Cells were fixed in chilled 4% PFA for 10 minutes, followed by washing with PBS. Cell permeabilization was achieved by adding PBS / 0.1% Triton X-100 for 5 minutes at room temperature. After the PBS wash, cells were blocked for 1 hour at room temperature using blocking buffer (PBS, 5% BSA, 0.1% Tween 20). Primary antibodies were diluted in blocking buffer, added to the cells, and incubated overnight at 4°C. Cells were then washed with PBS-T, and the appropriate secondary antibodies (diluted in blocking buffer) were added for 2 hours at room temperature in the dark. 10 μg / ml Hoechst 33258 stain was added along with the secondary antibodies, if necessary. Cells were washed again with PBS-T, followed by the addition of PBS containing 0.02% sodium azide. Cells were then stored at 4°C until ready for imaging. The primary antibodies used for immunofluorescence were: phospho-Ub Ser 65 (Sigma-Aldrich catalog no. ABS1513-I), TOM20 (Santa-Cruz catalog no. sc-17764), and the secondary antibodies and stains used for immunofluorescence were: anti-IgG2a Alexa Fluor™ 546 (ThermoFisher catalog no. A21133), anti-rabbit Alexa Fluor™ 647 (ThermoFisher catalog no. A21244), and Hoechst 33258 stain (Sigma-Aldrich catalog no. B2883).

[0068] Confocal microscopy was performed using a Zeiss LSM800 w / Airyscan fluorescence microscope equipped with lasers emitting at 405 nm, 488 nm, 561 nm, and 640 nm. All images were acquired using a 40x oil immersion objective and processed using Zeiss ZEN software. Images were analyzed and quantified using Columbus (PerkinElmer) and TIBCO Spotfire software.

[0069] To assess tetramethylrhodamine methyl ester perchlorate (TMRM) fluorescence, cell culture medium was removed and replaced with cell culture medium consisting of 5 nM TMRM (ThermoFisher) and 10 μg / ml Hoechst 33258 stain for 30 minutes at 37°C in the dark to allow for TMRM incorporation into mitochondria. After a PBS wash, FluoroBrite DMEM (Gibco) medium was added to the cells, followed by imaging at 37°C and 5% CO2 using a Zeiss LSM800 microscope.

[0070] PINK1 in vitro kinase assay The assay was performed by MRC PPU Reagents and Services (University of Dundee, UK). The assay was performed as follows: TcPINK1 (5–20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg / ml BSA, 0.1% mercaptoethanol) was assayed against GST PARK2 TV3 or ubiquitin-His in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EDTA, 10 mM DTT, protein substrate (0.3 mg / ml GST PARK2 TV3 or 1 mg / ml ubiquitin-His, 10 mM magnesium acetate, and 0.1 mM [33P-γ-ATP] (50–1000 cpm / pmole) in the presence of the relevant small molecule (serial dilution with the highest concentration being 100 μM) and incubated for 30 min at room temperature. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphate, followed by washing with 50 mM orthophosphate. Harvest onto Unifilter plates.

[0071] Example 1: PINK 1 activation (kinetin riboside) We previously reported that PINK1 activation by kinetin riboside 2 in cells was observed at high concentrations (50 μM), and we identified that detectable PINK1 activation by the nucleobase kinetin in cells was only observed in the presence of the mitochondrial uncoupler CCCP. Therefore, we first asked whether combining this nucleoside analog, kinetin riboside 2, with several known indirect PINK1 activators, such as niclosamide and CCCP, would result in synergistic and more significant activation of PINK1. To investigate this, we first treated Parkin-transfected HeLa cells with 50 μM kinetin, kinetin riboside, or kinetin riboside ProTide, a monophosphate prodrug of kinetin riboside, for 24 hours. Cells were then lysed or treated with 10 μM CCCP for 3 hours (Figure 1c). Niclosamide and CCCP treatment alone were used as controls. Cells were lysed and probed for Ub Ser65 (pUb), optic atrophy protein 1 (OPA1), and GAPDH. As expected, niclosamide and CCCP treatment alone resulted in robust phosphorylation of Ub at Ser65 (Figure 1c). Treatment of cells with 50 μM kinetin, kinetin riboside, or kinetin riboside ProTide alone did not result in significant phosphorylation of ubiquitin. Surprisingly, pretreatment of cells with kinetin riboside inhibited CCCP-induced phosphorylation of ubiquitin, whereas pretreatment with the related nucleobase, kinetin or kinetin riboside ProTide, did not. Treatment of cells with niclosamide and CCCP alone resulted in OPA1 cleavage, indicating mitochondrial membrane depolarization, whereas pretreatment with kinetin riboside did not prevent this effect (Figure 1c).

[0072] Surprised by this finding, we next investigated whether this same inhibition of CCCP-induced ubiquitin phosphorylation by kinetin riboside could also be observed with the mitochondrial uncoupler niclosamide. To this end, HeLa cells transfected with YFP-parkin were pretreated with 50 μM kinetin riboside for 24 h, and then lysed or treated with 10 μM niclosamide or CCCP for 1 h. Again, as expected, control samples in which cells were treated with niclosamide and CCCP alone induced strong ubiquitin phosphorylation, whereas pretreatment of cells with kinetin riboside inhibited both niclosamide- and CCCP-induced phosphorylation of ubiquitin (Figure 1d), similar to the results in Figure 1c.

[0073] We then investigated the ability of kinetin riboside to inhibit ubiquitin phosphorylation using immunofluorescence (Figure 1e). As shown in Figure 1d, HeLa cells transfected with YFP-parkin were left untreated or treated with 10 μM niclosamide or 10 μM CCCP alone for 1 hour, or pretreated with 50 μM kinetin riboside 24 hours before the addition of niclosamide or CCCP. Treatment of cells with niclosamide and CCCP again induced significant phosphorylation of ubiquitin, whereas pretreatment of cells with kinetin riboside inhibited ubiquitin phosphorylation (Figure 1e). Pretreatment with kinetin riboside inhibited ubiquitin phosphorylation but did not prevent the membrane potential collapse induced by niclosamide and CCCP (not shown), similar to the observations observed by investigating OPA1 cleavage (Figure 1c).

[0074] Example 2: N 6 Synthesis of substituted adenine and adenosine analogues. We were intrigued by the ability of kinetin riboside to inhibit niclosamide- and CCCP-induced ubiquitin phosphorylation and subsequently investigated the N-terminal amino acid sequence of kinetin and N-terminal amino acid sequence of kinetin riboside, which are structurally related to kinetin. 6 Substituted adenines and adenosines were designed and synthesized. In designing these nucleobases and nucleosides, the inventors focused on the N-substituted adenines and adenosines.6 We chose to modify position N and made a variety of small, medium and bulky substitutions at this position. 6 The substitutions were methyl, isopropyl, benzyl, tyramine, cyclopentylamine, neopentylamine, and furfuryl (8a–8f and 9a–9f, Figure 2a).

[0075] N 6 The synthesis of substituted adenine and adenosine compounds was carried out using one of two methods depending on the volatility of the nucleophile used in the reaction (Figure 2a). N2 Substitution was the preferred method (Method A) because it gave high yields after a simple purification step. This method involved heating 6-chloropurine or its nucleoliboside derivative with the corresponding nucleophile in ethanol in the presence of triethylamine. 6 Method B was used to synthesize adenine and adenosine analogs bearing either benzyl (8c and 9c), isopropyl (8d and 9d), methyl (8e and 9e), or tyramine (8f and 9f) modifications at the N-position. 6 The peptide coupling agent PyBOP was used to synthesize nucleobases and nucleosides bearing cyclopentylamine and neopentylamine at the nucleophilic positions. The choice of Method B, which uses neopentylamine (8a and 9a) and cyclopentylamine (8b and 9b) as nucleophiles, was dictated by the fact that these compounds are explosive when heated, precluding the use of Method A. For the synthesis of nucleobases and nucleosides in this case, hypoxanthine or inosine was added to PyBOP and partially dissolved in a mixture of acetonitrile and a substoichiometric amount of DMF. The corresponding nucleophile, cyclopentylamine or neopentylamine, was then added, and the reaction was allowed to proceed for 3 days.

[0076] Compared to Method A, Method B resulted in much lower yields and more complicated purification of the final compound.

[0077] Further details of the above synthesis can be found in the accompanying materials.

[0078] Example 3: PINK 1 activation (N 6 Substituted adenine and adenosine compounds Upon completion of the synthesis of Example 2, the ability of the synthesized nucleobases and nucleosides to activate PINK1 in cells was evaluated. HeLa cells, which endogenously express PINK1 but not parkin, were transiently transfected with parkin. Cells were then treated with nucleobases (8a–8f) and nucleosides (9a–9f) for 1 hour or with 10 μM CCCP as a control for 3 hours, and parkin Ser65 phosphorylation, total parkin, OPA1, and GAPDH as a loading control were examined. Results showed that CCCP resulted in significant activation of PINK1, as judged by parkin Ser65 phosphorylation, while nucleobases 8a–8f did not show significant activation of PINK1 at 50 μM, consistent with previous findings (Figure 2b). However, all of the tested nucleosides (9a–9f), except for compound 9f, showed significant activation of PINK1 (Figure 2b). Interestingly, activation of PINK1 by these nucleosides did not result in OPA1 cleavage, indicating that, in contrast to the CCCP-mediated activation of PINK1, which is associated with OPA1 cleavage, these nucleoside analogs activate PINK1 independently of mitochondrial depolarization. This is because the agent, CCCP, activates PINK1 indirectly through mitochondrial membrane depolarization, consistent with previous findings.

[0079] Example 4: CCCP / niclosamide-independent activation of PINK1 Next, we investigated the effects of kinetin riboside, nucleosides 9a and 9c, on mitochondria in the absence of niclosamide and CCCP. First, YFP-Parkin-transfected HeLa cells were left untreated or treated with 50 μM kinetin riboside, nucleosides 9a and 9c for 24 hours. Immunofluorescence analysis of TOM20 in these cells showed that these nucleoside analogs alone did not affect mitochondrial fragmentation, Parkin localization, or ubiquitin phosphorylation (Figure 3). YFP-Parkin-expressing HeLa cells were then treated with 50 μM kinetin riboside, nucleosides 9a or 9c for 24 hours, followed by treatment with 10 μM CCCP. Monitoring ubiquitin Ser65 phosphorylation using immunofluorescence revealed that CCCP treatment induced strong ubiquitin phosphorylation and promoted Parkin localization to mitochondria (Figure 3a). Notably, ubiquitin phosphorylation was inhibited by kinetin riboside pretreatment, consistent with the data (Figure 2c-e). Regarding novel compounds, pretreatment with nucleoside analog 9a followed by CCCP treatment did not significantly affect ubiquitin phosphorylation and Parkin localization to mitochondria compared to CCCP treatment alone (Figure 3a). However, pretreatment with nucleoside analog 9c resulted in a significant decrease in ubiquitin phosphorylation and Parkin localization to mitochondria, which appeared stronger than that induced by kinetin riboside (Figure 3a and Figure 3b).

[0080] Given the consistent observation that kinetin riboside and nucleoside 9c inhibit ubiquitin phosphorylation in cells, we investigated whether this effect was evident in astrocytes. Briefly, astrocytes were treated with 50 μM of the nucleobase kinetin, nucleosides 9a and 9c, or DMSO for 24 hours. Five hours before lysis, samples were treated with 50 nM of the mitochondrial uncoupler valinomycin. Ubiquitin Ser65 phosphorylation was then examined, demonstrating that treatment with the nucleobase kinetin or nucleosides 9a and 9c alone resulted in increased ubiquitin Ser65 phosphorylation (Figure 3c), consistent with the results observed for parkin Ser65 phosphorylation shown in Figure 2b. Interestingly, pretreatment of astrocytes with kinetin, nucleosides 9a, and 9c for 24 h, followed by treatment with valinomycin for 5 h, significantly suppressed ubiquitin Ser65 phosphorylation compared with astrocytes treated with valinomycin alone (black bars, Fig. 3c).

[0081] Example 5: PINK1-dependent mitophagy Using the inhibition of ubiquitin phosphorylation and Parkin localization to mitochondria by kinetin riboside and the nucleoside analog 9c, we next asked whether these compounds still induce mitophagy in a PINK1-dependent manner. To investigate this, we used the MitoQC assay, which is established to measure mitophagy in cells. 11、12Indeed, immortalized MEFs expressing either wild-type (WT) or knockout (KO) PINK1 were treated with 20 μM CCCP or 5 μM kinetin riboside or compound 9c for 24 hours. Consistent with previous reports, CCCP treatment induced increased mitophagy in MEFs expressing PINK1 wild-type but not in PINK1 KO MEFs (Figures 3d-e, enlarged view 3d). Interestingly, treatment of PINK1 WT MEFs with kinetin riboside or the nucleoside analog 9c also induced mitophagy, but this was not observed in PINK1 KO MEFs (Figures 3d-e). The data show that both kinetin riboside and nucleoside analog 9c induce low-level mitophagy in a PINK1-dependent manner, which is consistent with previous observations that kinetin riboside and nucleoside 9c (Fig. 2b) induce low-level PINK1 activation as judged by parkin phosphorylation.

[0082] Example 6: Molecular modeling studies In summary, the results of Examples 1 to 5 show that kinetin riboside and N 6 Substituted adenosine compounds, especially N 6 -benzyladenosine (9c) exhibits the ability to inhibit niclosamide- and CCCP-induced ubiquitin phosphorylation. To our knowledge, there are two examples where 9c inhibits the ability of PINK1 to phosphorylate ubiquitin. 13、14 The first involves the PD-associated mutations C125G and Q126P, which disrupt the intramolecular interaction between the N- and C-terminal extensions of human PINK1, leading to inhibition of ubiquitin phosphorylation. 14 The second is the oxidation of human PINK1 Cys166 and / or Cyc387. 13 Structural inspection and molecular modeling studies of the AlphaFold human PINK1 structure (Figure 5) showed that these cysteine ​​residues are distant from the nucleobase binding sites of nucleoside analogues and their triphosphate derivatives, and therefore, it is unlikely that they could be covalent inhibitors.

[0083] To further investigate this, we performed in vitro kinase assays using recombinant and constitutively active Tribolium castaneum PINK1 (TcPINK1) and either human parkin or ubiquitin as substrates in the presence of increasing concentrations (up to 100 μM) of kinetin riboside, benzyladenosine 9c, or its triphosphate derivatives (Figure 6). The results suggest that these compounds are unlikely to be covalent inhibitors of PINK1, although it should be noted that human PINK1 Cyc166 is not conserved in TcPINK1, whereas Cyc387 is conserved across human PINK1 and TcPINK1 (Figure 7). Furthermore, superposition of the ATP pockets of hPINK1 and TcPINK1 indicated that the TcPINK1 ATP pocket is more open than that of hPINK1, with the position of hPINK1 Cyc387 being closer to that of TcPINK1 Cys362, whereas the position of TcPINK1 Thr172 is farther away from that of hPINK1 Cyc166 (Figure 8). This likely affects the ability of hPINK1 to phosphorylate ubiquitin. Regarding the second possibility that kinetin riboside and 9c disrupt the intramolecular interaction between the N- and C-terminal extensions of PINK1, similar to the Q126P mutation, this may be due to these compounds or their phosphorylated species binding to the ATP pocket of PINK1 and inducing a conformational change that rearranges the N- and C-termini, rendering PINK1 unable to phosphorylate ubiquitin.

[0084] overview We describe herein nucleoside analogs as inducers of low-level PINK1-mitophagy, suppressing ubiquitin phosphorylation triggered by the mitochondrial uncouplers CCCP, valinomycin, and niclosamide. These nucleoside analogs represent useful tool compounds for deciphering the role of phosphoubiquitin signaling, including its Parkin-independent functions, such as inhibition of the E2 / E3 ubiquitin system and deubiquitinases. Importantly, postmortem analysis of the substantia nigra of patients with Lewy body dementia (the primary site of neuronal loss) as well as patients with age-related PD revealed elevated levels of Ub Ser65 phosphorylation compared with healthy age-matched controls. 9、10 Nucleoside analogs that exhibit inhibition of ubiquitin phosphorylation may have utility in the treatment of dementia with Lewy bodies or idiopathic PD.

[0085] appendix N 6 Synthesis and evaluation of substituted adenine and adenosine analogues. N-Methyl-9H-purin-6-amine (8a). Methylamine (0.40 mL, 9.01 mmol, 2.8 equiv.) and TEA (0.45 mL, 3.23 mmol, 1 equiv.) were added to a stirred solution of 6-chloropurine (500 mg, 3.23 mmol, 1 equiv.) in EtOH (15 mL). The reaction was then heated to 30° C. for 16 hours. The solvent was then removed under reduced pressure. The remaining crude oil was then purified by column chromatography using DCM / MeOH (19:1) as the eluent to give a white solid (200 mg, 41%). 1 H NMR(500MHz,DMSO)δ 12.93(1H,s,NH),8.25(1H,s,H-4),8.13(1H,s,H-1),7.60(1H,s,NH),3.02(3H,s,H-6);13C NMR(126MHz,DMSO)δ 152.90(C-4),152.85(C-3),27.40(C-6);HRMS-ES(m / z): Actual value [M+H] +[150.0781, [C6H7N5H] requires 150.0780. HPLC (reverse phase) 0.5 mL / min in 12 min with MeOH / H2O 80:20, λ = 254 nm, Rt = 4.88 min (99%).

[0086] N-Isopropyl-9H-purin-6-amine (8b). Isopropylamine (0.28 mL, 3.29 mmol, 1 equiv.) and TEA (0.45 mL, 3.23 mmol, 1 equiv.) were added to a stirred solution of 6-chloropurine (500 mg, 3.23 mmol, 1 equiv.) in EtOH (15 mL). The reaction was then heated to 40° C. for 16 hours. The solvent was then removed under reduced pressure. The remaining crude oil was then purified by column chromatography using DCM / MeOH (19:1) as the eluent to give a white solid (11.7 mg, 2%). 1 H NMR(500MHz,MeOD)δ 8.21(1H,s,H-4),8.06(1H,s,H-1),3.25-3.03(1H,m,H-6),1.32(6H,d,J=6.5Hz,H-7);13C NMR(126MHz,MeOD)δ 46.52(C-6),7.82(C-7). HPLC (reversed phase) 0.5 mL / min 12 min with MeOH / H2O 90:10, λ=254 nm, Rt=4.69 min (99%).

[0087] N-Benzyl-9H-purin-6-amine (8c). Benzylamine (0.42 mL, 3.88 mmol, 1.2 equiv.) and TEA (0.54 mL, 3.88 mmol, 1.2 equiv.) were added to a stirred solution of 6-chloropurine (500 mg, 3.24 mmol, 1 equiv.) in EtOH (15 mL). The reaction was then refluxed at 80° C. for 16 h. The product precipitated out of solution upon vigorous stirring in an ice-water bath. OL035 was then filtered off and further purified by column chromatography using DCM / MeOH (19:1) as the eluent to give a white solid (240 mg, 33%). 1H NMR(500MHz,MeOD)δ 8.25(1H,s,H-1),8.07(1H,s,H-4),7.39(2H,d,J=7.6Hz,H-8),7.32(2H,t,J=7.5Hz,H-9),7.25(1H,t,J=7.3Hz,H-10);13C NMR(126MHz,MeOD)δ 152.42(C-4),128.18(C-9),127.18(C-8),126.88(C-10)46.48(C-6);HRMS-ES(m / z):Actual value [M+H] + 226.1089, [C12H11N5H] requires 226.1093. HPLC (reverse phase) 0.5 mL / min in 12 min with MeOH / H2O 80:20, λ = 254 nm, Rt = 5.63 min (99%).

[0088] 4-(2-((9H-Purin-6-yl)amino)ethyl)phenol (8d). Tyramine (533 mg, 3.88 mmol, 1.2 equiv) and TEA (0.54 mL, 3.88 mmol, 1.2 equiv) were added to a stirred solution of 6-chloropurine (500 mg, 3.24 mmol, 1 equiv) in EtOH (15 mL). The reaction was then refluxed at 80° C. for 16 h. The product precipitated out of solution upon vigorous stirring in an ice-water bath. OL037 was then filtered off and further purified by column chromatography using DCM / MeOH (19:1) as eluent to give a white solid (141 mg, 17%). 1 H NMR(500MHz,DMSO)δ 13C NMR(126MHz,DMSO)δ 156.09(C-11),130.04(C-8),129.96(C-9),115.58(C-10),45.80(C-6),36.53(C-7);HRMS-ES(m / z):Actual value [M+H] +256.1205, [C13H13N5OH] requires 256.1198. HPLC (reverse phase) 0.5 mL / min in 12 min with MeOH / H2O 80:20, λ = 254 nm, Rt = 4.96 min (86%).

[0089] N-Cyclopentyl-9H-purin-6-amine (8e). Hypoxanthine (500 mg, 3.67 mmol, 1 eq.), cyclopentylamine (0.54 mL, 5.51 mmol, 1.5 eq.), and PyBOP (2.294 g, 4.41 mmol, 1.2 eq.) were dissolved in anhydrous ACN (20 mL) and a substoichiometric amount of DMF (2 mL) under an inert atmosphere. DIPEA (1.28 mL, 7.35 mmol, 2 eq.) was then added slowly over 5 min at RT, and the reaction was left stirring for 3 days. The solvent was then removed under reduced pressure. The remaining crude oil was then purified by column chromatography using DCM / MeOH (19:1) as the eluent to give a white solid (130 mg, 17%). 1 H NMR(500MHz,MeOD)δ 8.23(1H,s,H-4),8.07(1H,s,H-1),2.14-2.07(1H,m,H-6),1.85-1.60(8H,m,H-7,8);13C NMR(126MHz,DMSO)δ 152.80(C-4), 32.77(C-6), 23.91(C-7,8). HRMS-ES(m / z): Actual value [M+H] + 204.1244, [C10H13N5H] requires 204.1249. HPLC (reverse phase) 0.5 mL / min in 12 min with MeOH / H2O 80:20, λ = 254 nm, Rt = 5.78 min (99%).

[0090] N-Neopentyl-9H-purin-6-amine (8f). Hypoxanthine (300 mg, 2.20 mmol, 1 eq.), neopentylamine (0.39 mL, 3.31 mmol, 1.5 eq.), and PyBOP (1.721 g, 3.31 mmol, 1.5 eq.) were dissolved in anhydrous ACN (20 mL) and a substoichiometric amount of DMF (2 mL) under an inert atmosphere. DIPEA (0.77 mL, 4.41 mmol, 2 eq.) was then added slowly over 5 min at RT, and the reaction was left stirring for 3 days. The solvent was then removed under reduced pressure. The remaining crude oil was then purified by column chromatography using DCM / MeOH (19:1) as the eluent to give a white solid (31 mg, 7%). 1 H NMR(500MHz,MeOD)δ 8.74(1H,s,H-1),8.58(1H,s,H-4),3.19-3.15(2H,m,H-6),1.89-1.84(9H,m,H-8),13C NMR(126MHz,MeOD)δ 176.56 (C-5), 151.66 (C-4), 151.61 (C-3), 145.74 (C-1), 70.13 (C-6), 25.95 (C-8). HPLC (reversed phase) 0.5 mL / min 12 min with MeOH / H2O 90:10, λ=254 nm, Rt=4.72 min (99%).

[0091] (2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-(methylamino)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (9a). Methylamine (0.26 mL, 5.86 mmol, 2.8 equiv) and TEA (0.29 mL, 2.08 mmol, 1 equiv) were added to a stirred solution of 6-chloropurine riboside (600 mg, 2.09 mmol, 1 equiv) in EtOH (15 mL). The reaction was then heated to 30° C. for 16 hours. The solvent was then removed under reduced pressure. The remaining crude oil was then purified by column chromatography using DCM / MeOH (19:1) as the eluent to give a white solid (274 mg, 73%). 1H NMR(500MHz,MeOD)δ 8.23(2H,s,H-6,9),5.95(1H,d,J=6.5Hz,H-5),4.74(1H,dd,J=6.4,5.1Hz,H-4),4.32-4. 31(1H,m,H-3),4.16(1H,q,J=2.5Hz,H-2),3.90-3.72(2H,m,H-1),1.28(3H,m,H-11);13C NMR(126MHz,MeOD)δ 89.89(C-5),86.83(C-2),74.05(C-4),71.32(C-3),62.13(C-1),7.89(C-11);LCMS-ES(m / z):Actual value [M+H] + 282.10, [C11H15N5O4H] requires 282.11. HPLC (reverse phase) 0.5 mL / min in 12 min with MeOH / H2O 90:10, λ = 254 nm, Rt = 4.73 min (99%).

[0092] (2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-(isopropylamino)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (9b). Isopropylamine (0.18 mL, 2.11 mmol, 1 equiv.) and TEA (0.29 mL, 2.08 mmol, 1 equiv.) were added to a stirred solution of 6-chloropurine riboside (600 mg, 2.09 mmol, 1 equiv.) in EtOH (15 mL). The reaction was then heated to 40° C. for 16 hours. The solvent was then removed under reduced pressure. The remaining crude oil was then purified by column chromatography using DCM / MeOH (19:1) as the eluent to give a white solid (471 mg, 73%). 1H NMR(500MHz,MeOD)δ 8.24(1H,s,H-6),8.21(1H,s,H-9),5.94(1H,d,J=6.5Hz,H-5),4.74(1H,dd,J=6.4,5.1Hz,H-4),4.31(1H,m H-3),4.16(1H,m,H-2),3.81(2H,m,H-1),1.31(6H,d,J=6.5Hz,H-12);13C NMR(126MHz,MeOD)δ 152.17(C-9),139.98(C-6),89.91(C-5),86.84(C-2),74.05(C-4),71.32(C-3),62.12(C-1),21.43(C-12);LCMS-ES(m / z): Actual value [M+H] + 310.13, [C13H19N5O4H] requires 310.14. HPLC (reverse phase) 0.5 mL / min in 12 min with MeOH / H2O 80:20, λ = 254 nm, Rt = 5.03 min (99%).

[0093] (2R,3R,4S,5R)-2-(6-(benzylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (9c). Benzylamine (0.46 mL, 4.19 mmol, 1.5 equiv) and TEA (0.58 mL, 4.19 mmol, 1.5 equiv) were added to a stirred solution of 6-chloropurine riboside (800 mg, 2.79 mmol, 1 equiv) in EtOH (15 mL). The reaction was then refluxed at 80° C. for 16 hours. The product precipitated out of solution upon vigorous stirring in an ice-water bath. OL031 was then filtered off and dried under reduced pressure to give a white solid (948 mg, 95%). 1H NMR(500MHz,MeOD)δ 8.26(1H,s,H-6),8.23(1H,s,H-9),7.38(2H,d,J=7.6Hz,H-13),7.31(2H,t,J=7.5Hz,H-14),7.24(1H,t,J=7.3Hz,H-15),5.96(1H,d,J 13C NMR(126MHz,MeOD)δ 128.15(C-14),127.11(C-13),126.84(C-15),89.92(C-5),86.83(C-2),74.05(C-4),71.31(C-3),62.12(C-1);LCMS-ES(m / z):Actual value [M+H] + 358.13, [C17H19N5O4H] requires 358.14. HPLC (reverse phase) 0.5 mL / min in 12 min with MeOH / H2O 80:20, λ = 254 nm, Rt = 5.34 min (99%).

[0094] (2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-((4-hydroxyphenethyl)amino)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (9d). Inosine (500 mg, 1.86 mmol, 1 equiv.), tyramine (384 mg, 2.80 mmol, 1.5 equiv.), and PyBOP (1.46 g, 2.80 mmol, 1.5 equiv.) were dissolved in anhydrous ACN (20 mL) and a substoichiometric amount of DMF (2 mL) under an inert atmosphere. DIPEA (0.65 mL, 3.73 mmol, 2 equiv.) was then added slowly over 5 min at RT, and the reaction was left stirring for 3 days. The solvent was then removed under reduced pressure. The remaining crude oil was then purified by column chromatography using DCM / MeOH (19:1) as eluent to give a white solid (347 mg, 48%); 1H NMR(500MHz,DMSO)δ 8.34(1H,s,H-6),8.24(1H,s,H-9),7.04(2H,d,J=7.3Hz,H-14),6.68(2H,d,J=8.4Hz,H-15),5.89(1H,d,J=6.2Hz,H-5),4.63-4.60(1 13C NMR(126MHz,DMSO)δ 129.99(C-14),115.58(C-15),86.37(C-2),73.92(C-4),71.13(C-3),62.14(C-1),36.79(C-12);LCMS-ES(m / z):Actual value [M+H] + 388.20, [C18H21N5O5H] requires 388.15. HPLC (reverse phase) 0.5 mL / min in 12 min with MeCN / H2O 80:20, λ = 254 nm, Rt = 4.38 min (99%).

[0095] (2R,3R,4S,5R)-2-(6-(cyclopentylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (9e). Inosine (750 mg, 2.80 mmol, 1 eq.), cyclopentylamine (0.41 mL, 4.19 mmol, 1.5 eq.), and PyBOP (2.183 g, 4.19 mmol, 1.5 eq.) were dissolved in anhydrous ACN (20 mL) and a substoichiometric amount of DMF (2 ml) under an inert atmosphere. DIPEA (0.97 ml, 5.59 mmol, 2 eq.) was then added slowly over 5 min at RT, and the reaction was left stirring for 3 days. The solvent was then removed under reduced pressure. The remaining crude oil was then purified by column chromatography using DCM / MeOH (19:1) as eluent to give a white solid (749 mg, 80%); 1H NMR(500MHz,MeOD)δ 8.27(1H,s,H-6),8.23(1H,s,H-9),5.97(1H,d,J=6.5Hz,H-5),4.76(1H,dd,J=6.4,5.1Hz,H-4),4.34(1H,dd,J=5.1,2. 5Hz,H-3),4.19(1H,q,J=2.5Hz,H-2),3.92-3.75(2H,m,H-1),2.15-2.09(1H,m,H-11),1.86-1.61(8H,s,H-12,13);13C NMR(126MHz,DMSO)δ 152.84(C-9),140.06(C-6),88.42(C-5),86.37(C-2),73.92(C-4),71.12( C-3),62.14(C-1),32.49(C-11),23.93(C-12,13);LCMS-ES(m / z): Actual value [M+H] + 336.16, [C15H21N5O4H] requires 336.16. HPLC (reverse phase) 0.5 mL / min in 12 min with MeOH / H2O 80:20, λ = 254 nm, Rt = 5.43 min (95%).

[0096] (2R,3S,4R,5R)-2-(hydroxymethyl)-5-(6-(neopentylamino)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (9f). Inosine (300 mg, 1.12 mmol, 1 equiv.), neopentylamine (0.20 mL, 1.68 mmol, 1.5 equiv.), and PyBOP (873 mg, 1.68 mmol, 1.5 equiv.) were dissolved in anhydrous ACN (20 mL) and a substoichiometric amount of DMF (2 mL) under an inert atmosphere. DIPEA (0.39 mL, 2.24 mmol, 2 equiv.) was then added slowly over 5 min at RT, and the reaction was left stirring for 3 days. The solvent was then removed under reduced pressure. The remaining crude oil was then purified by column chromatography using DCM / MeOH (19:1) as eluent to give a white solid (305 mg, 81%); 11H NMR (500 MHz, MeOD) δ 8.27 (1H, s, H-6), 8.20 (1H, s, H-9), 5.95 (1H, d, J = 6.5 Hz, H-5), 4.75 (1H, dd, J = 6.4, 5.1 Hz, H-4), 4.33 (1H, dd, J = 5.1, 2.5 Hz, H-3), 4.17 (1H, q, J = 2.5 Hz, H-2), 3.90 - 3.73 (2H, m, H-1), 3.50 - 3.46 (2H, m, H-11), 1.00 (9H, s, H-13); 13C NMR (126 MHz, MeOD) δ 155.42 (C-10), 152.13 (C-9), 147.66 (C-8), 140.04 (C6), 119.84 (C-7), 89.94 (C-5), 86.86 (C-2), 74.06 (C-4), 71.33 (C-3), 62.13 (C-1), 51.06 (C-11), 31.94 (C-12), 26.28 (C-13); LCMS-ES (m / z): Measured value [M+H] + 338.18, [C15H23N5O4H] requires 338.18. HPLC (reverse phase) 0.5 mL / min for 12 min with MeCN / H2O 80:20, λ = 254 nm, Rt = 4.66 min (99%).

[0097] References 1. Valente EM, Abou-Sleiman PM, Caputo V, Muqit MM, Harvey K, Gispert S, Ali Z, Del Turco D, Bentivoglio AR, Healy DG, et al: Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 2004, 304:1158 - 1160. 2. Khalil B, El Fissi N, Aouane A, Cabirol-Pol MJ, Rival T, Lievens JC: PINK1-induced mitophagy promotes neuroprotection in Huntington’s disease. Cell Death Dis 2015, 6: e1617. 3.Witte ME,Mahad DJ,Lassmann H,van Horssen J:Mitochondrial dysfunction contributes to neurodegeneration in multiple sclerosis.Trends Mol Med 2014,20:179-187. 4.Reddy PH:Role of mitochondria in neurodegenerative diseases:mitochondria as a therapeutic target in Alzheimer’s disease.CNS Spectr 2009,14:8-13;discussion 16-18. 5.Billia F,Hauck L,Konecny F,Rao V,Shen J,Mak TW:PTEN-inducible kinase 1(PINK1) / Park6 is indispensable for normal heart function.Proc Natl Acad Sci U S A 2011,108:9572-9577. 6.Liu X,Ye B,Miller S,Yuan H,Zhang H,Tian L,Nie J,Imae R,Arai H,Li Y,et al:Ablation of ALCAT1 mitigates hypertrophic cardiomyopathy through effects on oxidative stress and mitophagy.Mol Cell Biol 2012,32:4493-4504. 7.Morais VA,Verstreken P,Roethig A,Smet J,Snellinx A,Vanbrabant M,Haddad D,Frezza C,Mandemakers W,Vogt-Weisenhorn D,et al:Parkinson’s disease mutations in PINK1 result in decreased Complex I activity and deficient synaptic function.EMBO Mol Med 2009,1:99-111. 8.Wilhelmus MM,van der Pol SM,Jansen Q,Witte ME,van der Valk P,Rozemuller AJ,Drukarch B,de Vries HE,Van Horssen J:Association of Parkinson disease-related protein PINK1 with Alzheimer disease and multiple sclerosis brain lesions.Free Radic Biol Med 2011,50:469-476. 9.Fiesel FC,et al.:(Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation.EMBO Rep 2015,16(9):1114-1130. 10.Hou X,et al.:Age-and disease-dependent increase of the mitophagy marker phospho-ubiquitin in normal aging and Lewy body disease.Autophagy 2018,14(8):1404-1418. 11.Allen,G.F.G.,et al.:Loss of iron triggers PINK1 / Parkin-independent mitophagy.EMBO Rep.2013,14(12):1127-35. 12.McWilliams,T.G.,et al.:mito-QC illuminates mitophagy and mitochondrial architecture in vivo.J Cell Biol.2016,214(3):333-45. 13.Gan,Z.Y.,et al.:Activation mechanism of PINK1.Nature.2022,602(7896):328-335. 14.Kakade,P.,et al.:Mapping of a N-terminal α-helix domain required for human PINK1 stabilization,Serine228 autophosphorylation and activation in cells.Open Biol.2022,12(1):210264.

Claims

1. Compounds of general formula (I), including all tautomers thereof, for use in the treatment of neurodegenerative diseases or conditions and / or cancers characterized by high levels of ubiquitin Ser65 phosphorylation 【Chemistry 1】 General formula (I) (In the formula, R １ OH, halo, nitro, C １～４ Alkyl, C １～４ haloalkyl, —O(C １～４ alkyl), —O(C １～４ haloalkyl), NH ２ , NH(C １～４ alkyl) and N(C １～４ alkyl) ２ C may be substituted with one or more substituents selected from １～１０ Alkyl, C ３～１０ Cycloalkyl, C ６～１０ is an aryl, heterocycloalkyl, or heteroaryl group; R ２ is the furanose moiety of general formula (II): 【Chemistry 2】 General formula (II) (In the formula, X is O, NH, S or CH ２ and R ３ H, OH, halo, nitro, C １～４ Alkyl, C １～４ haloalkyl, —O(C １～４ alkyl), —O(C １～４ haloalkyl), NH ２ , NH(C １～４ alkyl) and N(C １～４ alkyl) ２ or a mono-, di- or tri-phosphate derivative of general formula (VIII), １１ are independently selected from OH or aryloxy, amino acid ester, or pivaloyloxymethyl masking groups. 【Transformation 3】 General formula (VIII) and R ４ and R ５ H, OH, halo, nitro, C １～４ Alkyl, C １～４ haloalkyl, —O(C １～４ alkyl), —O(C １～４ haloalkyl), NH ２ , NH (C １～４ alkyl) and N(C １～４ alkyl) ２ (independently selected from is) or a pharmaceutically or veterinarily acceptable salt or hydrate thereof.

2. 2. A compound for use according to claim 1 for use in the treatment of idiopathic Parkinson's disease, dementia with Lewy bodies and / or cancer.

3. R1 is a group of general formula (III-A), (III-B), (III-C), (III-D), (III-E) or (III-F): 【Chemistry 4】 General formula (III-A); 【Transformation 5】 General formula (III-B); 【Transformation 6】 General formula (III-C); 【Transformation 7】 General formula (III-D); 【Transformation 8】 General formula (III-E); 【Chemistry 9】 General formula (III-F) (In the formula, n is 0, 1, 2 or 3; m is 0, 1, 2 or 3; Z is O, NH, S or CH ２ and preferably CH ２ and R ６ is OH or O(C １～４ alkyl) 3. A compound for use according to claim 1 or 2, wherein

4. R １ but, 【Chemistry 10】 4. The compound for use according to claim 3, selected from:

5. X is O and / or R ４ and R ５ are independently selected from H and OH, and / or R ３ A compound for use according to any one of claims 1 to 4, wherein is H, OH or a mono-, di- or tri-phosphate derivative of general formula (VIII).

6. R ３ , R ４ and R ５ 6. The compound for use according to claim 5, wherein each of is OH.

7. R ３ and R ４ are both OH, and R ５ 6. The compound for use according to claim 5, wherein is H.

8. The compound of the general formula (I) 【Chemistry 11】 3. A compound for use according to claim 1 or 2, selected from:

9. Compounds of formula (I), including all tautomers thereof 【Chemistry 12】 General formula (I) (In the formula, R １ OH, halo, nitro, C １～４ Alkyl, C １～４ haloalkyl, —O(C １～４ alkyl), —O(C １～４ haloalkyl), NH ２ , NH(C １～４ alkyl) and N(C １～４ alkyl) ２ C may be substituted with one or more substituents selected from １～１０ Alkyl, C ３～１０ Cycloalkyl, C ６～１０ is an aryl, heterocycloalkyl, or heteroaryl group; R ２ is the furanose moiety of general formula (II): 【Chemistry 13】 General formula (II) (In the formula, X is O, NH, S or CH ２ and R ３ H, OH, halo, nitro, C １～４ Alkyl, C １～４ haloalkyl, —O(C １～４ alkyl), —O(C １～４ haloalkyl), NH ２ , NH(C １～４ alkyl) and N(C １～４ alkyl) ２ or a mono-, di- or tri-phosphate derivative of general formula (VIII), １１ are independently selected from OH or aryloxy, amino acid ester, or pivaloyloxymethyl masking groups. 【Chemistry 14】 General formula (VIII) and R ４ and R ５ H, OH, halo, nitro, C １～４ Alkyl, C １～４ haloalkyl, —O(C １～４ alkyl), —O(C １～４ haloalkyl), NH ２ , NH (C １～４ alkyl) and N(C １～４ alkyl) ２ (independently selected from is) or a pharmaceutically or veterinarily acceptable salt or hydrate thereof, However, the compound is not kinetin riboside.

10. R １ is a group of general formula (III-A), (III-B), (III-C), (III-D), (III-E) or (III-F): 【Chemistry 15】 General formula (III-A); 【Chemistry 16】 General formula (III-B); 【Chemistry 17】 General formula (III-C); [Chemistry 18] General formula (III-D); 【Chemistry 19】 General formula (III-E); 【Chemistry 20】 General formula (III-F) (In the formula, n is 0, 1, 2 or 3; m is 0, 1, 2 or 3; Z is O, NH, S or CH ２ and preferably CH ２ and R ６ is OH or O(C １～４ alkyl) 10. The compound of claim 9, wherein:

11. R １ but, 【Chemistry 21】 10. The compound of claim 9 selected from:

12. X is O and / or R ４ and R ５ are independently selected from H and OH, and / or R ３ is H, OH or a mono-, di- or tri-phosphate derivative of general formula (VIII).

13. R ３ , R ４ and R ５ each of which is OH or R ３ and R ４ are both OH, and R ５ The compound of claim 12, wherein is H. 【Request Item 14】 【Chemistry 22】 10. The compound of claim 9 selected from:

15. (A) reacting a 6-halopurine derivative of general formula (V) with a nucleophilic compound of general formula (VI): 【Chemistry 23】 (V) 【Chemistry 24】 (VI); or (B) reacting a hypoxanthine derivative of general formula (VII) with a nucleophilic compound of general formula (VI) in the presence of benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate ("PyBOP"): 【Chemistry 25】 (VII) 【Chemistry 26】 (VI) (In the formula, R ７ is halo, preferably chloro, and R １ and R ２ is as defined for general formula (I) according to any one of claims 9 to 14.

15. A process for preparing a compound of general formula (I) according to any one of claims 9 to 14, comprising:

16. 15. A compound or kinetin riboside according to any one of claims 9 to 14 for use in the preparation of a medicament for treating cancer or a disorder or condition associated with high levels of phosphorylated ubiquitin.

17. A method for treating cancer or a disorder or condition associated with high levels of phosphorylated ubiquitin, comprising administering an effective amount of kinetin riboside or a compound described in any one of claims 9 to 14 to a patient in need of such treatment.

18. 18. The compound for use according to claim 16 or the method according to claim 17, wherein the disorder or condition associated with high levels of phosphorylated ubiquitin is selected from idiopathic Parkinson's disease and dementia with Lewy bodies.

19. 15. A pharmaceutical composition comprising kinetin riboside or a compound according to any one of claims 9 to 14 and a pharmaceutically or veterinarily acceptable excipient or carrier.

20. 20. The pharmaceutical composition of claim 19, formulated for oral delivery.

21. 15. A combination therapy comprising a dynamic riboside or a compound of any one of claims 9 to 14 and a further therapeutic agent used in the treatment of cancer or a neurodegenerative disease or condition characterized by high levels of ubiquitin Ser65 phosphorylation, for simultaneous, separate or sequential use in the treatment of cancer or a neurodegenerative disease or condition characterized by high levels of ubiquitin Ser65 phosphorylation.

Images