Adenosine derivatives for treatment of neurodegenerative disorders and cancer

Abstract

The present invention relates to compounds of general formula (I): (general formula I) wherein R1 and R2 are as defined herein; the compounds are useful for the treatment of neurodegenerative diseases or conditions characterized by elevated phosphorylation levels of ubiquitin Ser65 and / or for the treatment of cancer. The invention also relates to pharmaceutical compositions and combination therapeutics comprising compounds of formula (I) for the treatment of these disorders, and methods of using compounds of formula (I) in the treatment of such diseases and disorders. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a therapeutic agent for treating neurodegenerative diseases or disorders and / or cancer characterized by elevated levels of ubiquitin Ser65 phosphorylation, and a method for treating such diseases or disorders, wherein the therapeutic agent or composition or combination therapeutic agent according to the present invention is administered to a subject suffering from or suspected of suffering from a neurodegenerative disease or disorder and / or cancer characterized by elevated levels of ubiquitin Ser65 phosphorylation. Background Art

[0002] Parkinson's disease belongs to a class of disorders called movement system disorders and is the result of the loss of dopamine-producing brain cells. Parkinson's disease (PD) is the second leading cause of human neurodegeneration, and to date, there is no treatment that can slow or stop the clinical progression.

[0003] Mitochondria play an important role in the pathogenesis of PD as well as other neurodegenerative diseases and cardiomyopathies. In this regard, mitochondrial serine / threonine PTEN-induced kinase 1 (PINK1) has emerged as a key factor 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 the depolarization of the inner mitochondrial membrane, PINK1 stabilizes in its full-length form on the outer mitochondrial membrane (OMM). The accumulation of PINK1 leads to autophosphorylation and its subsequent activation. Then, active PINK1 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, resulting in the degradation of mitochondria by the autophagy mechanism, a process called mitophagy.

[0004] Therefore, activating PINK1 to trigger mitophagy has been generally recognized as a potential new therapeutic target for Parkinson's disease. Specifically, it has been emphasized that the activity of the PINK1 kinase is crucial in preventing the development of neurodegeneration, as evidenced by the fact that loss-of-function mutations in it lead to early-onset Parkinson's disease (PD). This observation has led to the discovery that when cells are exposed to the depolarizing agent carbonyl cyanide m-chlorophenylhydrazone (CCCP), a reagent used to stimulate mitochondrial damage, kinetin - N 6 -substituted adenine (1, Figure 1b ) enhances PINK1 activation in cells. It has been noted that the kinetin activation of PINK1 is due to its biotransformation into the active metabolite kinetin riboside triphosphate (3, Figure 1b), this metabolite is a new ATP-substrate of PINK1. In view of this observation, and in view of our interest in developing nucleoside analog therapeutics, we subsequently found that the nucleoside derivative of kinetin, also known as kinetin riboside 2, showed stronger CCCP-independent activation of PINK1 in cells compared to its nucleobase derivative kinetin, as demonstrated by parkin Ser65 phosphorylation.

[0005] Thus, upon mitochondrial depolarization, phosphorylation of ubiquitin by the mitochondrial protein kinase PINK1 is generally considered an important step in the repair and recycling of mitochondria by autophagy, and thus activators of PINK1 are considered promising therapies for treating various forms of PD.

[0006] However, recent evidence from autopsy analyses of brain tissues from PD and dementia patients without obvious genetic pathogenic factors for PINK1 (such as dementia with Lewy bodies and idiopathic forms of PD (i.e., elderly and sporadic PD patients)) has shown the opposite, with elevated levels of Ub Ser65 phosphorylation 9,10 . Thus, these subsets of PD and dementia patients show elevated levels of phosphorylated ubiquitin (ubiquitin Ser65 phosphorylation) in the brain. Therefore, this means that conventional PINK1 activators, and thus the associated enhanced ubiquitin phosphorylation, are ineffective in such patients with significantly elevated Ub Ser65 phosphorylation. In addition, although the role of mitophagy in tumorigenesis has not been fully elucidated, a large body of evidence indicates that mitophagy dysregulation is often associated with many types of cancer, and the accumulation of dysfunctional mitochondria can lead to tumorigenesis.

[0007] We disclose herein that in cells and astrocytes, N 6 -substituted adenosines, such as N 6 -(2-furfuryl)adenosine (known as kinetin riboside) and N 6 -benzyladenosine, unexpectedly inhibited ubiquitin phosphorylation induced by the established mitochondrial depolarizing agents (CCCP and niclosamide). Although these nucleoside analogs inhibited ubiquitin phosphorylation induced by niclosamide and CCCP, they did not prevent mitochondrial membrane depolarization. Notably, treatment of cells with these nucleoside analogs alone induced low levels of mitophagy without causing mitochondrial fragmentation. In summary, this study presents N 6-Substituted adenosines as novel inhibitors of PINK1-mediated ubiquitin phosphorylation, highlighting their potential use in the unexpected treatment of elderly and sporadic PD, as well as patients with Lewy body dementia (where elevated levels of phosphorylated ubiquitin are present and where conventional PINK1 activators have no effect) and / or in the treatment of cancer. Our findings suggest that these compounds and their analogs hold promise as therapeutic agents to arrest the neuronal death process in idiopathic Parkinson's disease and related disorders, and / or to arrest the tumorigenesis process caused by the accumulation of dysfunctional mitochondria. SUMMARY OF THE INVENTION

[0008] Aspects of the invention are set out in the appended claims.

[0009] According to a first aspect, the invention provides a compound of general formula (I), including all its tautomers:

[0010]

[0011] Wherein:

[0012] R 1 is a C 1-10 alkyl, C 3-10 cycloalkyl, C 6-10 aryl, heteroalkyl or heteroaryl group, optionally substituted with one or more substituents selected from: 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; and

[0013] R 2 is a furanosyl moiety of general formula (II):

[0014]

[0015] Wherein:

[0016] X is O, NH, S or CH2;

[0017] R 3 is H, OH, halo, nitro, C 1-4 alkyl, C 1-4 haloalkyl, -O(C 1-4 alkyl), -

[0018] O(C 1-4 haloalkyl), NH2, NH(C 1-4 alkyl) and N(C1-4 a (C1-C10)alkyl)2, or a monophoaphate, diphosphate or triphosphate derivative of formula (VIII), where q is 0, 1 or 2, each R 11 is independently selected from OH or aryloxy, amino ester or pivaloyloxymethyl masking group;

[0019]

[0020] and

[0021] R 4 and R 5 are independently selected from H, OH, halo, nitro, C 1-4 alkyl, C 1-4 haloalkyl, -

[0022] O(C 1-4 alkyl), -O(C 1-4 haloalkyl), NH2, NH(C 1-4 alkyl) and N(C 1-4 alkyl)2,

[0023] or a pharmaceutically or veterinarily acceptable salt or hydrate thereof,

[0024] for treating neurodegenerative diseases or disorders and / or cancer characterized by elevated levels of ubiquitin Ser65 phosphorylation.

[0025] Compounds of formula (I) have been shown to inhibit PINK1-mediated ubiquitin phosphorylation and can thus be used to treat disorders and conditions associated with elevated levels of phosphorylated ubiquitin for which conventional PINK1 activators would have no effect.

[0026] Compounds of formula (I) are particularly suitable for treating idiopathic (i.e., age-related and / or sporadic) Parkinson's disease, treating dementia with Lewy bodies and / or cancer. More specifically, compounds of formula (I) can be used to treat idiopathic Parkinson's disease and / or dementia with Lewy bodies.

[0027] In the context of the present invention, the term "C 1-10 alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon group having 1 to 10 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, tert-butyl and n-hexyl. It will be appreciated that other alkyl groups are as defined above but have different numbers of carbon atoms. For example, "C 1-4 alkyl" has 1 to 4 carbon atoms.

[0028] In the context of the present specification, the term "C 3-10 cycloalkyl" refers to a cyclic saturated hydrocarbon group containing 3 to 10 carbon atoms and containing a single ring or multiple fused rings.

[0029] The term "C 6-10 aryl" refers to a ring system having aromaticity, having 6 to 10 ring carbon atoms, and containing a monocyclic or multiple fused rings. When the aryl group contains two fused rings, both rings do not need to be completely aromatic. Examples of the aromatic moiety are phenyl and naphthyl.

[0030] In the context of this specification, the term "heterocycloalkyl" refers to a saturated ring system having 3 to 10 ring atoms (unless otherwise specified) and containing a monocyclic or multiple fused rings, wherein at least one, optionally two or three ring atoms are heteroatoms selected from N, O, and S.

[0031] In the context of this specification, the term "heteroaryl" refers to a ring system having aromaticity, having 6 to 10 ring atoms (unless otherwise specified) and containing a monocyclic or multiple fused rings, wherein at least one, optionally two or three ring atoms are heteroatoms selected from N, O, and S. When the heteroaryl group contains more than one ring, not all rings need to be completely aromatic. Examples of heteroaryl groups include pyridine, pyrimidine, indole, pyrrole, imidazole, triazole, tetrazole, oxazole, thiazole, benzofuran, benzimidazole, and dihydroindole.

[0032] In this specification, "halo" refers to fluoro, chloro, bromo, or iodo, more preferably chloro or bromo.

[0033] In this specification, "C 1-4 haloalkyl" refers to a C 1-4 alkyl group substituted by one or more halogen atoms up to complete substitution. Examples include chloromethyl, trifluoromethyl, 2-chloroethyl, 1-bromoethyl, etc.

[0034] In the context of this specification, the term "aryloxy" refers to a protecting group of the general formula (IX-A), wherein R 12 is a C 5-25 aryl or 5- to 25-membered heteroaryl group, either of which is optionally substituted by one or more functional groups selected from hydroxyl, mercapto, thioether, alkoxy, and amino:

[0035]

[0036] In the context of this specification, the term "amino ester" refers to a protecting group of the general formula (IX-B), wherein: R 13 is H, or a saturated or unsaturated hydrocarbon, preferably a C 1-4 alkyl chain, which is optionally substituted by one or more functional groups selected from mercapto, thioether, alkoxy, and amino; and R 14 is a saturated or unsaturated hydrocarbon, preferably a C 1-4An alkyl chain or a C6 aryl group, optionally substituted by one or more functional groups selected from hydroxy, mercapto, thioether, alkoxy and amino:

[0037]

[0038] In the context of the present specification, the term "pivaloyloxymethyl" refers to a protecting group of general formula (IX-C), where R 15 and R 16 each independently is hydrogen, a halogenated group or a C 1-10 alkyl group, optionally substituted by one or more substituents selected from hydroxy, mercapto, thioether, alkoxy and amino, or R 15 and R 16 , and together with the carbon to which R 15 and R 16 is attached form a C 3-4 cycloalkyl group or a 3- or 4-membered heterocycloalkyl group:

[0039]

[0040] The salts of the compounds of general formula (I) are pharmaceutically or veterinarily acceptable salts. Depending on the nature of R 1 to R 5 , these salts can be basic addition salts, such as sodium salts, potassium salts, calcium salts, aluminum salts, zinc salts, magnesium salts and other metal salts, as well as choline, diethanolamine, ethanolamine, ethylenediamine, meglumine and other well-known basic addition salts, as summarized in Paulekuhn et al., (2007) J. Med. Chem. 50:6665 - 6672 and / or known to those skilled in the art. Alternatively, when the compound of general formula (I) contains an amino group, it can be quaternized to form a salt with a counterion, such as halides, hydroxides, sulfates, nitrates, phosphates, formates, acetates, trifluoroacetates, fumarates, citrates, tartrates, oxalates, succinates, mandelates, methanesulfonates and p-toluenesulfonates.

[0041] In the compounds of general formula (I), R 1 is preferably a substituted C 1-6 alkyl, C 3-6 cycloalkyl or C6 aryl, optionally substituted by one or more substituents selected from OH and C 1-4 alkyl.

[0042] More preferably, R 1 is a group of general formula (III-A), (III-B), (III-C), (III-D), (III-E) or (III-F):

[0043]

[0044]

[0045] Wherein:

[0046] n is 0, 1, 2 or 3;

[0047] m is 0, 1, 2 or 3;

[0048] Z is O, NH, S or CH2, and preferably CH2; and

[0049] R 6 is OH or O(C 1-4 alkyl).

[0050] Most preferably, R 1 is selected from:

[0051]

[0052] As described above, the compound of general formula (I) comprises 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 monophosphate, diphosphate or triphosphate derivative of general formula (VIII).

[0053] In some compounds of the present invention, R 3 , R 4 and R 5 are each OH. As will be readily understood by the skilled reader, such compounds are adenosine ribonucleoside analogs.

[0054] In other suitable compounds, R 3 and R 4 are both hydroxyl groups, and R 5 is H. As will be readily understood by the skilled reader, such compounds are adenosine deoxyribonucleoside analogs.

[0055] Exemplary compounds of general formula (I) include the known compound kinetin riboside having the structure of formula (IV):

[0056]

[0057] Other exemplary compounds of general formula (I) include the following novel N 6 -substituted adenosine analogs:

[0058]

[0059] In a particularly preferred embodiment, the compounds of formula (I) are selected from the following structures:

[0060]

[0061] As described above, the known compound kinetin riboside is a compound of formula (I) as defined above. Thus, according to a second aspect, the present invention provides a compound of formula (I), including all its tautomers:

[0062]

[0063] wherein:

[0064] R 1 is a C 1-10 alkyl, C 3-10 cycloalkyl, C 6-10 aryl, heteroalkyl or heteroaryl group, optionally substituted by one or more substituents selected from: 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; and

[0065] R 2 is a furanosyl moiety of formula (II):

[0066]

[0067] wherein:

[0068] X is O, NH, S or CH2;

[0069] R 3 is H, OH, halo, nitro, C 1-4 alkyl, C 1-4 haloalkyl, -O(C 1-4 alkyl), -

[0070] O(C 1-4 haloalkyl), NH2, NH(C 1-4 alkyl) and N(C 1-4 alkyl)2, or a monophosphate, diphosphate or triphosphate derivative of formula (VIII), where q is 0, 1 or 2, and each R 11 is independently selected from OH or aryloxy, amino acid ester or pivaloyloxymethyl protecting group;

[0071]

[0072] and

[0073] R 4 and R 5 are independently selected from H, OH, halo, nitro, C 1-4 alkyl, C 1-4 haloalkyl, -

[0074] 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 pharmaceutically or veterinarily acceptable salt or hydrate thereof, provided that the compound is not kinetin riboside.

[0075] Preferred features relating to R 1 , R 2 , R 3 , R 4 , R 5 and X are as described above for the first aspect of the invention.

[0076] The compounds of general formula (I) can be prepared by the methods disclosed below.

[0077] Specifically, the N 6 -substituted adenosine analogues of general formula (I) can be prepared by coupling a nucleoside derivative of 6-halopurine with an amine nucleophile via a bimolecular nucleophilic substitution (S N2 ) reaction.

[0078] In this reaction, the compounds of general formula (I) are prepared in a one-step process by reacting a 6-halopurine derivative of general formula (V) with a nucleophilic compound of general formula (VI):

[0079]

[0080] wherein R7 is a halo group, preferably a chloro group, and R1 and R2 are as defined for general formula (I). In a preferred method, this nucleophilic substitution reaction is carried out in the presence of a tertiary amine (preferably triethylamine (‘TEA’)) and / or at an elevated temperature (i.e. 30 °C or higher).

[0081] Alternatively, the N 6 -substituted adenosine analogues of general formula (I) can be prepared by reacting a nucleoside derivative of hypoxanthine with an amine nucleophile in the presence of a peptide coupling reagent.

[0082] In this reaction, the compound of general formula (I) is prepared via a one-step method by reacting a hypoxanthine derivative of general formula (VII) with a nucleophilic compound of general formula (VI) in the presence of benzotriazol-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (‘PyBOP’):

[0083]

[0084] wherein R 1 and R 2 are as defined in general formula (I). In a preferred method, this peptide coupling reaction is carried out in the presence of a tertiary amine, preferably N,N-diisopropylethylamine (‘DIPEA’).

[0085] These methods for preparing the compound of general formula (I) represent the third aspect of the present invention.

[0086] Many compounds of general formula (V), (VI) and (VII) are well-known and readily available. Those skilled in the art can easily synthesize other compounds of general formula (V), (VI) and (VII) using standard methods.

[0087] According to a fourth aspect, the present invention provides the use of kinetin riboside or a compound according to the second aspect of the present invention in the preparation of a medicament for the treatment of cancer or a disorder or disease associated with elevated levels of phosphorylated ubiquitin.

[0088] According to a fifth aspect, the present invention extends to a method for treating cancer or a disorder or disease associated with elevated levels of phosphorylated ubiquitin, the method comprising administering to a patient in need thereof an effective amount of kinetin riboside or a compound according to the second aspect of the present invention.

[0089] In preferred embodiments of the fourth and fifth aspects, the disorder or disease 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 disease is selected from idiopathic Parkinson's disease and / or dementia with Lewy bodies.

[0090] It should be understood that kinetin riboside and / or the compound according to the second aspect of the present invention will generally be administered as part of a pharmaceutical composition. Accordingly, in a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising kinetin riboside or a compound according to the second aspect of the present invention and a pharmaceutically or veterinarily acceptable excipient or carrier.

[0091] Suitable pharmaceutical excipients are well known to those skilled in the art. The pharmaceutical compositions can be formulated for administration by any suitable route, such as oral administration, rectal administration, nasal administration, bronchial (inhalation) administration, topical (including eye drops, oral and sublingual) administration, vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, and can be prepared by any method well known in the pharmaceutical art.

[0092] The compositions can be prepared by associating kinetin riboside or a compound of the second aspect of the present invention with a carrier. Generally, the formulations are prepared by uniformly and intimately associating the compound with a liquid carrier or a finely divided solid carrier or both of the above carriers, and then shaping the product if necessary.

[0093] The oral administration formulations of the present invention can be presented in the following forms: discrete units, such as capsules, sachets or tablets, each containing a predetermined amount of the compound; powders or granules; aqueous or non-aqueous liquid solutions or suspensions of the compound; or oil-in-water liquid emulsions or water-in-oil liquid emulsions; or pills, etc.

[0094] For oral administration compositions (such as tablets and capsules), the term "acceptable carrier" includes carriers, such as common excipients, such as binders, such as syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, such as corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants, such as magnesium stearate, sodium stearate and other metal stearates, glyceryl stearate, stearic acid, silicone oil, talc wax, oil and colloidal silica. Flavoring agents, such as peppermint, wintergreen oil, cherry flavor, etc. can also be used. Colorants may be added to make the dosage form easily recognizable. Tablets can also be coated by methods well known in the art.

[0095] Tablets can be made by compression or molding, optionally using one or more accessory ingredients. Compressed tablets can be prepared by compressing the compound in a free-flowing form (such as powder or granules) in a suitable machine, optionally mixed with a binder, a lubricant, an inert diluent, a preservative, a surfactant or a dispersant. Molded tablets can be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine. Tablets can be optionally coated or scored and can be formulated to provide sustained release or controlled release of the active pharmaceutical agent.

[0096] Other suitable oral dosage forms include: lozenges, which contain the active agent in a flavored base, usually sucrose and gum arabic or tragacanth; pastilles, which contain the active agent in an inert base such as gelatin and glycerin or sucrose and gum arabic; and mouthwashes, which contain the active agent in a suitable liquid carrier.

[0097] Parenteral dosage forms are generally sterile.

[0098] For topical application to the skin, the composition can be formulated as a cream, ointment, gel, solution, or suspension, etc. Cream or ointment formulations for use in medicine are well-known conventional formulations in the art, for example, as described in pharmaceutical standard textbooks such as the British Pharmacopoeia.

[0099] In a preferred embodiment of this aspect of the invention, the composition is formulated for oral delivery.

[0100] One of ordinary skill in the art can readily determine the precise amount of the composition having a therapeutic effect as defined herein and the optimal route of administration of such a compound. Of course, these amounts will depend on the specific disorder being treated, the severity of the disorder, the parameters of the individual patient (including age, physical condition, body size, and weight), the duration of treatment, the nature of any concurrent therapies (if any), the specific route of administration, and similar factors within the knowledge and expertise of the health care provider. These factors are well known to those of ordinary skill in the art and can be addressed with routine experimentation. Generally, it is preferred to use the maximum dose of the individual component or combination thereof, i.e., the highest safe dose based on reasonable medical judgment. However, one of ordinary skill in the art will understand that a patient may, for medical, psychological, or almost any other reason, insist on using a lower or tolerable dose.

[0101] Depending on different parameters, in particular the mode of administration used and the condition of the subject, the dose of the compound or composition according to the invention to be administered to the subject can be selected. Other factors include the desired treatment time. If the subject does not respond adequately to the initial dose applied, higher doses (or higher effective doses by a different, more local delivery route) can be employed within the tolerance of the patient.

[0102] In some cases, the compounds of formula (I) can be used in combination with additional therapeutic agents, particularly therapeutic agents for the treatment of cancer or neurodegenerative diseases or disorders characterized by an elevated level of ubiquitin Ser65 phosphorylation.

[0103] Accordingly, in a seventh aspect of the present invention, there is provided a combination therapeutic agent comprising a compound of general formula (I) and an additional therapeutic agent for treating cancer or a neurodegenerative disease or disorder, for use simultaneously, separately or sequentially in the treatment of cancer or a neurodegenerative disease or disorder characterized by an elevated level of ubiquitin Ser65 phosphorylation.

[0104] When the compound of general formula (I) and the additional therapeutic agent are used simultaneously, they may be provided in the form of a pharmaceutical composition. Accordingly, the present invention also provides a pharmaceutical composition for treating a neurodegenerative disease or disorder characterized by an elevated level of ubiquitin Ser65 phosphorylation, comprising a compound of general formula (I), an additional therapeutic agent for treating a neurodegenerative disease or disorder, and a pharmaceutically acceptable excipient or carrier.

[0105] In some cases, the neurodegenerative disease or disorder is idiopathic (i.e., age-related or sporadic) Parkinson's disease and the other therapeutic agent is an agent for treating Parkinson's disease. Examples of therapeutic agents for treating Parkinson's disease include levodopa, ropinirole, rotigotine, pramipexole, and amantadine, as well as folic acid or deoxynucleosides and their monophosphates.

[0106] Throughout the description and claims of this specification, the words "comprising" and "containing" and variations of these words, such as "including" or "having", mean "including but not limited to" and do not exclude other moieties, additives, components, integers or steps. Unless the context requires otherwise, throughout the description and claims of this specification, the singular encompasses the plural. In particular, in the case of the use of an indefinite article, the specification is to be understood as contemplating both the plural and the singular unless the context requires otherwise.

[0107] All references cited in this specification, including any patents or patent applications, are incorporated herein by reference. No admission is made that any reference constitutes prior art. Furthermore, no admission is made that any prior art constitutes a part of the common general knowledge in the art.

[0108] Preferred features of each aspect of the present invention may be described as combined with any other aspect.

[0109] By the following examples, other features of the present invention will become apparent. Generally, the present invention is applicable to any novel feature or any combination of novel features disclosed in this specification (including the appended claims and drawings). Accordingly, features, integers, characteristics, compounds or chemical moieties that are combined with a particular aspect, embodiment or example of the present invention are to be understood as applicable to any other aspect, embodiment or example disclosed herein unless incompatible therewith.

[0110] In addition, unless otherwise specified, any feature disclosed herein may be replaced by alternative features that achieve the same or similar purpose. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] The present invention will now be described by way of example only, with reference to the following embodiments and the following drawings, in which:

[0112] Figure 1. Kinetin riboside inhibits niclosamide- and CCCP-induced Ub Ser65 phosphorylation. (A) Schematic representation of PINK1 / Parkin signaling in healthy and damaged mitochondria. (B) Chemical structure of kinetin and its metabolite PINK1 ATP-new substrate kinetin riboside triphosphate. (C) Parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 h, then lysed or treated with 10 μM CCCP for 3 h. Cell lysates were probed for Ub Ser65 phosphorylation, OPA1, and GAPDH. UU: untreated and untransfected cells; UT: untreated and transfected cells. (D) YFP-parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 h, then lysed or treated with 10 μM niclosamide or CCCP for 1 h. Cell lysates were probed for Ub Ser65 phosphorylation, LC3B-I / -II, and α-tubulin. N.T.: untreated cells. (E) As in (d), but samples were studied for Ub Ser65 phosphorylation, YFP-parkin expression, and TOM20 using immunofluorescence. Scale bar = 40 μM.

[0113] Figure 2. N 6 -substituted adenines and adenosines: chemical synthesis and their activation of PINK1 in cells. (A) Method A reagents and conditions: triethylamine, ethanol, heat, 16 h. Method B reagents and conditions: PyBOP, DIPEA, acetonitrile / DMF, 3 days, room temperature (rt). (B) Parkin-transfected HeLa cells were treated with 50 μM 1, 2, 8a-8f, and 9a-9f for 1 h. CCCP was used at 10 μM and treated for 3 h. Cells were then lysed and probed for anti-phosphorylated Ser65 Parkin (pS65 Parkin), total parkin, OPA1, and GAPDH. UU: untreated and untransfected HeLa cells. UT: untreated and Parkin-transfected HeLa cells. The data represent three replicates.

[0114] Figure 1. N 6-Benzyladenosine induces mitophagy in cells and astrocytes and inhibits Ub Ser65 phosphorylation. (A) YFP-parkin transfected HeLa cells were pretreated with 50 μM kinetin riboside or N 6 -benzyladenosine for 24 h and then lysed or treated with 10 μM CCCP for 1 h. Immunofluorescence data probed for Ub Ser65 phosphorylation, YFP-parkin expression, and TOM20. Scale bar = 40 μM. (B) Quantification of the localization of Ub pSer65 in mitochondria. (C) Astrocytes were treated with 50 μM kinetin (1), N 6 -methyladenosine (9a), and N 6 -benzyladenosine (9c) and then treated with 10 nM valinomycin for 5 h. Samples were probed for phosphorylated Ub. (D) Mitophagy was induced in MEF expressing wild-type PINK1 and PINK1 knockout MEF after treatment with 5 μM CCCP, N 6 -benzyladenosine (9c), and kinetin riboside (KR) for xx h. n = 3. e. Quantification of MEF undergoing mitophagy in d.

[0115] Figure 4 . Kinetin riboside and nucleosides 9a and 9c do not affect mitochondrial fragmentation, parkin localization, or ubiquitin phosphorylation.

[0116] Figure 5. Docking of kinetin riboside and nucleoside 9c to the human AlphaFold structure of human PINK1. (A) Docking of kinetin riboside to human PINK1. (B) 2D interaction between kinetin riboside and human PINK1. (C) Docking of nucleoside 9c to human PINK1. (D) 2D interaction between nucleoside 9c and human PINK1.

[0117] Figure 6. In vitro effects of kinetin riboside, nucleoside 9c, and their triphosphate derivatives on TcPINK1, using ubiquitin (A) or parkin (B) as substrates.

[0118] Figure 7.Protein sequence alignment of human PINK1 (hPINK1; UniProt ID Q9BXM7) and Tribolium castaneum PINK1 (TcPINK1; UniProt ID D6WMX4). The data was obtained using UniProt Align. Human PINK1 Cyc166 (equivalent to TcPINK1 T172) and Cys387 (equivalent to TcPINK1 Cys362) are marked with red rectangles. The data shows that hPINK1 Cyc166 is not conserved, while hPINK1 Cyc387 is conserved.

[0119] Figure 8. (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 pocket of hPINK1 and (C) TcPINK1. Detailed implementation

[0120] Materials and methods

[0121] All reagents and solvents were of general or analytical grade and were purchased from Sigma-Aldrich Ltd. (Merck), Fisher Scientific, Fluorochem or Acros. 1H and 13C NMR data were recorded on a Bruker AVANCE DPX500 spectrometer operating at 202, 500 and 125 MHz, respectively. Chemical shifts (δ) are reported in ppm and coupling constants (J) in Hz. The following abbreviations were used when reporting spectral data: s (singlet), d (doublet), t (triplet), q (quartet), dd (doubledoublet), td (tripledoublet) and m (multiplet). All reactions were carried out under a nitrogen atmosphere and monitored using analytical thin layer chromatography on precoated silica gel plates (kieselgel 60F254, BDH). Compounds were visualized by irradiation with UV light (254 nm) or by using a KMnO4 stain followed by heating. Flash column chromatography was performed using silica gel 60 (230 - 400 mesh) (Merck). HPLC was carried out on a SHIMADZU Prominence-i quaternary low pressure gradient pump equipped with a Prominence-i UV detector (190 to 700 nm). All solvents for HPLC were HPLC grade solvents purchased from Fisher Scientific. HPLC data analysis was performed using the SHIMADZU Lab solutions software package. The purity of the prodrugs tested was determined by HPLC and, unless otherwise stated, they had a purity of ≥ 95%.

[0122] Cell culture

[0123] Both HeLa and HeLa cells overexpressing parkin were maintained at 37 °C and 5% CO2 in high glucose DMEM medium (Gibco) supplemented with 10% FBS (Sigma-Aldrich). For experiments, cells were counted and seeded into a series of culture plates. Depending on the experiment, cell counting was performed using TrypanBlue (Gibco) with a Cellometer Auto T4. HeLa cells were incubated in T75 flasks (Corning) at 5% CO2 and 37 °C until they reached 70 - 80% confluence, at which point the cells were used for experiments. Alternatively, HeLa cells were seeded in well plates and then transfected with 0.2 μg ml -1 of parkin cDNA using the PEI method. After 6 h, the medium was changed. Passage was carried out when the cells reached approximately 90% confluence.

[0124] Preparation of total protein lysates and measurement of protein concentration

[0125] Lyse cell cultures at 70 - 80% confluence by the following method: Wash the cells with phosphate buffered saline (PBS) (Sigma). Use 150 μl of lysis buffer per well, each well containing:

[0126] · 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 sodium pyrophosphate and fresh 1 mM benzamidine, 1% NP-40 and 0.1; or

[0127] · 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 0.27

[0128] M sucrose, 1 mM Na3VO4, 50 mM NaF, 10 mM sodium pyrophosphate with fresh 1 mM benzamidine,

[0129] 1% Triton X-100, protease inhibitor cocktail completely free of EDTA, phosphatase inhibitor cocktail 3 and 100 μM 2-chloroacetamide.

[0130] Scrape the cells and transfer them to a microtube, and finally spin at 12,000 rpm for 15 minutes at 4°C. Finally, transfer the supernatant to a new microtube and store at -20°C. Measure the protein concentration using the Bradford assay. Bovine serum albumin (BSA) (Sigma-Aldrich) at serial concentrations of 0.125; 0.25; 0.5 and 1 mg / ml is used as a standard. Samples are boiled in SDS sample loading buffer at 90°C for 5 minutes.

[0131] Antibody

[0132] Anti-GAPDH antibody (1:1000 5% BSA / TBS-T, Cell Signaling), anti-PARKIN phosphorylated Ser65 antibody (2 μg / ml, 5% milk / TBS-T, S210D, second blood collection, University of Dundee), anti-PARKIN phosphorylated Ser65 antibody (2 μg / ml, 5% milk / TBS-T, S210D, third blood collection, University of Dundee), anti-PARKIN total antibody (2 μg / ml, 5% milk / TBS-T, S966C, second blood collection, University of Dundee), anti-PARKIN phosphorylated Ser65 antibody (1:10000, 5% BSA / TBS-T, rabbit monoclonal antibody, MJF foundation), non-phosphopeptide PARKIN Ser65 antibody (2 mg / ml, 5% milk / TBS-T, University of Dundee), anti-PINK1 total antibody (2 μg / ml, 5% milk / TBS-T, S085D, third blood collection, University of Dundee), anti-Bcl-xL total antibody (1:1000, 5% BSA / TBS-T, Cell Signaling), anti-Bcl-xL phosphorylated Ser62 antibody (1:1000, 5% BSA / TBS-T, Invitrogen), anti-PINK1 phosphorylated Thr257 antibody (2 μg / ml, 5% milk / TBS-T, S114D, third blood collection, University of Dundee), non-phosphopeptide PINK1 Thr257 antibody (2 mg / ml, 5% milk / TBS-T, University of Dundee), anti-OPA1 antibody (1:1000, 5% BSA / TBS-T, BD Biosciences). Anti-rabbit IgG-HRP conjugated antibody (1:1000 5% BSA / TBS-T, Cell Signaling), anti-goat IgG HRP conjugated antibody (1:5000 5% milk / TBS-T, abcam), anti-mouse IgG HRP conjugated antibody (1:1000 5% BSA / TBS-T, Cell Signaling).

[0133] Phosphorylated-Ub immunoblot

[0134] The cells were pretreated with kinetin riboside for 23 hours and then treated with 10 μM CCCP or niclosamide for 1 hour. The 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 mixture (Roche), 1X phosphatase inhibitor mixture (Roche)). The denatured proteins were loaded onto a Mini-PROTEAN TGX 4-12% precast gel and then protein separation was performed. The proteins were then transferred to a methanol-activated PVDF membrane using a Trans-Blot Turbo System (Bio-rad), and subsequently the membrane was blocked with a 5% non-fat milk solution in TBS / 0.1% Tween (TBST) at room temperature for 1 hour. After blocking, the membrane was washed in TBST and incubated overnight at 4 °C with agitation with a primary antibody diluted in 5% BSA / TBST or 5% milk / TSBT. The next day, the membrane was washed in TBST and incubated for 1 hour at room temperature with agitation with an antibody conjugated to horseradish peroxidase (HRP). After washing with TBST, the proteins were visualized using an Amersham TM ECL TM Prime kit (GE Biosciences) and imaged using a Bio-rad ChemicDoc. Primary antibodies for Western blotting: GAPDH antibody (Abcam product number ab9485), phosphorylated Ub Ser 65 antibody (Sigma-Aldrich product number ABS1513-I), LC3B antibody (CellSignaling product number 3868S). Secondary antibodies for Western blotting: anti-rabbit HRP antibody (Agilent Dako product number P0399), anti-mouse HRP antibody (Agilent Dako product number P0260).

[0135] Immunofluorescence

[0136] Cells were seeded on PerkinElmer PhenoPlate 96-well plates and then treated with compounds. The cells were fixed in cooled 4% PFA for 10 minutes and then washed in PBS. Permeabilization of the cells was achieved by adding PBS / 0.1% TritonX-100 and treating at room temperature for 5 minutes. After PBS washing, the cells were blocked with blocking buffer (PBS, 5% BSA, 0.1% Tween 20) at room temperature for 1 hour. The primary antibodies were diluted in the blocking buffer and added to the cells, and incubated overnight at 4°C. Subsequently, the cells were washed with PBS-T and appropriate secondary antibodies (diluted with blocking buffer) were added and treated for 2 hours in the dark at room temperature. When needed, 10 μg / ml of Hoechst 33258 stain was added together with the secondary antibody. The cells were washed again with PBS-T and then PBS containing 0.02% sodium azide was added. Then the cells were stored at 4°C until ready for imaging. The primary antibodies used for immunofluorescence were as follows: phosphorylated Ub Ser 65 antibody (Sigma-Aldrich product number ABS1513-I), TOM20 antibody (Santa-Cruz product number sc-17764). Secondary antibodies and stains used in immunofluorescence: anti-IgG2a Alexa Fluor TM 546 antibody (ThermoFisher product number A21133), anti-rabbit Alexa Fluor TM 647 antibody (ThermoFisher product number A21244), Hoechst 33258 stain (Sigma-Aldrich product number B2883).

[0137] Confocal microscopic analysis 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. The images were analyzed and quantified using Columbus (PerkinElmer) and TIBCO Spotfire software.

[0138] To evaluate the fluorescence of tetramethylrhodamine methyl ester perchlorate (TMRM), the cell medium was removed and replaced with cell medium consisting of 5 nM TMRM (ThermoFisher) and 10 μg / ml Hoechst 33258 stain, and treated for 30 minutes at 37°C in the dark to allow TMRM to enter the mitochondria. After PBS washing, FluoroBrite DMEM (Gibco) medium was added to the cells, and then imaging was performed using a Zeiss LSM800 microscope at 37°C and 5% CO2.

[0139] PINK1 in vitro kinase assay

[0140] This assay was performed by MRC PPU Reagents and Services (University of Dundee, U.K.). The assay was carried out 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 solution with a final volume of 25.5 μl in the presence of the relevant small molecule (serial dilutions, highest concentration 100 μM) and incubated at room temperature for 30 minutes. The final volume contained 50 mM Tris pH 7.5, 0.1 mM EDTA, 10 mM DTT, protein substrate (0.3 mg / ml of GST PARK2 TV3 or 1 mg / ml of ubiquitin - His), 10 mM magnesium acetate, and 0.1 mM [33P - γ - ATP] (50 - 1000 cpm / pmole). The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with 50 mM orthophosphoric acid wash buffer.

[0141] Example 1: PINK1 activation (kinetin riboside)

[0142] We found that the previously reported activation of PINK1 by kinetin riboside 2 in cells was observed at high concentration (50 μM) and that detectable activation of PINK1 in cells by the nucleobase kinetin was only observed in the presence of the mitochondrial uncoupler CCCP. Therefore, we first explored whether combining the nucleoside analogue (kinetin riboside 2) with some known indirect activators of PINK1 (such as niclosamide and CCCP) would produce a synergistic and more significant activation of PINK1. To explore 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. Subsequently, the cells were lysed or treated with 10 μM CCCP for 3 hours ( Figure 1c ). Treatment with niclosamide and CCCP alone was used as a control. When probing for UbSer65 (pUb), optic atrophy 1 (OPA1), and GAPDH after cell lysis, as expected, treatment with niclosamide and CCCP alone caused strong 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 any significant ubiquitin phosphorylation. Notably, pretreatment of cells with kinetin riboside inhibited CCCP-induced ubiquitin phosphorylation, whereas pretreatment with the related nucleobases 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 ).

[0143] Surprised by this finding, we next explored whether the same inhibitory effect of kinetin riboside on CCCP-induced ubiquitin phosphorylation could also be observed with the mitochondrial uncoupler niclosamide. To this end, YFP-parkin-transfected HeLa cells were pretreated with 50 μM kinetin riboside for 24 h, and then the cells were 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 robust ubiquitin phosphorylation, whereas pretreatment of cells with kinetin riboside inhibited both niclosamide- and CCCP-induced ubiquitin phosphorylation ( Figure 1d ),and Figure 1c The results in are similar.

[0144] Immunofluorescence was then used to probe the ability of kinetin riboside to inhibit ubiquitin phosphorylation ( Figure 1e ).like Figure 1d As shown, YFP-parkin transfected HeLa cells were either untreated, treated with 10 μM niclosamide or 10 μM CCCP alone for 1 hour, or pretreated with 50 μM kinetin riboside for 24 hours before the addition of niclosamide or CCCP. Treatment of cells with niclosamide and CCCP again induced significant ubiquitin phosphorylation, whereas pretreatment of cells with kinetin riboside inhibited ubiquitin phosphorylation ( Figure 1e Although pretreatment with kinetin riboside inhibited ubiquitin phosphorylation, it did not prevent the membrane potential collapse induced by niclosamide and CCCP (not shown), similar to what was observed by probing OPA1 cleavage ( Figure 1c ).

[0145] Example 2: Synthesis of N 6 -Substituted adenine and adenosine analogs

[0146] Inspired by the ability of kinetin riboside to inhibit niclosamide- and CCCP-induced ubiquitin phosphorylation, we subsequently designed and synthesized N-terminal riboside structurally related to kinetin and kinetin riboside. 6 -Replace adenine and adenosine. In the design of these nucleobases and nucleosides, we chose to modify the N of adenine and adenosine 6Position and select to perform various small, medium, and large substitutions at this position. Specifically, N 6 -substitutions are methyl, isopropyl, benzyl, tyramine, cyclopentylamine, neopentylamine, and furfuryl (8a-f and 9a-f, Figure 2a ).

[0147] Depending on the volatility of the nucleophile used in the reaction, one of two methods is used for the synthesis of N 6 -substituted adenine and adenosine compounds ( Figure 2a ). Standard nucleophilic S N2 substitution is the preferred method (Method A) because high yields are obtained after a simple purification step. This method involves heating 6-chloropurine or its riboside derivative with the corresponding nucleophile in ethanol in the presence of triethylamine. This method is employed in the synthesis of adenine and adenosine analogs modified with benzyl (8c and 9c), isopropyl (8d and 9d), methyl (8e and 9e), or tyramine (8f and 9f) at the N 6 position. In Method B, the peptide coupling agent PyBOP is used for the synthesis of nucleobases and nucleosides bearing cyclopentylamine and neopentylamine at the N 6 position. The reason for choosing to use Method B for the synthesis with neopentylamine (8a and 9a) and cyclopentylamine (8b and 9b) as nucleophiles is that these compounds explode upon heating, precluding the use of Method A. In this case, for the synthesis of nucleobases and nucleosides, hypoxanthine or inosine is added to PyBOP and partially dissolved in a mixture of acetonitrile and sub-stoichiometric amounts of DMF. Then the corresponding nucleophile - cyclopentylamine or neopentylamine - is added and the reaction is allowed to stand for three days.

[0148] Compared with Method A, the yield of Method B is much lower and the purification of the final compound is also more complex.

[0149] For more details of the above synthesis, refer to the appendix.

[0150] Example 3: PINK 1 activation (N 6 -substituted adenine and adenosine compounds)

[0151] After the synthesis of Example 2 was completed, the ability of the synthesized nucleobases and nucleosides to activate PINK1 in cells was evaluated. HeLa cells that endogenously express PINK1 but do not express parkin were transiently transfected with parkin. Subsequently, the cells were treated with nucleobases (8a - 8f) and nucleosides (9a - 9f) for 1 hour, or treated with 10 μM CCCP for 3 hours as a control, and parkin Ser65 phosphorylation, total Parkin, OPA1, and GAPDH (as a loading control) were probed. The results showed that CCCP significantly activated PINK1 as judged by parkin Ser65 phosphorylation, while 50 μM of nucleobases 8a - 8f did not show any significant PINK1 activation, which is consistent with previous findings ( Figure 2b ). However, all of the nucleosides (9a - 9f) studied, except for compound 9f, exhibited significant PINK1 activation ( Figure 2b ). Interestingly, the activation of PINK1 by these nucleosides did not lead to OPA1 cleavage. This indicates that the activation of PINK1 by these nucleoside analogs is independent of mitochondrial depolarization, contrary to the activation of PINK1 by CCCP (which is associated with OPA1 cleavage). This is because the agent CCCP indirectly activates PINK1 through mitochondrial membrane depolarization, which is consistent with previous observations.

[0152] Example 4: CCCP / Niclosamide - independent Activation of PINK1

[0153] 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 probing of TOM20 in these cells showed that these nucleoside analogs alone had no effect on mitochondrial fragmentation, parkin localization, or ubiquitin phosphorylation (Figure 3). Subsequently, HeLa cells expressing YFP - parkin were treated with 50 μM kinetin riboside, nucleoside 9a, or 9c for 24 hours and then treated with 10 μM CCCP. Monitoring ubiquitin Ser65 phosphorylation using immunofluorescence, CCCP treatment induced strong ubiquitin phosphorylation and promoted parkin localization to mitochondria ( Figure 3a ). Notably, pretreatment with kinetin riboside inhibited ubiquitin phosphorylation, which is consistent with the data (Figure 2c - e). For the new compounds, pretreatment with nucleoside analog 9a followed by CCCP treatment had no significant effect on ubiquitin phosphorylation and parkin localization to mitochondria compared to CCCP treatment alone ( Figure 3a)。 However, pretreatment with nucleoside analogue 9c significantly reduced ubiquitin phosphorylation and parkin mitochondrial localization, and its effect seemed stronger than that induced by kinetin riboside ( Figure 3a and b).

[0154] Since kinetin riboside and nucleoside 9c were consistently observed to 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 h and 5 h before lysis, and the samples were treated with 50 nM valinomycin, a mitochondrial uncoupler. Subsequently, probing for ubiquitin Ser65 phosphorylation showed that treatment with the nucleobase kinetin, nucleosides 9a and 9c alone led to enhanced ubiquitin Ser65 phosphorylation, which was consistent with Figure 2b the observed results of parkin Ser65 phosphorylation shown in Figure 3c ). Interestingly, pretreatment of astrocytes with kinetin, nucleosides 9a and 9c for 24 h and then treatment with valinomycin for 5 h significantly inhibited ubiquitin Ser65 phosphorylation compared to treatment of astrocytes with valinomycin alone (black bars, Figure 3c ).

[0155] Example 5: PINK1-dependent mitophagy

[0156] Since kinetin riboside and nucleoside analogue 9c inhibit ubiquitin phosphorylation and parkin mitochondrial localization, our next question was whether these compounds still induced mitophagy in a PINK1-dependent manner. To investigate this, we employed the established MitoQC assay to measure mitophagy in cells 11 , 12. Indeed, immortalized MEFs expressing PINK1 wild type (WT) or knockout (KO) were treated with 20 μM CCCP or 5 μM of kinetin riboside or compound 9c for 24 h. In MEFs expressing PINK1 wild type, CCCP treatment induced increased mitophagy, but not in PINK1 KO MEFs, which was consistent with previous reports ( Figure 3d -e. Magnification Figure 3d ). Interestingly, treatment of PINK1 WT MEFs with kinetin riboside or nucleoside analogue 9c also induced mitophagy, which was not observed in PINK1 KO MEFs ( Figure 3d -e). This data indicates that both kinetin riboside and nucleoside analogue 9c induce low levels of mitophagy in a PINK1-dependent manner, which is consistent with previous observations that kinetin riboside and nucleoside 9c ( Figure 2b ) induce low levels of PINK1 activation, as judged by parkin phosphorylation.

[0157] Example 6: Molecular Modeling Study

[0158] Collectively, the results of Examples 1 to 5 demonstrated the ability of kinetin riboside and N 6 -substituted adenosine compounds (specifically N 6 -benzyladenosine (9c)) to inhibit the phosphorylation of ubiquitin induced by niclosamide and CCCP. To our knowledge, there are two instances of the ability to inhibit the phosphorylation of ubiquitin by PINK1 13,14 . The first instance involves PD-related mutations C125G and Q126P, which disrupt the intramolecular interaction between the N-terminal and C-terminal extensions of human PINK1, resulting in the inhibition of ubiquitin phosphorylation 14 , while the second instance 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 far from the nucleobase binding sites of nucleoside analogs and their triphosphate derivatives, and are therefore unlikely to be covalent inhibitors

[0159] To further investigate this, we performed in vitro kinase assays (Figure 6) using recombinant and constitutively active Tribolium castaneum PINK1 (TcPINK1) and human parkin or ubiquitin as substrates in the presence of increasing concentrations (up to 100 μM) of kinetin riboside, benzyladenosine 9c, or their triphosphate derivatives. Although the results suggest that these compounds are unlikely to be covalent inhibitors of PINK1, it must be noted that human PINK1 Cyc166 is not conserved in TcPINK1, while Cyc387 is conserved in both human PINK1 and TcPINK1 ( Figure 7 ). In addition, superimposing the ATP pockets of hPINK1 and TcPINK1 showed that the TcPINK1 ATP pocket is more open than the hPINK1 ATP pocket, and although the position of hPINK1 Cyc387 is close to TcPINK1 Cys362, the position of TcPINK1 Thr172 seems to be far from the position of hPINK1 Cyc166 (Figure 8), which seems to affect 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 the binding of these compounds or their phosphorylated species to the ATP pocket of PINK1, leading to conformational changes that rearrange the N- and C-terminal conformations, resulting in the inability of PINK1 to phosphorylate ubiquitin

[0160] Summary

[0161] We describe herein nucleoside analogs as inducers of low-level PINK1-mediated mitophagy and inhibitors of ubiquitin phosphorylation induced by mitochondrial uncouplers (CCCP, valinomycin, and niclosamide). These nucleoside analogs are useful tool compounds for deciphering the role of phosphorylated ubiquitin signaling, including its parkin-independent functions such as inhibition of the E2 / E3 ubiquitin system and deubiquitinating enzymes. Importantly, postmortem analysis of the substantia nigra in patients with Lewy body dementia (the major site of neuronal loss) and elderly PD patients revealed elevated levels of Ub Ser65 phosphorylation compared to healthy age-matched controls. 9,10 , and thus, nucleoside analogs that exhibit inhibition of ubiquitin phosphorylation may be useful for the treatment of Lewy body dementia or idiopathic PD.

[0162] Appendix

[0163] N 6 Synthesis and Evaluation of N-Substituted Adenines and Adenosine Analogs

[0164] 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 ethanol (15 mL). The reaction was then heated to 30 °C for 16 h. The solvent was then removed under reduced pressure. The remaining crude oil was purified by column chromatography using DCM / MeOH (19:1) as the eluent to afford a white solid (200 mg, 41%); 1 1H NMR (500 MHz, 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); 13 13C NMR (126 MHz, DMSO) δ 152.90 (C-4), 152.85 (C-3), 27.40 (C-6); HRMS-ES (m / z): found [M + H] + [150.0781, [C6H7N5H] requires 150.0780. HPLC (reverse phase) 0.5 mL / min MeOH / H2O 80:20, 12 min, λ = 254 nm, Rt = 4.88 min (99%).

[0165] 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 ethanol (15 mL). The reaction was then heated to 40 °C for 16 h, and the solvent was removed under reduced pressure. The remaining crude oil was purified by column chromatography using DCM / MeOH (19:1) as the eluent to afford a white solid (11.7 mg, 2%); 1 1H NMR (500 MHz, 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 = 6.5 Hz, H-7); 13 13C NMR (126 MHz, MeOD) δ 46.52 (C-6), 7.82 (C-7). HPLC (reverse phase) 0.5 mL / min MeOH / H2O 90:10, 12 min, λ = 254 nm, Rt = 4.69 min (99%).

[0166] 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 ethanol (15 mL). The reaction was then refluxed at 80 °C for 16 h. The product precipitated from the solution when the reaction mixture was placed in an ice-water bath and stirred vigorously. OL035 was then filtered off, and the product was further purified by column chromatography using DCM / MeOH (19:1) as the eluent to afford a white solid (240 mg, 33%); 1 1H NMR (500 MHz, MeOD) δ 8.25 (1H, s, H-1), 8.07 (1H, s, H-4), 7.39 (2H, d, J = 7.6 Hz, H-8), 7.32 (2H, t, J = 7.5 Hz, H-9), 7.25 (1H, t, J = 7.3 Hz, H-10); 13 13C NMR (126 MHz, 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): found [M + H] + 226.1089, [C 12 H 11N5H] Claim 226.1093. HPLC (reverse phase) 0.5 mL / min MeOH / H2O 80:20, 12 min, λ = 254 nm, Rt = 5.63 min (99%).

[0167] 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 ethanol (15 mL). The reaction was then refluxed at 80 °C for 16 h. The product precipitated from the solution when the reaction mixture was placed in an ice-water bath and stirred vigorously. OL037 was then filtered off and the product was further purified by column chromatography using DCM / MeOH (19:1) as the eluent to give a white solid (141 mg, 17%); 1 1H NMR (500 MHz, DMSO) δ 8.20 (1H, s, H-1), 8.07 (1H, s, H-4), 7.05 (2H, d, J = 7.8 Hz, H-9), 6.69 (2H, d, J = 8.5 Hz, H-10), 3.64 - 3.63 (2H, m, H-6) 2.81 - 2.78 (2H, m, H-7); 13 13C NMR (126 MHz, 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): found [M+H] + 256.1205, [C 13 H 13 N5OH] Claim 256.1198. HPLC (reverse phase) 0.5 mL / min MeOH / H2O 80:20, 12 min, λ = 254 nm, Rt = 4.96 min (86%).

[0168] N-Cyclopentyl-9H-purin-6-amine (8e). Under an inert atmosphere, hypoxanthine (500 mg, 3.67 mmol, 1 equiv), cyclopentylamine (0.54 mL, 5.51 mmol, 1.5 equiv), and PyBOP (2.294 g, 4.41 mmol, 1.2 equiv) were dissolved in anhydrous ACN (20 mL) and substoichiometric DMF (2 mL). Then DIPEA (1.28 ml, 7.35 mmol, 2 equiv) was slowly added over 5 minutes at room temperature, and the reaction was stirred for 3 days. Then the solvent was removed under reduced pressure. The remaining crude oil was purified by column chromatography using DCM / MeOH (19:1) as the eluent to give a white solid (130 mg, 17%); 1 1H NMR (500 MHz, 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); 13 13C NMR (126 MHz, DMSO) δ 152.80 (C-4), 32.77 (C-6), 23.91 (C-7, 8). HRMS-ES (m / z): found [M + H] + 204.1244, [C 10 H 13 N5H] requires 204.1249. HPLC (reverse phase) 0.5 mL / min MeOH / H2O 80:20, 12 min, λ = 254 nm, Rt = 5.78 min (99%).

[0169] N-Neopentyl-9H-purin-6-amine (8f). Under an inert atmosphere, hypoxanthine (300 mg, 2.20 mmol, 1 equiv), neopentylamine (0.39 mL, 3.31 mmol, 1.5 equiv), and PyBOP (1.721 g, 3.31 mmol, 1.5 equiv) were dissolved in anhydrous ACN (20 mL) and substoichiometric DMF (2 ml). Then DIPEA (0.77 ml, 4.41 mmol, 2 equiv) was slowly added over 5 minutes at room temperature, and the reaction was stirred for 3 days. Then the solvent was removed under reduced pressure. The remaining crude oil was purified by column chromatography using DCM / MeOH (19:1) as the eluent to give a white solid (31 mg, 7%); 1 1H NMR (500 MHz, 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), 1313C NMR (126 MHz, 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 (reverse phase) 0.5 mL / min MeOH / H2O 90:10, 12 min, λ = 254 nm, Rt = 4.72 min (99%).

[0170] (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 ethanol (15 mL). The reaction was then heated to 30 °C for 16 h, and the solvent was 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%); 1 1H NMR (500 MHz, MeOD) δ 8.23 (2H, s, H-6,9), 5.95 (1H, d, J = 6.5 Hz, H-5), 4.74 (1H, dd, J = 6.4, 5.1 Hz, H-4), 4.32 - 4.31 (1H, m, H-3), 4.16 (1H, q, J = 2.5 Hz, H-2), 3.90 - 3.72 (2H, m, H-1), 1.28 (3H, m, H-11); 13 13C NMR (126 MHz, 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): found [M+H] + 282.10, [C 11 H 15 N5O4H] requires 282.11. HPLC (reverse phase) 0.5 mL / min MeOH / H2O 90:10, 12 min, λ = 254 nm, Rt = 4.73 min (99%).

[0171] (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-chloropurin riboside (600 mg, 2.09 mmol, 1 equiv) in ethanol (15 mL). The reaction was then heated to 40 °C for 16 h, and the solvent was 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%); 1 1H NMR (500 MHz, MeOD) δ 8.24 (1H, s, H-6), 8.21 (1H, s, H-9), 5.94 (1H, d, J = 6.5 Hz, H-5), 4.74 (1H, dd, J = 6.4, 5.1 Hz, 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 = 6.5 Hz, H-12); 13 13C NMR (126 MHz, 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): found [M+H] + 310.13, [C 13 H 19 N5O4H] requires 310.14. HPLC (reverse phase) 0.5 mL / min MeOH / H2O 80:20, 12 min, λ = 254 nm, Rt = 5.03 min (99%).

[0172] (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, 4.17 mmol, 1.5 equiv) were added to a stirred solution of 6-chloropurin riboside (800 mg, 2.79 mmol, 1 equiv) in ethanol (15 mL). The reaction was then refluxed at 80 °C for 16 h. The product precipitated from the solution when the product was placed in an ice-water bath and stirred vigorously. OL031 was then filtered off and dried under reduced pressure to give a white solid (948 mg, 95%); 1HNMR(500 MHz, MeOD) δ 8.26 (1H, s, H-6), 8.23 (1H, s, H-9), 7.38 (2H, d, J = 7.6 Hz, H-13), 7.31 (2H, t, J = 7.5 Hz, H-14), 7.24 (1H, t, J = 7.3 Hz, H-15), 5.96 (1H, d, J = 6.5 Hz, H-5), 4.75 (1H, dd, J = 6.4, 5.1 Hz, H-4), 4.32 (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); 13 C NMR(126 MHz, 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): Observed value [M + H] + 358.13, [C 17 H 19 N5O4H] requires 358.14. HPLC (reverse phase) 0.5 mL / min MeOH / H2O 80:20, 12 min, λ = 254 nm, Rt = 5.34 min (99%);

[0173] (2R,3S,4R,5R)-2-(Hydroxymethyl)-5-(6-((4-hydroxyphenethyl)amino)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (9d). Under an inert atmosphere, 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 substoichiometric amounts of DMF (2 mL). Then DIPEA (0.65 mL, 3.73 mmol, 2 equiv) was slowly added at room temperature over 5 minutes, and the reaction was stirred for 3 days. Then the solvent was removed under reduced pressure. Then the remaining crude oil was purified by column chromatography using DCM / MeOH (19:1) as the eluent to give a white solid (347 mg, 48%); 11H NMR (500 MHz, DMSO) δ 8.34 (1H, s, H-6), 8.24 (1H, s, H-9), 7.04 (2H, d, J = 7.3 Hz, H-14), 6.68 (2H, d, J = 8.4 Hz, H-15), 5.89 (1H, d, J = 6.2 Hz, H-5), 4.63 - 4.60 (1H, m, H-4), 4.16 - 4.14 (1H, m, H-3), 3.98 - 3.96 (1H, m, H-2), 3.70 - 3.64 (2H, m, H-1) 3.58 - 3.53 (2H, m, H-11), 2.81 - 2.78 (2H, m, H-12); 13 13C NMR (126 MHz, 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): found [M+H] + 388.20, [C 18 H 21 N5O5H] requires 388.15. HPLC (reverse phase) 0.5 mL / min MeCN / H2O 80:20, 12 min, λ = 254 nm, Rt = 4.38 min (99%).

[0174] (2R,3R,4S,5R)-2-(6-(Cyclopentylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (9e). Under an inert atmosphere, inosine (750 mg, 2.80 mmol, 1 equiv), cyclopentylamine (0.41 mL, 4.19 mmol, 1.5 equiv) and PyBOP (2.183 g, 4.19 mmol, 1.5 equiv) were dissolved in anhydrous ACN (20 mL) and substoichiometric amounts of DMF (2 mL). Then DIPEA (0.97 ml, 5.59 mmol, 2 equiv) was added slowly at room temperature over 5 minutes and the reaction was stirred for 3 days. Then the solvent was removed under reduced pressure. The remaining crude oil was purified by column chromatography using DCM / MeOH (19:1) as the eluent to give a white solid (749 mg, 80%); 11H NMR (500 MHz, MeOD) δ 8.27 (1H, s, H-6), 8.23 (1H, s, H-9), 5.97 (1H, d, J = 6.5 Hz, H-5), 4.76 (1H, dd, J = 6.4, 5.1 Hz, H-4), 4.34 (1H, dd, J = 5.1, 2.5 Hz, H-3), 4.19 (1H, q, J = 2.5 Hz, 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); 13 13C NMR (126 MHz, 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): found [M+H] + 336.16, [C 15 15 21 N5O4H] requires 336.16. HPLC (reverse phase) 0.5 mL / min MeOH / H2O 80:20, 12 min, λ = 254 nm, Rt = 5.43 min (95%).

[0175] (2R,3S,4R,5R)-2-(Hydroxymethyl)-5-(6-(neopentylamino)-9H-purin-9-yl)tetrahydrofuran-3,4-diol (9f). Under an inert atmosphere, 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 sub-stoichiometric amounts of DMF (2 mL). Then DIPEA (0.39 mL, 2.24 mmol, 2 equiv) was slowly added at room temperature over 5 min and the reaction was stirred for 3 days. Then the solvent was removed under reduced pressure. The remaining crude oil was purified by column chromatography using DCM / MeOH (19:1) as the eluent to afford a white solid (305 mg, 81%); 1HNMR (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); 13 CNMR (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): Observed value [M + H] + 338.18, [C 15 H 23 N5O4H] requires 338.18. HPLC (reverse phase) 0.5 mL / min MeCN / H2O 80:20, 12 min, λ = 254 nm, Rt = 4.66 min (99%).

[0176] References

[0177] 1. Valente EM, Abou-Sleiman PM, Caputo V, Muquit 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.

[0178] 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.

[0179] 3.Witte ME,Mahad DJ,Lassmann H,van Horssen J:Mitochondrialdysfunction contributes to neurodegeneration in multiple sclerosis.Trends MolMed 2014,20:179-187.

[0180] 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.

[0181] 5.Billia F,Hauck L,Konecny F,Rao V,Shen J,Mak TW:PTEN-induciblekinase 1(PINK1) / Park6 is indispensable for normal heart function.Proc NatlAcad Sci U S A 2011,108:9572-9577.

[0182] 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 througheffects on oxidative stress and mitophagy.Mol Cell Biol 2012,32:4493-4504.

[0183] 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.

[0184] 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.

[0185] 9. Fiesel FC, et al.: (Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation. EMBO Rep 2015, 16(9):1114-1130.

[0186] 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.

[0187] 11.Allen, G.F.G., et al.: Loss of iron triggers PINK1 / Parkin-independent mitophagy. EMBO Rep. 2013, 14(12): 1127-35.

[0188] 12.McWilliams, T.G., et al.: mito-QC illuminates mitophagy and mitochondrial architecture in vivo. J Cell Biol. 2016, 214(3): 333-45.

[0189] 13.Gan, Z.Y., et al.: Activation mechanism of PINK1. Nature. 2022, 602(7896): 328-335.

[0190] 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 their tautomers: Wherein: R 1 is C 1-10 alkyl, C 3-10 cycloalkyl, C 6-10 aryl, heteroalkyl or heteroaryl group, optionally substituted by one or more substituents selected from: 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; and R 2 is a furanose moiety of the general formula (II): Wherein: X is O, NH, S or CH2; R 3 is H, OH, a halogen group, 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 monophoaphate, diphosphate or triphosphate derivative of the general formula (VIII), where q is 0, 1 or 2 and each R 11 is independently selected from OH or an aryloxy, amino acid ester or pivaloyloxymethyl protecting group; And; R 4 and R 5 are independently selected from H, OH, a halogen group, 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 pharmaceutically or veterinarily acceptable salt or hydrate thereof, for the treatment of neurodegenerative diseases or disorders and / or cancer characterized by elevated levels of ubiquitin Ser65 phosphorylation.

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

3. The compound as used according to claim 1 or 2, wherein R1 is a group of general formula (III-A), (III-B), (III-C), (III-D), (III-E) or (III-F): Wherein: n is 0, 1, 2 or 3; m is 0, 1, 2 or 3; Z is O, NH, S or CH2, and is preferably CH2; and R 6 is OH or O(C 1-4 alkyl).

4. The compound as used in claim 3, wherein R 1 is selected from:

5. The compound as used in any one of the foregoing claims, wherein 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 monophosphate, diphosphate or triphosphate derivative of the general formula (VIII).

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

7. The compound as used in claim 5, wherein R 3 and R 4 are both OH, and R 5 is H.

8. The compound as used according to claim 1 or 2, wherein the compound of general formula (I) is selected from:

9. Compounds of general formula (I), including all their tautomers: Wherein: R 1 is C 1-10 alkyl, C 3-10 cycloalkyl, C 6-10 aryl, heteroalkyl or heteroaryl group, optionally substituted by one or more substituents selected from: 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; and R 2 is a furanose moiety of the general formula (II): Wherein: X is O, NH, S or CH2; R 3 is H, OH, a halogen group, a nitro group, 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 monoposphate, diphosphate or triphosphate derivative of the general formula (VIII), where q is 0, 1 or 2 and each R 11 is independently selected from OH or an aryloxy group, an amino acid ester or a pivaloyloxymethyl protecting group; And R 4 and R 5 are independently selected from H, OH, a halogen group, 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 pharmaceutically or veterinarily acceptable salt or hydrate thereof, Provided that the compound is not kinetin riboside.

10. The compound according to claim 9, wherein R 1 is a group of the general formula (III-A), (III-B), (III-C), (III-D), (III-E) or (III-F): Wherein: n is 0, 1, 2 or 3; m is 0, 1, 2 or 3; Z is O, NH, S or CH2, and is preferably CH2; and R 6 is OH or O(C 1-4 alkyl).

11. The compound according to claim 9, wherein R 1 is selected from:

12. A compound according to any one of claims 9 to 11, wherein 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 monophosphate, diphosphate or triphosphate derivative of the general formula (VIII).

13. The compound according to claim 12, wherein: R 3 , R 4 and R 5 Each of which is OH, or where R 3 and R 4 are OH, and R 5 It's H.

14. The compound according to claim 9, which is selected from:

15. A method for preparing a compound of general formula (I) according to any one of claims 9 to 14, the method comprising: (A) Reacting a 6-halopurine derivative of general formula (V) with a nucleophilic compound of general formula (VI): (B) Reacting a hypoxanthine derivative of general formula (VII) with a nucleophilic compound of general formula (VI) in the presence of benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (‘PyBOP’): Wherein R7 is a halogen group, preferably a chloro group, and R1 and R2 are as defined in general formula (I) in any one of claims 9 to 14.

16. The compound according to any one of claims 9 to 14 or kinetin riboside, for the preparation of a medicament for the treatment of cancer or disorders or diseases associated with elevated levels of phosphorylated ubiquitin.

17. A method for treating cancer or disorders or diseases associated with elevated levels of phosphorylated ubiquitin, the method comprising administering to a patient in need thereof an effective amount of kinetin riboside or a compound according to any one of claims 9 to 14.

18. The compound as used according to claim 16 or the method according to claim 17, wherein the disorders or diseases associated with elevated levels of phosphorylated ubiquitin are selected from idiopathic Parkinson's disease and dementia with Lewy bodies.

19. 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. The pharmaceutical composition according to claim 19, which is formulated for oral delivery.

21. A combination therapeutic agent comprising kinetin riboside or a compound according to any one of claims 9 to 14 and an additional therapeutic agent for treating cancer or a neurodegenerative disease or disorder, for simultaneous, separate or sequential use in treating cancer or a neurodegenerative disease or disorder characterized by elevated levels of ubiquitin Ser65 phosphorylation.

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