Novel compounds for the diagnosis, treatment and prevention of diseases associated with the aggregation of alpha-synuclein

Abstract

The present invention relates to compounds represented by general formula (Ia), (Ib), (IIa), or (IIb). These compounds are suitable for imaging α-synuclein and diagnosing diseases associated with α-synuclein aggregation. The compounds are also useful for treating and preventing diseases associated with α-synuclein aggregation. TIFF2023502794000057.tif78144

Description

[Technical field]

[0001] The present invention refers to novel compounds suitable for imaging α-synuclein and for diagnosing diseases associated with the aggregation of α-synuclein. The compounds are also useful for treating and preventing diseases associated with the aggregation of α-synuclein. [Background technology]

[0002] Numerous neurological and neurodegenerative diseases are known, many of which are currently incurable and difficult to diagnose. All common neurodegenerative diseases are characterized by the misfolding, aggregation and deposition of certain proteins in the brain. These diseases include medical conditions such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, Creutzfeldt-Jakob disease and many others.

[0003] Synucleinopathies are a group of diseases characterized by the accumulation and deposition of aggregated and misfolded α-synuclein protein (αSYN). Synucleinopathies include Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). These diseases differ in terms of the distribution of the accumulation and deposition of aggregated and misfolded α-synuclein protein in the central and peripheral nervous systems. Neuropathologically, they can be distinguished from other neurodegenerative diseases by disease-specific accumulation and deposition of aggregated and misfolded α-synuclein protein, while other neurodegenerative diseases are characterized by the accumulation and deposition of aggregated and misfolded other proteins. For example, Alzheimer's disease is characterized by aggregated and misfolded Abeta (Aβ) and tau proteins, progressive supranuclear palsy and corticobasal degeneration are characterized by aggregated and misfolded tau proteins, as are some cases of frontotemporal dementia, and Creutzfeldt-Jakob disease is characterized by aggregated and misfolded prion proteins.

[0004] Disease-specific accumulation and deposits of aggregated and misfolded proteins provide targets for both therapy and diagnosis with compounds that bind to these aggregated and misfolded protein deposits.

[0005] One option for diagnostic detection of disease-specific accumulation and deposition of aggregated and misfolded proteins is the use of detectably labeled compounds that exhibit specific and selective high affinity binding to said deposits of aggregated proteins. This can be done, for example, with compounds that are labeled with a suitable radioisotope and with PET imaging for detection. Compounds are available for clinical use for PET imaging of Aβ and tau, but no compound has been invented so far that can be used for diagnostic PET imaging of α-synuclein deposits in synucleinopathies (Kotzbauer, PT, Tu, Z. and Mach, RH, Current status of the development of PET radiotracers for imaging alpha synuclein aggregates in Lewy bodies and Lewy neurites. Clin. Transl. Imaging, 2017.5: p.3-14). Compounds developed and tested by other groups to date have lacked a suitable combination of properties, including high binding affinity for aggregated and misfolded α-synuclein compared to other aggregated and misfolded proteins, especially Aβ and tau, sufficient selectivity in binding affinity (required for the differential diagnosis of various diseases characterized by disease-specific accumulation and deposition of aggregated and misfolded proteins, as well as the ability to accurately detect the presence of aggregated and misfolded α-synuclein in patients with one or more diseases at the same time, namely dementia with Lewy bodies and Alzheimer's disease). Additionally, required properties include the ability to cross the blood-brain barrier and bind to intracellular deposits, low non-specific binding to brain tissue, and rapid washout of unbound compounds from the brain.

[0006] WO2009 / 146343 refers to certain pyrazoles, 1,2,4-oxadiazoles and 1,3,4-oxadiazoles that are tracers for positron emission tomography (PET) imaging to study amyloid deposits in the brain in vivo to enable the diagnosis of Alzheimer's disease. Alzheimer's disease is characterized by the aggregation of Aβ and tau proteins, and the PET tracers mentioned bind to the aggregates of these proteins. In contrast, the present invention targets diseases characterized by the aggregation of α-synuclein. Selective imaging diagnosis of aggregated α-synuclein requires selective binding to α-synuclein and no or low binding to aggregated Aβ and tau.

[0007] WO00 / 66578 describes certain NPY antagonists that are useful for treating NPY-mediated diseases / conditions, such as obesity. In addition to the "direct" effect of the compounds of WO00 / 66578 on the NPY5 subtype, it mentions that there are diseases / conditions that benefit from weight loss, such as insulin resistance, impaired glucose tolerance, type II diabetes, hypertension, hyperlipidemia, cardiovascular disease, gallstones, certain cancers, sleep apnea syndrome, etc.

[0008] WO2010 / 000372 relates to certain compounds that are useful for the treatment or prevention of diseases associated with protein aggregation and / or neurodegenerative diseases. The compounds of WO2010 / 000372 are particularly suitable for treating diseases associated with protein aggregation, including Parkinson's disease. The compounds have been shown to bind to several different aggregated proteins, including Aβ and tau. Thus, they can be used to diagnose disorders associated with protein aggregation, but cannot reliably distinguish between disorders associated with α-synuclein aggregation and other disorders associated with amyloid protein aggregation, such as tauopathies or Alzheimer's disease. However, this is important because the clinical symptoms of disorders associated with protein aggregation are very similar, and it is highly desirable, for example, to distinguish between various disorders and adapt treatment accordingly. Furthermore, the molecules described in WO2010 / 000372 have high non-specific binding to lipids and hydrophobic proteins, which leads to strong non-specific binding in brain tissue and other tissues. Therefore, the molecule does not achieve the necessary specific binding to α-synuclein and signal-to-noise ratio required for a PET tracer. Summary of the Invention [Problem to be solved by the invention]

[0009] In view of the foregoing, there is a need for compounds with improved diagnostic properties. In particular, the specificity of binding to α-synuclein needs to be improved. Non-specific binding in brain tissue and other tissues needs to be reduced, binding affinity needs to be increased, and physiological half-life needs to be decreased.

[0010] US Patent Application Publication No. 2005 / 0075375 discloses certain heterocyclic compounds for treating Hepatitis C virus. [Brief description of the drawings]

[0011] [Figure 1]FIG. 1 shows autoradiography using human brain tissue from a patient suffering from dementia with Lewy bodies using [3H]-Compound 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention relates to compounds of the general formula Ia, Ib, IIa or IIb [ka] The present invention relates to a compound represented by the formula:

[0013] X 1 , X 2 and X 3 CR 2 , N and NR 1 are independently selected from, where X 1 , X 2 and X 3 At least two of the are N or NR 1 N and NR 1 exists where valence permits, i.e., N = X 1 -, =X 2 -or=X 3 -position, NR 1 -X 1 -or-X 2 It is understood that the cation can only be present in the -position.

[0014] [ka] Examples include: [ka] Examples include:

[0015] [ka] Examples include: [ka] Examples include:

[0016] In a preferred embodiment, [ka] teeth [ka] (More preferably, [ka] ) or [ka] teeth [ka] (More preferably, [ka] ).

[0017] Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 CR 3 and N, where Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 At least one of is N.

[0018] In a preferred embodiment, in formula Ia or Ib, Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 CR 3and N, where Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 At least one of Y is N. In a more preferred embodiment, 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 and even more preferably one, two or three of 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 and even more preferably one or two of Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 is N. In a preferred embodiment, Y 1 is N.

[0019] In a preferred embodiment, in formula Ia or Ib, Y 1 , Y 3 , Y 4 and Y 6 In a further preferred embodiment, in formula Ia or Ib, at least one of Y 1 is N and Y 3 , Y 4 and Y 6 At least one of is N.

[0020] In a preferred embodiment, in formula Ia or Ib, Y 1 , Y 3 , Y 4 and Y 6 At least one of them is N and the other is Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6is CR 3 (e.g., CH). In a further preferred embodiment, in formula Ia or Ib, Y 1 is N and Y 3 , Y 4 and Y 6 At least one of them is N and the other is Y 2 , Y 3 , Y 4 , Y 5 and Y 6 is CR 3 (e.g. CH).

[0021] In a preferred embodiment, in formula Ia or Ib, Y 1 is N, and the other Y 2 , Y 3 , Y 4 , Y 5 and Y 6 is CR 3 and more preferably Y 1 is N and Y 2 , Y 3 , Y 4 , Y 5 and Y 6 is CH.

[0022] In a preferred embodiment, in formula IIa or IIb, Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 CR 3 and N, where Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 At least one of Y is N. In a more preferred embodiment, 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y7 and Y 8 and even more preferably one, two or three of 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 and even more preferably one or two of Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 is N. In a preferred embodiment, Y 1 is N.

[0023] In a preferred embodiment, in formula IIa or IIb, Y 1 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 At least one of Y is N. In a preferred embodiment, in formula IIa or IIb, Y 1 , Y 3 , Y 4 , Y 5 and Y 7 In a further preferred embodiment, in formula IIa or IIb, at least one of Y 1 is N and Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 At least one of is N.

[0024] In a preferred embodiment, in formula IIa or IIb, Y 1 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8At least one of them is N and the other is Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 is CR 3 (e.g., CH). In a preferred embodiment, in formula IIa or IIb, Y 1 , Y 3 , Y 4 , Y 5 and Y 7 At least one of them is N and the other is Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 is CR 3 (e.g., CH). In a further preferred embodiment, in formula IIa or IIb, Y 1 is N and Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 At least one of them is N and the other is Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 is CR 3 (e.g., CH). In a preferred embodiment, in formula IIa or IIb, Y 1 is N and Y 3 , Y 4 , Y 5 and Y 7 At least one of them is N and the other is Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 is CR 3 (e.g. CH).

[0025] In a preferred embodiment, in formula IIa or IIb, Y 1 is N, and the other Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 is CR 3 and more preferably, Y 1 is N and Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 is CH.

[0026] Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 It has surprisingly been found that the introduction of one or more nitrogen atoms as a strongly enhances the selectivity of binding to α-synuclein, such that corresponding disorders associated with α-synuclein aggregation, such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, can be distinguished from disorders due to the aggregation of other proteins, such as Alzheimer's disease, in which mainly Aβ and tau aggregate. Furthermore, the signal-to-noise ratio is improved.

[0027] R 1 is hydrogen, C 1~4 alkyl and -(CH2)-OP(=O)(OR)(OR), 1~4 The alkyl may be optionally substituted with one or more halogens. In a preferred embodiment, R 1 is hydrogen, methyl or 2-fluoroethyl. When present, -(CH2)-OP(=O)(OR)(OR) is X 1 or X 2 It is preferred that the .alpha.-

[0028] R is hydrogen or a cation. The cation may be any cation that is pharma- ceutically acceptable. Preferably, the cation is a monovalent cation. Examples include sodium, lithium, potassium, ammonium, and the protonated forms of ethanolamine, choline, lysine, meglumine, piperazine, and tromethamine. Preferably, the cation is sodium. In the compounds of the present invention, both R can be hydrogen, both R can be cations (same or different cations), or one R can be hydrogen and the other a cation. Preferably, both R are sodium. Divalent cations, such as Ca 2+ , Mg 2+ and Zn 2+ , or trivalent cations, e.g. Al 3+ is possible but not preferred due to the low water solubility of the resulting salt. 1 The compound where is -(CH2)-OP(=O)(OR)(OR) and its synthesis are described in WO2017 / 102893, which is incorporated herein by reference.

[0029] R 2 is hydrogen, halogen and C 1~4 independently selected from alkyl, C 1~4 The alkyl may be optionally substituted with one or more halogens. In a preferred embodiment, R 2 is hydrogen.

[0030] R 3 is hydrogen, halogen, C 1~4 Alkyl, OH and C 1~4 Alkoxy, C 1~4 Alkyl and C 1~4 The alkoxy may be optionally substituted with one or more halogens. In a preferred embodiment, R 3 is hydrogen or fluorine. Even more preferably, R 3 is hydrogen.

[0031] R 4 and R 5 H and C 1~4independently selected from alkyl, C 1~4 The alkyl may be optionally substituted with one or more halogens or R 4 and R 5 together with the nitrogen atom to which they are attached form a 4- to 6-membered saturated heterocyclic ring, which is represented by R 4 and R 5 In addition to the nitrogen atom to which is attached, optionally contains one or more heteroatoms selected from O and N, and the 4-6 membered saturated heterocycle contains one or more R 6 may be optionally replaced with

[0032] In one embodiment, R 4 and R 5 H and C 1~4 independently selected from alkyl, C 1~4 The alkyl may be optionally substituted with one or more halogens. Preferably, R 4 and R 5 H and C 1~4 In a preferred embodiment, R 4 and R 5 At least one of them is C 1~4 Alkyl, C 1~4 The alkyl may be optionally substituted with one or more halogens. In a more preferred embodiment, R 4 is hydrogen, and R 5 is C 1~4 Alkyl, C 1~4 The alkyl may be optionally substituted with one or more halogens. In a more preferred embodiment, R 4 and R 5 At least one of them is C 1~4 In a more preferred embodiment, R 4 is hydrogen, and R 5 is C 1~4 It is an alkyl.

[0033] In a further embodiment, R 4 and R 5together with the nitrogen atom to which they are attached form a 4- to 6-membered saturated heterocyclic ring, which is represented by R 4 and R 5 In addition to the nitrogen atom to which is attached, the 4-6 membered saturated heterocycle optionally contains one or more (e.g., one) heteroatoms selected from O and N, and the 4-6 membered saturated heterocycle may further include one or more R 6 In a preferred embodiment, the 4-6 membered saturated heterocycle is selected from azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl and piperazinyl, and the azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl and piperazinyl are optionally substituted with one or more R 6 More preferably, the 4-6 membered saturated heterocycle is selected from azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl and piperazinyl, and the N atom of the piperazinyl is optionally substituted with R 6 may be optionally replaced with

[0034] R 6 is a halogen, C 1~4 Alkyl, OH and C 1~4 alkoxy, 1~4 Alkyl and C 1~4 The alkoxy may be optionally substituted with one or more halogens, preferably R 6 is C 1~4 It is alkyl or fluorine.

[0035] Hal is a halogen, such as Br, Cl and F, preferably Hal is Br or F.

[0036] m is Y 1 , Y 2 , Y 3 and Y 4 R other than hydrogen in the ring containing 3 The number of groups. m is 0 to m 最大 is an integer, m 最大 is Y 1 , Y 2 , Y 3 and Y 4 is the number of carbon atoms in the ring containing. Preferably, m is 0.

[0037] n is Y 5 , Y 6 , Y 7 and Y 8 R other than hydrogen in the ring containing 3 is the number of groups (Formulas IIa and IIb). n is 0 to n 最大 is an integer, n max is Y 5 , Y 6 , Y 7 and Y 8 Preferably, n is 0.

[0038] p is Y 5 and Y 6 R other than hydrogen in the ring containing 3 is the number of groups (formulas Ia and Ib). p is 0 to p 最大 is an integer, and p 最大 is Y 5 and Y 6 Preferably, p is 0.

[0039] The compounds of the present invention may also exist in the form of their prodrugs, solvates or salts.

[0040] The compounds of the present invention form salts, which are also within the scope of the present invention.Unless otherwise indicated, it is understood that in this specification, reference to the compounds of the present invention includes reference to their salts.Pharmacologically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, but other salts are also useful, for example, in the isolation or purification steps that may be used during preparation or in in vitro methods.The salts of the compounds of the present invention can be formed, for example, by reacting the compound with an amount, for example, an equivalent amount, of an acid in a medium, for example, in a medium in which the salt precipitates or in an aqueous medium, and then lyophilizing.

[0041] Compounds containing a basic moiety can form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetates (e.g., those formed with acetic acid or trihaloacetic acids, such as trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, hydroxyethanesulfonates (e.g., hydroxyethanesulfonates ... For example, 2-hydroxyethanesulfonates), lactates, maleates, methanesulfonates, naphthalenesulfonates (e.g. 2-naphthalenesulfonate), nicotinates, nitrates, oxalates, pectinates, persulfates, phenylpropionates (e.g. 3-phenylpropionate), phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (e.g. formed with sulfuric acid), sulfonates (e.g. those mentioned herein), tartrates, thiocyanates, toluenesulfonates, e.g. tosylates, undecanoates, and the like.

[0042] Prodrugs and solvates of the compounds of the invention are also contemplated herein. As used herein, the term "prodrug" refers to a compound that, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to produce a compound of the invention or a salt and / or solvate thereof.

[0043] Solvates of the compounds of the present invention include, for example, hydrates.

[0044] All stereoisomers of the compounds of the present invention, including enantiomeric and diastereomeric forms (e.g., those that may exist due to asymmetric carbons on various substituents), are contemplated within the scope of the present invention. Individual stereoisomers of the compounds of the present invention may, for example, be substantially free of other isomers (e.g., pure or substantially pure optical isomers with a particular activity), or may be mixed, for example, as a racemate, or with all other or selected other stereoisomers. The chiral centers of the compounds of the present invention may have the S or R configuration as defined by the IUPAC 1974 Recommendations.

[0045] The racemic forms can be resolved by physical methods, such as fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. The individual optical isomers can be obtained from the racemates by any suitable method, including, but not limited to, formation of a salt with an optically active acid followed by crystallization.

[0046] All configurational isomers of the compounds of the present invention are contemplated, either in admixture or in pure or substantially pure form. The definition of the compounds of the present invention encompasses both cis (Z) and trans (E) alkene isomers, as well as cis and trans isomers of cyclic hydrocarbons or heterocycles.

[0047] Deuterated versions of the claimed compounds may also be provided. The position at which the deuteration occurs may be, but is not limited to, for example, R 4 and R 5 C 1~4 It can be alkyl.

[0048] Throughout the specification, groups and substituents thereof may be chosen to provide stable moieties and compounds.

[0049] The compounds of the present invention may be provided in the form of diagnostic or pharmaceutical compositions, optionally containing a pharma- ceutically acceptable carrier or excipient.

[0050] According to the present invention, the term "diagnostic composition" relates to a composition for the determination of the presence of aggregated α-synuclein that underlies a disease associated with α-synuclein aggregation.

[0051] In a preferred embodiment, the compound of the present invention is detectable or detectably labeled.According to the present invention, it is understood that compound is detectable or detectably labeled if the presence of compound can be monitored by conventional techniques, such as NMR spectroscopy, single photon emission computed tomography (SPECT), light detection, positron emission tomography (PET), electron microscopy, magnetic resonance imaging (MRI), spectroscopy, chromatography, ELISA assay, detection of radioactive emission, preferably by PET, scintillation counting or gamma counting, more preferably by PET.

[0052] When the compounds of the invention are used as probes for imaging aggregated α-synuclein, they must be labeled. The specific nature of the label will depend on the method used for imaging. Typically, radioactive labels that emit positrons (PET) and have a short half-life, e.g. 18 F, 11 C. 125 I, 123 I, 131 I, 77 Br and 76 Br, especially 18 F and 11 C is useful. Due to its short half-life, the labeled compound of the present invention needs to be prepared immediately before it is used in the test. Therefore, the diagnostic composition of the present invention may also be provided in the form of a kit consisting of at least two precursors of the compound of the present invention, which react to form the desired compound of the present invention.

[0053] Those skilled in the art will be able to devise methods by which detectable labels can be attached to the compounds of the invention. The following schemes may be utilized as illustrative examples.

[0054] 18Labeling with F 2-[ 18 [F]Fluoroethyl tosylate can be prepared via fluoroethylation of compounds containing nitrogen, oxygen and sulfur nucleophiles as described in WO 2010 / 000372, page 32, scheme A for the preparation of compounds 21 and 22 starting from compound 23. 18 Compounds A and B are useful precursors for incorporating F. Compounds A and B can be prepared in the same manner. Alternative methods include the conversion of C to D (see J. Med. Chem. 2013, 56, 4568-4579, Scheme 2) or direct nucleophilic displacement of a suitable leaving group, as shown for the conversion of compound 24 to compound 12 in Scheme 1. [ka]

[0055] 11 Labeling with C 11 The compounds of the present invention labeled with C can be prepared by the reaction of the compound 27 and the compound 28 starting from the compound 23 as described in WO2010 / 000372, page 32, scheme B, 11 Compound 1 of the present invention can be prepared by direct nucleophilic alkylation of a suitable precursor with [C]Mel. Compound 1 of the present invention can be synthesized analogously starting from compound 2. In the same manner, compounds 18 and 19 can be synthesized starting from compound 9 as shown in Scheme 2. Alternatively, compounds 18 and 19 can be synthesized under mild conditions without loss of specific activity. 11 C] Methyl iodide [ 11 C] formaldehyde as a solution in dimethylformamide. 11 A simple and available method for the generation of [C]formaldehyde (JM Hooker et al., Angew. Chem. Int. Ed. 2008, 47, 5989-5992) may be used for the conversion of compound 2 to compound 1 via reductive amination. [ka]

[0056] The present invention provides a method for imaging aggregated alpha-synuclein deposits, comprising the steps of: (i) introducing into a subject a detectable amount of a composition comprising a detectably labeled compound of the invention; (ii) allowing a sufficient time for the compound to associate with aggregated alpha-synuclein; (iii) detecting a compound associated with aggregated alpha-synuclein; The present invention provides a method comprising:

[0057] A composition comprising a detectably labeled compound may be introduced into a subject by any of the administration routes described below, such as orally or parenterally. A labeled compound may be introduced into a patient, and after a sufficient period of time for the compound to become associated with aggregated α-synuclein, the labeled compound may be detected non-invasively within the patient. Alternatively, a labeled compound may be introduced into a patient, and after a sufficient period of time for the compound to become associated with aggregated α-synuclein, a tissue sample from the patient is then taken, and the labeled compound may be detected in the tissue remote from the patient. The tissue sample may also be removed from the patient before introducing the labeled compound into the tissue sample. After a sufficient period of time for the compound to become bound to aggregated α-synuclein, the compound may be detected.

[0058] Imaging of aggregated alpha-synuclein may also be performed quantitatively so that the amount of aggregated alpha-synuclein can be determined.

[0059] The present invention further relates to the compounds of the invention, and their prodrugs, solvates or salts, for use in the treatment or prevention of diseases associated with the aggregation of α-synuclein.

[0060] Further embodiments are the use of the compounds of the invention for the preparation of a pharmaceutical composition for treating or preventing a disease associated with the aggregation of alpha-synuclein, and a method for treating or preventing a disease associated with the aggregation of alpha-synuclein, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the invention, including the application of the compounds as a "pharmaceutical composition" as described below.

[0061] According to the present invention, the term "aggregation" refers to the formation of oligomeric or multimeric complexes of α-synuclein, which may involve the incorporation of additional biomolecules such as carbohydrates, nucleic acids, lipids and / or metal ions into the complex.

[0062] As used herein, the term "diseases associated with aggregation of α-synuclein" refers to diseases characterized by the presence of aggregated α-synuclein. Such aggregated α-synuclein can form deposits in certain tissues, more preferably in neural or brain tissue. The extent of aggregation depends on the particular disease.

[0063] According to the present invention, the term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a mammal, more preferably a human patient. The pharmaceutical composition of the present invention comprises the compounds listed above, as well as optional additional molecules that can change the properties of the compound of the present invention, thereby, for example, stabilizing, regulating and / or activating its function. The composition may be in solid, liquid or gas form, in particular in the form of a powder, tablet, solution or aerosol. The pharmaceutical composition of the present invention may optionally and additionally comprise a pharma-ceutically acceptable carrier or excipient. Examples of suitable pharmaceutical carriers and excipients are well known in the art, and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, organic solvents, including DMSO, and the like. Compositions containing such carriers can be formulated by well-known conventional methods.

[0064] Pharmaceutical compositions are formulated and administered in a manner consistent with good medical practice, taking into consideration the clinical condition of individual patients, the delivery site of pharmaceutical compositions, administration method, administration schedule and other factors known to practitioners.Therefore, the "effective amount" of pharmaceutical compositions for the purposes of this specification is determined by such considerations.Those skilled in the art know that the effective amount of pharmaceutical compositions administered to individuals depends on, among other things, the nature of the compound.

[0065] The pharmaceutical compositions of the present invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (such as by powder, ointment, drops or transdermal patch), bucally or as an oral or nasal spray. Preferably, they are administered intravenously when used as PET tracers for diagnosis, or orally when used to treat or prevent disease.

[0066] "Pharmaceutically acceptable carrier" means a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0067] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0068] For therapeutic purposes, pharmaceutical compositions may also be suitably administered by sustained release systems.Suitable examples of sustained release compositions include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules.Sustained release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58481), copolymers of L-glutamic acid and γ-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly(2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981) and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., ibid.) or poly-D-(-)-3-hydroxybutyric acid (EP 133988). The sustained release pharmaceutical composition may also contain the compound encapsulated in liposome.The liposome containing pharmaceutical composition is prepared by a method known per se: DE3218121;Epstein et al., Proc.Natl.Acad.Sci.(USA)82:3688-3692(1985);Hwang et al., Proc.Natl.Acad.Sci.(USA)77:4030-4034(1980);EP52322;EP36676;EP88046;EP143949;EP142641;JP83-118008;US Patent Nos. 4,485,045 and 4,544,545;and EP102324. Typically, the liposomes are small (approximately 200-800 angstroms) unilamellar, with lipid content exceeding approximately 30 mol% cholesterol, with the selected percentage being adjusted for optimal therapeutic application.

[0069] For parenteral administration, a pharmaceutical composition is generally formulated by mixing it with the desired degree of purity, in a unit dosage injectable form (solution, suspension or emulsion), and with a pharma- ceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with the other ingredients of the formulation.

[0070] In general, the formulation is prepared by uniformly and intimately contacting the components of the pharmaceutical composition with liquid carriers or finely divided solid carriers or both.Then, if necessary, the product is shaped into the desired formulation.Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient.Examples of such carrier vehicles include water, saline, Ringer's solution and dextrose solution.Non-aqueous vehicles such as fixed oils and ethyl oleate, and liposomes are also useful herein.The carrier preferably contains minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Such substances are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate, succinate, acetate and other organic acids or salts thereof; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) (poly)peptides such as polyarginine or tripeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides and other carbohydrates, including cellulose or its derivatives, glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and / or non-ionic surfactants such as polysorbates, poloxamers or PEG.

[0071] The components of the pharmaceutical composition used for therapeutic administration must be sterile. Sterility is easily achieved by filtration through a sterile filtration membrane (e.g., 0.2 μm membrane). The therapeutic components of the pharmaceutical composition are generally placed into a container with a sterile access port, such as an intravenous bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0072] The components of pharmaceutical compositions are usually stored in unit or multi-dose containers, such as sealed ampoules or vials, as aqueous solutions or as lyophilized formulations for reconstitution.As an example of lyophilized formulations, 10ml vials are filled with 5ml of sterile filtered 1% (w / v) aqueous solution, and the resulting mixture is lyophilized.Infusion solutions are prepared by reconstituting lyophilized compounds with bacteriostatic water for injection.

[0073] The present invention further relates to a method for treating or preventing a disease associated with α-synuclein aggregation, comprising the step of administering a therapeutically effective amount of a compound of the present invention to a patient in need thereof.

[0074] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to induce a desired biological response, in the present invention, the desired biological response being inhibition of α-synuclein aggregation and / or a reduction in the amount of aggregated α-synuclein present in a tissue.

[0075] The present invention also relates to the use of the compounds as defined above for inhibiting α-synuclein aggregation in vitro or ex vivo.

[0076] The disease associated with α-synuclein aggregation is not particularly limited, and is typically selected from Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy.

[0077] The following examples are intended to illustrate the invention, but should not be construed as limiting. EXAMPLES

[0078] Example 1: Synthesis and testing Chemical synthesis procedure The following methods provide details for the preparation of the compounds of the invention and illustrative examples. The compounds of the invention can be prepared by one skilled in the art of organic synthesis from known or commercially available starting materials and reagents.

[0079] All starting materials and solvates were commercial grade and used as received unless otherwise noted.

[0080] Thin layer chromatography (TLC) was performed using Macherey-Nagel precoated sheets, 0.25 mm ALUGRAM® SIL G / UV254 plates, detection by UV and / or by charring with 10 wt % ethanolic phosphomolybdic acid reagent followed by heating at 200° C.

[0081] Flash column chromatography was performed using Merck silica gel 60 (0.063-0.100 mm).

[0082] Analytical high-performance liquid chromatography (HPLC) was performed using a Waters HPLC system equipped with a Waters 996 photodiode array detector. All separations involved mobile phases of 0.1% trifluoroacetic acid (TFA) in water (v / v) and 0.1% TFA in acetonitrile. HPLC was performed at a flow rate of 1 mL min -1 The separation was carried out at RT using a reversed-phase (RP) column Eurospher RP18, 100 Å, 5 μm, 250×4.6 mm.

[0083] Electrospray ionization mass spectrometry (ESI-MS) and liquid chromatography / mass spectrometry (LC / MS) analyses were obtained using a Waters Micromass ZQ4000 mass spectrometer coupled to a Waters HPLC instrument as described above.

[0084] NMR spectra were recorded using a 400 MHz Bruker Avance spectrometer equipped with a TXI HCN z-gradient probe (Bruker AG, Rheinstetten, Germany). All spectra were processed using TOPSPIN3.1 (Bruker AG, Karlsruhe, Germany). 1H NMR chemical shifts (δ) are reported in percentages (ppm) relative to CHCl3, DMSO-d5 and TFA-d1 as internal standards. Data are reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, qi=quintet, dd=doublet of doublets, dt=doublet of triplets, b=broad, m=multiplet), coupling constant (J, given in Hz), integration. 13 C NMR chemical shifts (δ) are reported in percentages (ppm) relative to CDCl3, DMSO-d6 and TFA-d1 as internal standards. The following experimental methods were used to record the resonances of the compounds: 1 H-1D, 13 C-1D NMR spectrum and 13 C-APT (attached proton study with a single J-development time of 1 / 145 sec, spectra were processed such that quaternary and methylene groups have a positive sign and methyl and methine groups have a negative sign). 1 To resolve the overlapping resonances in the H and APT spectra and recover undetectable resonances, 2D-[ 13 C, 1 H]-HSQC (heteronuclear single quantum coherence), 2D-[ 13 C, 1 H]-HMBC (heteronuclear multibond correlation) and 2D-NOESY were recorded for several compounds.

[0085] Selected compounds (compound 1, compound 2, compound 12, compound 18) were tritium labeled for binding assays by RC TRITEC AG, Teufen, Switzerland using 99% tritium gas / Kerr catalyzed H / T exchange labeling. Compounds were supplied as ethanol solutions in 185 MBq packages, at a concentration of 37 MBq / mL and specific activities ranging from 1.1 to 2.3 GBq / mmol.

[0086] Method A: Synthesis of 1H-pyrazoles Those skilled in the art will recognize that compound 11A and compound 11B shown below are two tautomeric forms of the same compound. All such tautomeric forms are considered to be part of the present invention. By way of illustration, all tautomeric forms of the pyrazole moiety shown, for example, for compound 11 below, are included in the present invention. In a related manner, those skilled in the art will recognize that the compound names contained herein are based on nomenclature conventions, and the configurations of tautomers are shown as for compound 11A below. Thus, the 1,3-benzodioxole substituent is at the 3-position. An alternative nomenclature convention is based on tautomeric compound 11B below, in which the 1,3-benzodioxole substituent is at the 5-position. [ka]

[0087] Illustrative Example: 2-[3-(1,3-benzodioxol-5-yl)-1H-pyrazol-5-yl]-6-fluoropyridine Compound 11 [ka] Sodium hydride (FW24.00, 60% in oil, 3.9 mmol, 156 mg) was added to a solution of 1-(1,3-benzodioxol-5-yl)ethanone (FW164.16, 492 mg, 3.00 mmol) and methyl 6-fluoropyridine-2-carboxylate (FW155.13, 605 mg, 3.9 mmol) in DMSO (7.5 mL) and THF (1.9 mL) and the reaction mixture was stirred at 20° C. for 15 h. The reaction mixture was poured into 60 mL of ice water containing AcOH (450 μL). The mixture was stirred for 1 h. The resulting precipitate was filtered off, washed with water (10 mL), hexane:EtOH=5:1 (10 mL), hexane (10 mL), and air-dried to give the crude intermediate 1-(1,3-benzodioxol-5-yl)-3-(6-fluoropyridin-2-yl)propane-1,3-dione (594 mg) as a yellow solid. To a suspension of this crude intermediate in EtOH (20 mL) was added hydrazine hydrate (146 μL, 150 mg, 3 mmol). The reaction mixture was stirred at 70° C. for 5 h, cooled, and concentrated in vacuo. The residue was suspended in MeOH (10 mL), boiled with stirring for 5 min, cooled, filtered off, washed with MeOH (10 mL), and dried under high vacuum at 20° C. for 15 h to give the pure product compound 11 (471 mg, 1.66 mmol, 55% over two steps) as a white solid.

[0088] Method B: Synthesis of 1H-pyrazoles Illustrative Example: 4-[3-(1,3-benzodioxol-5-yl)-1H-pyrazol-5-yl]-2-bromopyridine Compound 9 [ka] A solution of potassium tert-butoxide (FW112.21, 281 mg, 2.5 mmol) in dry THF (5 mL) was added under nitrogen to a stirred solution of 1-(1,3-benzodioxol-5-yl)ethanone (FW164.16, 328 mg, 2 mmol) and methyl 2-bromopyridine-4-carboxylate (FW216.03, 518 mg, 2.4 mmol) in dry THF (5 mL). The reaction mixture was stirred at 20 °C for 15 h. An aliquot of 20 μL was sampled, quenched with 1 M phosphate buffer pH 7, extracted with EtOAc and analyzed by TLC. Complete conversion of the ketone was observed. The mixture was poured into 1 M phosphate buffer pH 7 (15 mL) and ice water (15 mL) and stirred at 0 °C for 30 min. The resulting yellow solid was filtered off, washed with water (5×10 mL) and air-dried to give 656 mg (1.88 mmol, 94%) of the crude intermediate 1-(1,3-benzodioxol-5-yl)-3-(2-bromopyridin-4-yl)propane-1,3-dione, which was used in the next step without purification. A mixture of this intermediate and hydrazine monohydrate (FW50.06, d1.03; 274 μL, 282 mg, 5.64 mmol) in THF (10 mL) was stirred at 50° C. for 15 h. The cooled mixture was poured into water (40 mL) and stirred at 0° C. for 30 min. The resulting precipitate was filtered off, washed with water and air-dried. The crude product was crystallized from n-BuOH (10 mL) and DMF (1 mL) to give the pure product compound 9 (458 mg, 1.33 mmol, 67% over two steps) as a white powder.

[0089] Method C: Removal of the Boc protecting group Illustrative Example: 4-[5-(2-bromopyridin-4-yl)-1H-pyrazol-3-yl]aniline Compound 7 [ka] Trifluoroacetic acid (2 mL, 2.96 g, 26 mmol) was added to a suspension of crude tert-butyl {4-[5-(2-bromopyridin-4-yl)-1H-pyrazol-3-yl]phenyl}carbamate (FW415.28, 865 mg, 2.08 mmol), prepared from tert-butyl N-(4-acetylphenyl)carbamate and methyl 2-bromopyridine-4-carboxylate according to method B. The mixture was stirred at room temperature for 15 h and concentrated in vacuo. 1 M phosphate buffer pH 7 (20 mL) was added and the resulting precipitate was filtered off, washed with water (2×10 mL) and air-dried to give 543 mg (1.72 mmol, 69% over three steps) of the desired product as a yellow-orange solid.

[0090] Method D: Synthesis of 1H-pyrazoles Illustrative Example: 4-[3-(1,3-benzodioxol-5-yl)-1H-pyrazol-5-yl]-2-fluoropyridine Compound 12 [ka] iPr2NEt (1.79 mL, 1.33 g, 10.26 mmol) was added to a stirred mixture of 1-(1,3-benzodioxol-5-yl)ethanone (561 mg, 3.42 mmol) and MgBr2·Et2O (1.56 g, 6.04 mmol) in CHCl2 (35 mL). The resulting suspension was stirred for 5 min, then pentafluorophenyl 2-fluoropyridine-4-carboxylate (1.37 g, 4.45 mmol) in CHCl2 (7 mL) was added dropwise. The reaction mixture was stirred for 48 h. 1N aqueous HCl (20 mL) was then added and stirring was continued for 5 min. The aqueous layer was extracted with CHCl2 (20 mL) and the combined organic extracts were dried (MgSO4) and concentrated in vacuo. The residue was triturated with Et2O (10 mL) and filtered. The solid was washed with Et2O and air-dried to give the crude product (1.14 g, orange solid). The crude product was recrystallized from EtOAc (8 mL) and hexanes (4 mL) to give the pure intermediate 1-(1,3-benzodioxol-5-yl)-3-(2-fluoropyridin-4-yl)propane-1,3-dione (900 mg, 3.13 mmol, 92%) as an orange solid. This intermediate was suspended in THF (20 mL) and hydrazine monohydrate (292 μL, 300 mg, 6 mmol) was added. The reaction mixture was stirred at 70 °C for 5 h, cooled, and concentrated in vacuo. The residue was suspended in EtO (10 mL), boiled with stirring for 5 min, cooled, filtered off, washed with EtO (2×10 mL), and dried under high vacuum at 20° C. for 15 h to give the product compound 12 (722 mg, 2.55 mmol, 76% over two steps) as a white solid.

[0091] Method E: Synthesis of pentafluorophenyl esters Illustrative Example: Pentafluorophenyl 2-fluoropyridine-4-carboxylate Compound 59 [ka] DCC (FW206.33, 2.27 g, 11 mmol) was added to a stirred suspension of 2-fluoropyridine-4-carboxylic acid (1.41 g, 10 mmol) and pentafluorophenol (1.84 g, 10 mmol) in 1,4-dioxane (40 mL). Stirring was continued for 15 h, by which time a colorless precipitate had formed. The mixture was filtered through Celite® and evaporated to give a semi-solid. Chromatography on silica gel afforded the ester compound 59 (2.21 g, 7.2 mmol, 72%) as a pure colorless oil.

[0092] Method F: Synthesis of 1H-pyrazoles Illustrative Example: 6-[3-(3-bromophenyl)-1H-pyrazol-5-yl]-1,3-dioxolo[4,5-c]pyridine Compound 20 [ka]

[0093] Step 1 A suspension of 1,3-dioxolo[4,5-c]pyridine-6-carboxaldehyde (WO / 2019 / 208509) (35 mg, 0.23 mmol) and 1-(3-bromophenyl)ethanone (46 mg, 0.23 mmol) in methanol (0.7 mL) was added to the reaction mixture with Ba(OH) * 8H2O (5 mg) and NaOH (0.5 mg) were added and the resulting mixture was stirred at room temperature (RT) overnight. After evaporation of methanol in vacuo, the residue was triturated in water (5 mL) and the solid was collected by filtration, washed with cold methanol (0.5 mL) and dried to give the intermediate compound 1-(3-bromophenyl)-3-([1,3]dioxolo[4,5-c]pyridin-6-yl)prop-2-en-1-one (67 mg, 88%) as a white solid.

[0094] Step 2 To a vigorously stirred suspension of 1-(3-bromophenyl)-3-([1,3]dioxolo[4,5-c]pyridin-6-yl)prop-2-en-1-one (67 mg, 0.2 mmol) in DMSO (0.8 mL) was added an aqueous solution of H2O2 (30%, 45 mg, 0.4 mmol), followed by dropwise addition of aqueous NaOH (10%, 16 μL, 0.04 mmol). The yellow mixture was stirred at RT for 1.5 h and poured into cold phosphate buffer (20 mL, 0.1 M, pH 7). The oily precipitate was extracted with ethyl acetate (2 * The mixture was extracted with 15 mL of ethyl acetate (2 M, 100 mL) and the combined organic fractions were dried over Na2SO4, concentrated in vacuo, and the residue was resuspended in toluene (0.8 mL). The suspension in toluene was treated with hydrazine hydrate (35 mg, 0.7 mmol) and PTSA hydrate (5 mg) and the mixture was dried under reflux for 1.5 h. After cooling, phosphate buffer (0.3 M, 20 mL) was added and the product was extracted with ethyl acetate (2 M, 100 mL). * The crude residue was purified by column chromatography (15 g, silica gel 63-100, CHCl3 / MeOH=100 / 1) to give a yellow solid, which was washed with Et2O (1 mL) to give compound 20 (25 mg, 36%) as a white solid.

[0095] Method G: Synthesis of 1H-pyrazoles Illustrative examples: 4-[3-(1,3-benzodioxol-5-yl)-1H-pyrazol-5-yl]-3,6-dichloropyridazine compound 48 [ka] A mixture of 1-(1,3-benzodioxol-5-yl)ethanone (138 mg, 0.84 mmol), MgBr2·Et2O (542 mg, 2.1 mmol) in DCM (5 mL) was treated with DIPEA (323 mg, 426 μL, 2.5 mmol) and stirred at RT for 10 min. Then, a crude mixture of 1H-benzotriazol-1-yl(3,6-dichloropyridazin-4-yl)methanone, prepared separately by stirring 3,6-dichloropyridazine-4-carboxylic acid (203 mg, 1.05 mmol), benzotriazole (125 mg, 1.05 mmol) and DCC (216 mg, 1.05 mmol) in dry DCM (5 mL) at 25 °C for 3 h, was added dropwise over 5 min. The resulting mixture was stirred at 25° C. for 12 h, then treated with aqueous 0.5 M HCl (2 mL) and stirred at 25° C. for an additional 10 min. After the addition of water (20 mL), the mixture was diluted with DCM ( * The mixture was extracted with 15 mL of toluene (HCl, 200 rpm, 15 ml). The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated in vacuo. The crude product was purified by column chromatography to give intermediate 1-(1,3-benzodioxol-5-yl)-3-(3,6-dichloropyridazin-4-yl)propane-1,3-dione (190 mg, 67%) as an orange solid, which was used in the next step without further purification. This intermediate was suspended in THF (4 mL) and hydrazine monohydrate (40 mg, 0.8 mmol) was added. The reaction mixture was stirred at 40 °C overnight, cooled, and concentrated in vacuo. The crude product was purified by column chromatography (silica gel 63-100, 20 g, chloroform / methanol = 100 / 1) to give compound 48 (100 mg, 36% over two steps) as a light yellow solid.

[0096] Method H: Synthesis of 1H-imidazoles Illustrative Example: 4-[4-(1,3-benzodioxol-5-yl)-1H-imidazol-2-yl]-2-bromopyridine Compound 33 [ka]

[0097] Step 1 A suspension of 1-(1,3-benzodioxol-5-yl)-2-bromoethanone (729 mg, 3 mmol), 2-bromopyridine-4-carboxylic acid (606 mg, 3 mmol) and K2CO3 (414 mg, 3 mmol) in DMF (6 mL) was stirred at 55-60 °C for 6 h. After cooling, the mixture was poured into water (60 mL), stirred for 10 min, and the resulting precipitate was collected by filtration, washed on the filter with water (20 mL) and dried to give the intermediate compound 2-(1,3-benzodioxol-5-yl)-2-oxoethyl 2-bromopyridine-4-carboxylate (899 mg, 87%), which was used in the next step without further purification.

[0098] Step 2 A suspension of the intermediate compound 2-(1,3-benzodioxol-5-yl)-2-oxoethyl 2-bromopyridine-4-carboxylate (364 mg, 1 mmol) and AcONH4 (924 mg, 12 mmol) in toluene (7 mL) was heated at 100 °C with intensive stirring for 4 h. After cooling, the reaction mixture was treated with phosphate buffer (50 mL, 0.25 M, pH 7) and ethyl acetate (2 * The mixture was extracted with 50 mL of ethyl acetate (50 ml). The combined organic fractions were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (silica gel 63-100, 30 g, CHCl3 / MeOH=100 / 1→100 / 3). The purified product was recrystallized from aqueous ethanol (90%) to give compound 33 (140 mg, 41%) as a pale solid.

[0099] Method I: Synthesis of 1H-1,2,4-triazoles Illustrative Example: 4-[3-(1,3-benzodioxol-5-yl)-1H-1,2,4-triazol-5-yl]-2-bromopyridine Compound 30 [ka] To a solution of t-BuOK (84 mg, 0.75 mmol) in n-BuOH (2 mL) was added benzo[1,3]dioxole-5-carboxamidine hydrochloride (100 mg, 0.5 mmol) at 0° C. and the mixture was stirred at RT for 20 min. After addition of 2-bromoisonicotinic acid hydrazide (108 mg, 0.5 mmol), the yellow suspension was stirred at 85° C. for 3 h. After cooling to room temperature, ethanol (4 mL) was added and CO2 was bubbled through the suspension for 5 min. After removal of the solvent in vacuo, the residue was triturated in water (10 mL) and the resulting precipitate was collected by filtration, washed with water (5 mL) and dried to give compound 30 (85 mg, 49%) as a grey solid.

[0100] Method J: Synthesis of 1H-1,2,4-triazoles Illustrative example: 4-[3-(2-bromopyridin-4-yl)-1H-1,2,4-triazol-5-yl]-N,N-dimethylaniline compound 44 [ka] A mixture of 2-bromoisonicotinic acid hydrazide (151 mg, 0.7 mmol), 4-(dimethylamino)benzonitrile (307 mg, 2.1 mmol) and K2CO3 (48 mg, 0.5 mmol) in n-BuOH (2 mL) was stirred at 145 °C for 8 h. The mixture was concentrated in vacuum and the crude product was purified by two column chromatography (silica gel 63-100, 20 g, CHCl3 / MeOH = 100 / 1) and (silica gel 63-100, 20 g, acetone / hexane = 1 / 5) to give compound 44 (20 mg, 8%) as a beige solid.

[0101] Method K: Synthesis of 4-chloro-1H-pyrazole 4-[3-(1,3-benzodioxol-5-yl)-4-chloro-1H-pyrazol-5-yl]-2-bromopyridine, compound 28 [ka] A suspension of compound 9 (100 mg, 0.29 mmol) and NCS (80 mg, 0.6 mmol) in water (3 mL) was stirred at 70° C. for 16 h. TLC indicated consumption of starting material. After cooling to RT, the precipitate was collected by filtration and washed with water (2 * The extract was washed with 5 mL of ethyl acetate and recrystallized from ethanol (8 mL) to give compound 28 (52 mg, 47%) as a grey solid.

[0102] Method L: Synthesis of 1-[4-(4-R-piperazin-1-yl)phenyl]ethanone Illustrative Example: 1-{4-[4-(2-methoxyethyl)piperazin-1-yl]phenyl}ethenone [ka] A mixture of 1-[4-(piperazin-1-yl)phenyl]ethanone (408 mg, 2 mmol), 1-bromo-2-methoxyethane (417 mg, 3 mmol) and Cs2CO3 (1304 mg, 4 mmol) in DMF (5 mL) was stirred at 25 °C overnight. After evaporation of DMF in vacuum, the residue was partitioned between water (25 mL) and ethyl acetate (35 mL), the aqueous phase was extracted with ethyl acetate (25 mL) and the combined organic fractions were washed with brine, dried over Na2SO4 and concentrated in vacuum. The crude product was purified by column chromatography (30 g silica gel 63-100, chloroform → chloroform / MeOH = 100 / 2) to give 1-{4-[4-(2-methoxyethyl)piperazin-1-yl]phenyl}ethenonone (525 mg, 99%) as a pale yellow solid.

[0103] 2-Methoxyethyl 2,6-dibromopyridine-4-carboxylate and 2-methoxyethyl 2,6-dichloropyridine-4-carboxylate were prepared according to published protocols (Journal of Medicinal Chemistry (2012), 55, 10564-10571).

[0104] tert-Butyl(4-acetylphenyl)methylcarbamate, tert-Butyl(4-acetylphenyl)-N-( 2H3) Methyl carbamate, tert-butyl(4-acetylphenyl)ethyl carbamate and tert-butyl(4-acetylphenyl)(2-fluoroethyl)carbamate were prepared according to published protocols (Organic Letters (2020), 22, 5522-5527).

[0105] 1-[4-(4-fluoropiperidin-1-yl)phenyl]ethanone and 1-[4-(3-fluoroazetidin-1-yl)phenyl]ethanone were prepared according to published protocols (WO2011071570A1).

[0106] Pentafluorophenyl 2-fluoropyridine-4-carboxylate and pentafluorophenyl 2-chloropyridine-4-carboxylate were prepared according to Method E. 1-{4-[4-(2-methoxyethyl)piperazin-1-yl]phenyl}ethenone and 1-{4-[4-(2-fluoroethyl)piperazin-1-yl]phenyl}ethenone were prepared according to Method L.

[0107] Exemplary compounds of the present invention are shown in Tables 1 and 2. Table 1 shows the name, structural formula, IUPAC name, starting material for preparation, synthesis method, and chemical yield of certain exemplary compounds. Table 2 shows the high performance liquid chromatography (HPLC) retention time, molecular weight measured using HPLC plus low resolution mass spectrometry, and proton nuclear magnetic resonance ( 1 H-NMR) is shown.

[0108] [Table 1-1]

[0109] [Table 1-2]

[0110] [Table 1-3]

[0111] [Table 1-4]

[0112] [Table 1-5]

[0113] [Table 1-6]

[0114] [Table 2-1]

[0115] [Table 2-2]

[0116] [Table 2-3]

[0117] [Table 2-4]

[0118] [Table 2-5]

[0119] Example 2: Binding studies with aggregated α-synuclein, tau and Aβ To analyze the binding affinity and target selectivity of the compounds, two types of in vitro fiber binding assays were used: a saturation assay and a competition assay.

[0120] 1) Preparation of fibers α-Synuclein and tau46 were purified from E. coli according to established protocols (Nuscher B et al., J. Biol. Chem., 2004;279(21):21966-75), and Aβ 1~42 was obtained from rPeptide, Watkinsville, GA, USA. Fibrils were prepared by aggregating the recombinant protein with constant stirring. In detail, 70 μM α-synuclein mixed with 100 mM NaCl in 50 mM Tris, pH 7.0 + 0.02% NaN3 was incubated at 1400 rpm, 37 °C for 96 h. 10 μM tau46 was incubated with 0.03 mg / ml heparin in 50 mM Tris, pH = 7.0, 1,000 rpm, 37 °C for 72 h. Aβ 1~42 20 mM NaP i , pH=8.0 + 0.2 mM EDTA + 0.02% NaN3 (Deeg AA et al., Biochim. Biophys. Acta, 2015;1850(9):1884-90; Goedert M et al., Nature, 1996;383(6600):550-3).

[0121] 2a) Saturation binding assay Sonicated αSYN (0.04 μM) or tau46 (0.4 μM) or Aβ diluted in PBS 1~42 (6 μM) fibers were treated with decreasing concentrations of [ 3 H-labeled compounds (48 nM or 24 nM–23 pM) in 50 mM Tris base, 10% ethanol, 0.05% Tween 20, pH 7.4. To determine nonspecific binding, each unlabeled compound (400 nM) was added to a duplicate set of binding reactions.

[0122] The assay plates were covered with plastic foil (Reseal Tape, PerkinElmer, Waltham, MA, USA) and incubated at 37°C for 2 h with agitation. Prior to harvesting, the filters (Printed Filtermat B, PerkinElmer, Waltham, MA, USA) were incubated with 5 mg / mL polyethyleneimine (PEI, poly(ethyleneimine) solution, Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) for 30 min at 4°C. After incubation, bound and free ligand were separated by vacuum filtration using a harvester (Filtermate harvester, PerkinElmer, Waltham, MA, USA). The filters were washed three times with buffer cooled to 4°C and then dried in a microwave oven at medium power for 2 min. Melt-on scintillator sheets (MeltiLex™ B / HS, PerkinElmer, Waltham, MA, USA) were melted onto the filters using a heating plate set at 120°C. After solidification at room temperature, the filters were sealed in plastic bags (MicroBeta® sample bags, PerkinElmer, Waltham, MA, USA). Tritium accumulation was rapidly counted in a liquid scintillation counter (Wallac MicroBeta® TriLux, 1450 LSC & Luminescence Counter, PerkinElmer, Waltham, MA, USA). Radioactivity was plotted against increasing tritiated compound or cold compound concentration. Data points were fitted using nonlinear regression analysis in GraphPad Prism (GraphPad Software, Inc., version 7.03, La Jolla, CA, USA).

[0123] 2b) Modified saturation binding assay For saturation binding assays, fixed concentrations of sonicated human recombinant αSYN (15 nM / well) or tau46 (250 nM / well) or Aβ were diluted in phosphate-buffered saline (PBS). 1~42(1 μM / well) fibrils were incubated with increasing concentrations (0.05 nM–12 nM / 24 nM) of [ 3 H]-Compound 1 or [ 3 H]-Compound 2 in a total volume of 200 μL / well of 30 mM Tris-HCl, 10% ethanol, 0.05% Tween 20, pH 7.4 (hereafter referred to as incubation buffer). Nonspecific binding of the radiotracer was determined by co-incubation with 400 nM unlabeled Compound 1 or Compound 2. Optimal fibril concentrations were determined using a concentration determination assay.

[0124] Plates covered with removable sealing tape (PerkinElmer) were incubated for 2 h at 37°C at 45 rpm on a shaker (MaxQ™ 6000, orbit diameter 1.9 cm, Thermo Fisher Scientific Inc., Marietta, OH, USA). After incubation, bound and free radioligand were separated by vacuum filtration through glass fiber filter mats B (PerkinElmer) using a filter mat harvester (PerkinElmer). αSYN and Aβ 1~42 To harvest plates containing fibers, filter mats were further incubated with 5 mg / mL polyethyleneimine for 30 min at 4° C. before harvesting. Filters were washed three times with 100 mL (approximately 1 mL / well) of cold incubation buffer and then dried in a microwave oven at medium power for 2.5 min. Melt-on scintillator sheets (MeltiLex™ B / HS, PerkinElmer) were melted onto the filters using a heating plate set at 120° C. After curing at room temperature, filters were sealed in plastic sample bags (PerkinElmer). Tritium accumulation was rapidly counted in a Wallac MicroBeta® TriLux liquid scintillation counter (PerkinElmer). Radioactivity was determined by: 3The data were plotted against the H-labeled compound concentration. Data points were fitted using nonlinear regression analysis in GraphPad Prism (GraphPad Software, Inc., version 7.03, La Jolla, Calif., USA).

[0125] 3a) Competitive binding assays Constant concentrations of sonicated human recombinant αSYN (200 nM / well) or tau46 (208 nM / well) fibrils diluted in phosphate-buffered saline (PBS) were incubated in low-binding plates (96-well microtest plates, Ratiolab GmbH, Dreieich, Germany) at 1 nM [ 3 H]-Compound 1 and 1:4 serial dilutions of the cold compound of interest starting at 1 μM in 50 mM Tris-base, 10% EtOH, 0.05% Tween 20, pH 7.4. i For calculation of K values, the K of compound 1 on sonicated human recombinant αSYN fibrils was d The value was set at 3 nM.

[0126] Assay plates were covered with plastic foil (Reseal Tape, PerkinElmer, Waltham, MA, USA) and incubated at 37°C for 2 h with agitation. Prior to harvesting, filters (Printed Filtermat B, PerkinElmer, Waltham, MA, USA) were incubated with 5 mg / mL polyethylenimine (PEI, poly(ethylenimine) solution, Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) for 30 min at 4°C. After incubation, bound and free ligand were separated by vacuum filtration using a harvester (Filtermate harvester, PerkinElmer, Waltham, MA, USA). Filters were washed three times with buffer cooled to 4°C and then dried in a microwave oven at medium power for 2 min. Filters were sealed in plastic bags (MicroBeta® sample bags, PerkinElmer, Waltham, MA, USA) with added scintillator (BETAPLATE SCINT, PerkinElmer, Waltham, MA, USA). Tritium accumulation was rapidly counted in a liquid scintillation counter (Wallac MicroBeta® TriLux, 1450 LSC & Luminescence Counter, PerkinElmer, Waltham, MA, USA). Radioactivity was plotted against increasing tritium-labeled compound or cold compound concentration. Data points were fitted using nonlinear regression analysis in GraphPad Prism (GraphPad Software, Inc., version 7.03, La Jolla, CA, USA).

[0127] 3b) Modified competitive binding assay In competitive binding experiments, a fixed concentration of recombinant αSYN fibrils (sonicated, 15 nM / well) was added to the αSYN fibril buffer at 1 nM [ 3 H]-Compound 1 or [ 3Plates were incubated with [H]-compound 2 and decreasing concentrations of serial dilutions of unlabeled competitor (serial dilutions 1:4 or 1:3.5 or 1:3 providing a concentration range of unlabeled competitor 1 μM-1 pM or 1 μM-4 nM or 1 μM-17 pM, respectively) in a total volume of 200 μL / well in 30 mM Tris-HCl, 10% ethanol, 0.05% Tween 20, pH 7.4. Plates were covered with removable sealing tape (PerkinElmer, Waltham, MA, USA) and incubated at RT for 4.5 h.

[0128] Filtration and readout were performed as described for the saturation binding assay. 3 H]-labeled ligand binding [in CMP] was plotted against increasing competitor concentrations and the data points were fitted using nonlinear regression analysis to obtain IC 50 and K. i Values ​​were calculated (GraphPad Software, Inc., version 7.03, La Jolla, CA, USA). In competitive binding assays with sonicated recombinant αSYN fibrils, K i The K values ​​were calculated to be 0.6 nM and 0.2 nM for compound 1 and compound 2, respectively. D Based on the values.

[0129] Fiber binding saturation assay D This assay was performed using directly radiolabeled compounds (e.g. 3 H labeling). D The values ​​are shown in Tables 3a and 3b. Saturation binding assays were performed with recombinant human α-synuclein, Aβ 1-42 and using fibrillar aggregates generated from tau46 as the target structure.

[0130] The results show high affinity for α-synuclein fibrils. Compounds 1, 2 and 12 showed very high affinity, with K Dvalues ​​were less than 10 nM. Compounds 1 and 2 were also tested for binding to Aβ and tau46 fibrils and showed good to excellent selectivity, demonstrating their suitability for the diagnostic detection of aggregated α-synuclein.

[0131] [Table 3]

[0132] [Table 4]

[0133] Fiber competition assay i Provide the value. i is the binding affinity of 50% of the reference compound, in this case [ 3 H]-Compound 1 (1 nM) or [ 3 H]-Compound 2 (1 nM). This assay is suitable for screening non-radioactive ligands. The K obtained for the test compound i The values ​​are shown in Tables 4a, 4b and 5.

[0134] [Table 5]

[0135] [Table 6]

[0136] [Table 7-1]

[0137] [Table 7-2]

[0138] [Table 7-3]

[0139] The results of various competition assays show high binding affinity of the test compounds to α-synuclein fibrils (K i (Lower values ​​indicate higher binding affinity.) In the competition assay for compound 1 (Table 4a), it showed similar K i The K values ​​showed similar selectivity to compound 1, with the K i For compounds with higher values, the data suggest further improvements in selectivity.

[0140] Additionally, compounds 63, 64 and 65 were tested in direct comparison with the corresponding compounds having a nitrogen atom in one of the phenyl rings. In Table 6 for compound 1, a K i The K value was obtained for compound 65 (which lacks a nitrogen atom in the phenyl ring). i The value is much higher (1.9 nM), indicating a significant improvement in the binding affinity of compound 1. i Analysis of the binding affinity based on the values ​​revealed a significant improvement in the binding affinity of the N-containing compounds. 3 H]-Compound 1 (1 nM) i The values ​​are shown in Table 6.

[0141] [Table 8]

[0142] Example 3: Therapeutic Efficacy of Cell Cultures To identify potentially therapeutically useful effects, compounds were tested in two cellular models of aggregated α-synuclein-dependent toxicity in H4 cells. These cellular models allow for inducible overexpression of α-synuclein hemi-Venus fusion constructs (V1S+SV2) or full-length α-synuclein (models are described in detail in Bartels M, Weckbecker D, Kuhn PH, Ryazanov S, Leonov A, Griesinger C, Lichtenthaler SF, Botzel K, Giese A: Iron-mediated aggregation and toxicity in a novel neuronal cell culture model with inducible alpha-synuclein expression, Sci. Rep. 2019 Jun 24;9(1):9100). Furthermore, addition of DMSO and FeCl3 promotes α-synuclein aggregation, which is associated with increased cytotoxicity. Cell culture assays use these effects by determining changes in cell number and the percentage of condensed nuclei, indicative of apoptosis, to obtain information about which compounds show the most promising effects and therefore may be therapeutically useful.

[0143] The data are summarized in Table 7 below. In these experiments, cells were incubated with 100 μM FeCl3 and 0.75% DMSO in the presence of 10 μM anle138c as a positive control, and compounds 1-19 (10 μM) or DMSO as negative controls. After 48 hours, cells were imaged on an OPERA high-throughput imaging system and analyzed using Acapella software (PerkinElmer). The table shows the change in cell number and the change in the fraction of cells with condensed nuclei (i.e. apoptotic cells) compared to the DMSO control. A decrease in the fraction of condensed nuclei (without a significant decrease in cell number) indicates a beneficial therapeutic effect.

[0144] [Table 9]

[0145] Example 4: 11 In vivo biodistribution of [C]-labeled Compound 1 and Compound 2 [ 11 [C]-labeled compound 1 and compound 2 were each injected intravenously into the tail vein of mice. The mice were then imaged with a small animal PET device. Good blood-brain barrier penetration was observed for both compounds with SUV values ​​>1.5. Furthermore, rapid washout from the brain was observed. 11 The clearance half-lives of [C]-labeled Compound 1 and Compound 2 were 12 and 9 min, respectively.

[0146] Example 5: Autoradiography using human brain tissue Autoradiography was performed on frozen brain tissue with tritium-labeled Compound 1 and on histological sections of brain tissue from the cingulate cortex of patients with dementia with Lewy bodies. 3 When [H]-Compound 1 was used at a concentration of 3 nM for tissue incubation followed by a washing step, preferential binding to gray matter could be observed, a pattern identical to the known distribution of aggregated α-synuclein (Figure 1, left panel). When specific binding was blocked by excess non-tritiated (i.e., "cold") Compound 1, non-specific binding of Compound 1 to brain tissue was found to be absent or very low (Figure 1, right panel).

[0147] These findings indicate that compound 1 binds specifically and with high affinity to pathological aggregated α-synuclein present in human synucleinopathic patients, enabling the diagnostic detection of aggregated α-synuclein.

Claims

1. General formula Ia, Ib, IIa or IIb 【Chemistry 1】 (In the formula, 【Chemistry 2】 but, 【Chemistry 3】 and 【Chemistry 4】 but, 【Chemistry 5】 and Y １ , Y ２ , Y ３ , Y ４ , Y ５ , Y ６ , Y ７ and Y ８ is CR ３ and N, where Y １ , Y ２ , Y ３ , Y ４ , Y ５ , Y ６ , Y ７ and Y ８ at least one of is N; wherein in formula Ia or Ib, at least one of Y 1 , Y 3 , Y 4 and Y 6 is N; wherein in formula IIa or IIb, Y 1 is N and at least one of Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 is N; or in formula IIa or IIb, Y 1 is N and Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 are CR 3 ; R １ is hydrogen, C １～４ Alkyl and -(CH ２ )-O-P(=O)(OR)(OR), １～４ The alkyl may be optionally substituted with one or more halogens; R ２ is hydrogen, halogen and C １～４ alkyl; １～４ The alkyl may be optionally substituted with one or more halogens; R is hydrogen or a cation; R ３ is hydrogen, R ４ and R ５ is H and C １～４ alkyl; １～４ The alkyl may be optionally substituted with one or more halogens or R ４ and R ５ together with the nitrogen atom to which they are attached form a 4-6 membered saturated heterocyclic ring, which is ４ and R ５ In addition to the nitrogen atom to which is attached, the 4-6 membered saturated heterocycle optionally contains one or more heteroatoms selected from O and N, and the 4-6 membered saturated heterocycle may contain one or more R ６ may be optionally replaced with R ６ is halogen, C １～４ Alkyl, OH and C １～４ alkoxy; １～４ Alkyl and C １～４ The alkoxy may be optionally substituted with one or more halogens; Hal is a halogen. or a solvate or salt thereof. 【Request 2】 【Chemical 6】 but, 【Chemistry 7】 or 【Chemistry 8】 but, 【Chemistry 9】 and Here, R １ and R ２ 2. The compound according to claim 1, or a solvate or salt thereof, wherein: 【Request 3】 【Chemical 10】 but, 【Chemistry 11】 or 【Chemistry 12】 but, 【Chemistry 13】 and Here, R １ and R ２ 3. The compound according to claim 2, or a solvate or salt thereof, wherein:

4. Y １ , Y ２ , Y ３ , Y ４ , Y ５ , Y ６ , Y ７ and Y ８ The compound according to any one of claims 1 to 3, wherein one, two or three of are N, or a solvate or salt thereof.

5. In formula Ia or Ib, Y １ is N and Y ３ , Y ４ and Y ６ The compound according to any one of claims 1 to 4, or a solvate or salt thereof, wherein at least one of:

6. Y １ is N and Y ２ , Y ３ , Y ４ , Y ５ , Y ６ , Y ７ and Y ８ is CR ３ The compound according to any one of claims 1 to 4, or a solvate or salt thereof.

7. The compound according to claim 6, or a solvate or salt thereof, wherein Y 1 is N and Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 and Y 8 are CH.

8. R ２ The compound according to any one of claims 1 to 7, or a solvate or salt thereof, wherein

9. R ４ and R ５ At least one of １～４ alkyl, C １～４ 9. The compound according to any one of claims 1 to 8, or a solvate or salt thereof, wherein the alkyl may be optionally substituted with one or more halogens.

10. R ４ and R ５ together with the nitrogen atom to which they are attached form a 4- to 6-membered saturated heterocyclic ring, which is represented by R ４ and R ５ In addition to the nitrogen atom to which is attached, the 4-6 membered saturated heterocycle optionally contains one or more heteroatoms selected from O and N, and the 4-6 membered saturated heterocycle may contain one or more R ６ 9. The compound according to any one of claims 1 to 8, or a solvate or salt thereof, optionally substituted with

11. The 4-6 membered saturated heterocycle is selected from azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl and piperazinyl, and the azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl and piperazinyl are each independently selected from one or more R ６ 11. The compound of claim 10, or a solvate or salt thereof, which may be optionally substituted with:

12. The compound according to any one of claims 1 to 11, or a solvate or salt thereof, wherein the compound is detectably labeled.

13. The compound of claim 12, or a solvate or salt thereof, wherein the compound is detectably labeled with 18 F, 11 C, 125 I, 123 I, 131 I, 77 Br and 76 Br.

14. The compound according to claim 13, or a solvate or salt thereof, wherein the compound is detectably labeled with 18 F or 11 C.

15. A diagnostic composition comprising a compound according to any one of claims 1 to 14, or a solvate or salt thereof, and optionally a pharma- ceutically acceptable carrier.

16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11, or a solvate or salt thereof, and optionally a pharma- ceutically acceptable carrier.

17. A compound according to any one of claims 1 to 14, or a solvate or salt thereof, for use in the diagnosis of a disease associated with α-synuclein aggregation.

18. A compound according to any one of claims 1 to 11, or a solvate or salt thereof, for use in the treatment of a disease associated with alpha-synuclein aggregation.

19. 19. The compound for use according to claim 17 or 18, or a solvate or salt thereof, wherein the disease associated with alpha-synuclein aggregation is selected from Parkinson's disease, dementia with Lewy bodies and multiple system atrophy.

20. Use of a compound according to any one of claims 1 to 14, or a solvate or salt thereof, for inhibiting α-synuclein aggregation in vitro or ex vivo.

21. A compound according to any one of claims 1 to 14, or a solvate or salt thereof, for use in imaging alpha-synuclein aggregation.

22. 15. A kit for preparing a detectably labeled compound according to any one of claims 12 to 14, or a solvate or salt thereof, comprising at least two precursor compounds which upon reaction form a compound according to any one of claims 12 to 14, or a solvate or salt thereof.

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