RU2822486C1 - Novel compounds for diagnosing, treating and preventing diseases associated with alpha-synuclein aggregation - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of general formulas Ia, Ib, IIa or IIb:

,

a pharmaceutical composition based thereon, as well as the use and method of imaging alpha-synuclein and for diagnosing diseases which are associated with alpha-synuclein aggregation.

EFFECT: obtaining novel compounds which are suitable for treating and preventing diseases associated with alpha-synuclein aggregation.

20 cl, 5 ex, 7 tbl, 1 dwg

Description

The present invention provides novel compounds that are useful for imaging alpha-synuclein and for diagnosing diseases associated with alpha-synuclein aggregation. The compounds are also useful for the treatment and prevention of diseases that are associated with alpha-synuclein aggregation.

BACKGROUND OF THE ART

There are a large number of neurological and neurodegenerative diseases, many of which are currently incurable and difficult to diagnose. All common neurodegenerative diseases are characterized by the misfolding, aggregation and deposition of specific 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 other.

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

Pathospecific accumulation and deposition of aggregated and misfolded proteins is a target for both therapy and diagnosis through compounds that bind to these aggregated and misfolded protein deposits.

One option for the diagnostic detection of pathospecific accumulation and deposition of aggregated and misfolded proteins is the use of detectably labeled compounds that exhibit specific and selective high-affinity binding to these aggregated protein deposits. This can be done, for example, by using compounds that are labeled with suitable radioactive isotopes and using PET imaging for detection. Although compounds are available for clinical use for PET imaging of A beta and tau, no compounds have yet been developed that can be used for diagnostic PET imaging of alpha-synuclein deposits in synucleinopathies (Kotzbauer, P.T., Tu, Z. and Mach, R.H., Current status of the development of PET radiotracers for imaging alpha synuclein aggregates in Lewy bodies and Lewy neurites Transl. Imaging, 2017. 5: p. Compounds developed and tested by other groups have so far lacked the appropriate combination of properties, including high binding affinity for aggregated and misfolded alpha-synuclein, sufficient binding affinity selectivity over aggregated and misfolded other proteins, especially A beta and tau ( required for the differential diagnosis of various diseases characterized by pathospecific accumulation and deposition of aggregated and misfolded proteins, and for the ability to accurately detect the presence of aggregated and misfolded alpha-synucleins also in patients suffering from more than one disease simultaneously, i.e. dementia with Lewy bodies and disease Alzheimer's). Additionally, desired properties include the ability to cross the blood-brain barrier and bind to intracellular deposits, low nonspecific binding to brain tissue, and rapid clearance of the unbound compound from the brain.

WO 2009/146343 relates to certain pyrazoles, 1,2,4-oxadiazoles and 1,3,4-oxadiazoles, which are labeled substances in positron emission tomography (PET) imaging to study amyloid deposits in the brain in vivo for disease diagnosis Alzheimer's. Alzheimer's disease is characterized by aggregation of A beta and tau proteins, and the PET labeled substances mentioned bind to aggregates of these proteins. In contrast, the present invention is aimed at diseases characterized by alpha-synuclein aggregation. Selective diagnostic imaging of aggregated alpha-synuclein requires selective binding to alpha-synuclein and no or weak binding to aggregated A beta and tau.

WO 00/66578 describes specific NPY antagonists that can be used to treat NPY-mediated diseases/conditions such as obesity. It is mentioned that in addition to the "direct" effect of the compounds from WO 0066578 on the NPY5 subtype, there are diseases/conditions that may benefit from weight loss, such as insulin resistance, impaired glucose tolerance, type II diabetes, hypertension, hyperlipidemia, cardiac -vascular diseases, gallstones, some types of cancer, sleep apnea, etc.

WO 2010/000372 relates to a specific compound that is used for the treatment or prevention of protein aggregation diseases and/or neurodegenerative diseases. The compounds of WO 2010/000372 are particularly suitable for the treatment of diseases associated with protein aggregation, including Parkinson's disease. They have been shown to bind to several different aggregated proteins, including A beta and tau. Therefore, they can be used to diagnose protein aggregation disorders, but cannot reliably distinguish alpha-synuclein aggregation disorders from other disorders that are associated with amyloid protein aggregation, such as tauopathies or Alzheimer's disease. This would, however, be important because the clinical manifestations of protein aggregation disorders are very similar and it would be very desirable to distinguish, for example, between different disorders to tailor treatment accordingly. Moreover, the molecules described in WO 2010/000372 have high non-specific binding to lipids and hydrophobic proteins, which results in strong non-specific binding in brain tissue and other tissues. Thus, they do not achieve the required specific binding and signal-to-noise ratio to alpha-synuclein as required for trace PET imaging.

In connection with the above, there is a need for compounds with improved diagnostic properties, in particular, it is necessary to improve the specificity of binding to alpha-synuclein. Non-specific binding in brain and other tissues must be reduced, binding affinity must be increased, and physiological half-life must be decreased.

US 2005/0075375 describes specific heterocyclic compounds for the treatment of hepatitis C virus.

brief description of the DRAWING

Figure 1: Autoradiography using brain tissue from a patient suffering from dementia with Lewy bodies using [3H]-compound 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a compound represented by the general formula Ia, Ib, IIa or IIb.

X ¹ , X ² and X ³ are independently selected from CR ² , N and NR ¹ , provided that at least two of X ¹ , X ² and X ³ are N or NR ¹ . It is clear that N and NR ¹ are present as valency allows, i.e., N can only be present at the =X ¹ -, =X ² - or =X ³ - position, and NR ¹ can only be present at the -X ¹ position - or -X ² -.

Examples include

Examples include

In a preferred embodiment, represents or (more preferably ) or represents or (more preferably ).

Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ are independently selected from CR ³ and N, provided that at least one of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represents N.

In a preferred embodiment, in formulas Ia or Ib, Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ and Y ⁶ are independently selected from CR ³ and N, provided that at least one of Y ¹ Y ² , Y ³ , Y ⁴ , Y ⁵ and Y ⁶ are N. In a more preferred embodiment, one or two or three of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ and Y ⁶ are N even more preferably, one or two of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ and Y ⁶ is N, even more preferably one of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ and Y ⁶ is N. In a preferred embodiment, Y ¹ is N.

In a preferred embodiment, in formulas Ia or Ib, at least one of Y ¹ , Y ³ , Y ⁴ and Y ⁶ is N. In another preferred embodiment, in formulas Ia or Ib, Y ¹ is N and at least one of Y ³ , Y ⁴ and Y ⁶ is N.

In a preferred embodiment, in formulas Ia or Ib, at least one of Y ¹ , Y ³ , Y ⁴ and Y ⁶ is N and the others of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ and Y ⁶ represent CR ³ (such as CH). In another preferred embodiment, in formulas Ia or Ib, Y ¹ is N, at least one of Y ³ , Y ⁴ and Y ⁶ is N, and the others of Y ² , Y ³ , Y ⁴ , Y ⁵ and Y ⁶ are CR ³ (such as CH).

In a preferred embodiment, in formulas Ia or Ib, Y ¹ is N and the others of Y ² , Y ³ , Y ⁴ , Y ⁵ and Y ⁶ are CR ³ , more preferably Y ¹ is N and Y ² . Y ³ , Y ⁴ , Y ⁵ and Y ⁶ are CH.

In a preferred embodiment, in formulas IIa or IIb, Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ are independently selected from CR ³ and N, provided that at least one of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ is N. In a more preferred embodiment, one or two or three of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represent N, even more preferably one or two of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represent is N, even more preferably one 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.

In a preferred embodiment, in formulas IIa or IIb, at least one of Y ¹ , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ is N. In a preferred embodiment, in formulas IIa or IIb, at least one of Y ¹ , Y ³ , Y ⁴ , Y ⁵ and Y ⁷ is N. In another preferred embodiment, in formulas 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 represents N.

In a preferred embodiment, in formulas IIa or IIb, at least one of Y ¹ , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ is N, and the others are Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represent CR ³ (such as CH). In a preferred embodiment, in formulas IIa or IIb, at least one of Y ¹ , Y ³ , Y ⁴ , Y ⁵ and Y ⁷ is N and the others of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represent CR ³ (such as CH). In another preferred embodiment, in formulas IIa or IIb, Y ¹ is N, at least one of Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ is N, and the others are Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represent CR ³ (such as CH). In a preferred embodiment, in formulas IIa or IIb, Y ¹ is N, at least one of Y ³ , Y ⁴ , Y ⁵ and Y ⁷ is N and the others of Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represent CR ³ (such as CH).

In a preferred embodiment, in formulas IIa or IIb, Y ¹ is N and the others of Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ are CR ³ , more preferably Y ¹ is N and Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represent CH.

Surprisingly, it has been found that the introduction of one or more nitrogen atoms as Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ greatly increases the selectivity of binding to alpha-synuclein, thus disrupting the corresponding disorders associated with alpha-synuclein aggregation, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, can be distinguished from disorders with aggregation of other proteins, such as Alzheimer's disease, in which A beta and tau aggregate predominantly. The signal to noise ratio improves.

R ¹ is selected from hydrogen, C _1-4 alkyl and -(CH ₂ )-OP(=O)(OR)(OR), wherein C _1-4 alkyl may be optionally substituted with one or more halogens. In a preferred embodiment, R ¹ is hydrogen, methyl or 2-fluoroethyl. If present, it is preferred that -(CH ₂ )-OP(=O)(OR)(OR) be attached to X ¹ or X ² .

R represents hydrogen or a cation. The cation may be any pharmaceutically acceptable cation. Preferably, the cation is a monovalent cation. Examples are sodium, lithium, potassium, ammonium, and protonated forms of ethanolamine, choline, lysine, meglumine, piperazine, and tromethamine. Preferably, the cation is sodium. In the compounds of the present invention, both R's may be hydrogen, both R's may be cations (the same or different cations), or one R may be hydrogen and the other may be a cation. Preferably, both R's are sodium. Divalent cations such as Ca ²⁺ , Mg ²⁺ and Zn ²⁺ or trivalent cations such as Al ³⁺ are possible but not preferred since the resulting salts are less soluble in water. Compounds wherein R ¹ is -(CH ₂ )-OP(=O)(OR)(OR) and their synthesis are described in WO 2017/102893, which is incorporated herein by reference.

R ² is independently selected from hydrogen, halogen and C _1-4 alkyl, wherein the C _1-4 alkyl may be optionally substituted with one or more halogens. In a preferred embodiment, R ² is hydrogen.

R ³ represents hydrogen, halogen, C _1-4 alkyl, OH and C _1-4 alkoxy, wherein C _1-4 alkyl and C _1-4 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.

R⁴ and R⁵ independently selected from H and C_1-4 alkyl, where C_1-4 alkyl may be optionally substituted with one or more halogens, or where R⁴ and R⁵ together with the nitrogen atom to which they are bonded, form a 4-6 membered saturated heterocyclic ring, which optionally contains one or more heteroatoms selected from O and N, in addition to the nitrogen atom to which R are bonded⁴ and R⁵, where 4-6-membered saturated heterocyclic the ring may optionally be replaced by one or more R⁶.

In one embodiment, R ⁴ and R ⁵ are independently selected from H and C _1-4 alkyl, wherein C _1-4 alkyl may be optionally substituted with one or more halogens. Preferably, R ⁴ and R ⁵ are independently selected from H and C _1-4 alkyl. Preferably, at least one of R ⁴ and R ⁵ is C _1-4 alkyl, wherein C _1-4 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, wherein C _1-4 alkyl may be optionally substituted with one or more halogens. More preferably, at least one of R ⁴ and R ⁵ is C _1-4 alkyl. More preferably, R ⁴ is hydrogen and R ⁵ is C _1-4 alkyl.

In another embodiment, R ⁴ and R ⁵ together with the nitrogen atom to which they are bonded form a 4-6 membered saturated heterocyclic ring, which optionally contains one or more (for example, one) heteroatoms selected from O and N, in addition to the nitrogen atom to which R ⁴ and R ⁵ are bonded, wherein the 4-6 membered saturated heterocyclic ring may be optionally substituted with one or more R ⁶ . In a preferred embodiment, the 4-6 membered heterocyclic ring is selected from azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl and piperazinyl, wherein azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl and piperazinyl may be optionally substituted with one or more R ⁶ . More preferably, the 4-6 membered saturated heterocyclic ring is selected from azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl and piperazinyl, wherein the piperazinyl N atom may be optionally substituted with R ⁶ .

R ⁶ is independently selected from halogen, C _1-4 alkyl, OH and C _1-4 alkoxy, wherein C _1-4 alkyl and C _1-4 alkoxy may be optionally substituted with one or more halogens, preferably R ⁶ is C _{1 -4} alkyl or fluorine.

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

m represents the number of R ³ groups other than hydrogen in the ring that contains Y ¹ , Y ² , Y ³ and Y ⁴ . m is an integer from 0 to m _max , where m _max represents the number of carbon atoms in the ring that contains Y ¹ , Y ² , Y ³ and Y ⁴ . Preferably m is 0.

n represents the number of R ³ groups other than hydrogen in the ring that contains Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ (formulas IIa and IIb). n is an integer from 0 to _nmax , where _nmax is the number of carbon atoms in the ring that contains ^Y5 , ^Y6 , ^Y7 and ^Y8 . Preferably n is 0.

p represents the number of R ³ groups other than hydrogen on the ring that contains Y ⁵ and Y ⁶ (formulas Ia and Ib). p is an integer from 0 to _nmax , where _nmax is the number of carbon atoms in the ring that contains ^Y5 and ^Y6 . Preferably, p is 0.

The compounds of the present invention may also be present in the form of prodrugs, solvates or salts thereof.

The compounds of the present invention form salts, which are also included within the scope of the present invention. It is understood that reference to a compound of the present invention herein includes reference to its salts unless otherwise indicated. Pharmaceutically acceptable (ie, non-toxic, physiologically acceptable) salts are preferred, although other salts may also be used, for example, in isolation or purification steps that may be used during preparation or in in vitro methods. Salts of the compounds of the present invention can be prepared, for example, by reacting the compound with an amount of acid, such as an equivalent amount, in a medium, such as a salt-precipitating medium, or in an aqueous medium, followed by lyophilization.

Compounds that contain a basic group can form salts with various organic and inorganic acids. Examples of acid addition salts include acetates (for example, formed with acetic acid or trihaloacetic acid, e.g. trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentane propionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, hydroxyethanesulfonates (for example, 2-hydroxyethanesulfonates), lactates, maleates, methanesulfonates, naphthalene sulfonates (for example, 2-naphtha linsulfonates), nicotinates, nitrates, oxalates, pectinates, persulfates, phenylpropionates (for example, 3-phenylpropionates), phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (for example, those formed with sulfuric acid), sulfonates (for example, those mentioned herein), tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates and the like.

Also discussed herein are prodrugs and solvates of the compounds of the present invention. The term “prodrug” as used herein means a compound that, when administered to a subject, undergoes a chemical transformation through metabolic or chemical processes to produce a compound of the present invention or salts and/or solvates thereof.

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

All stereoisomers of the compounds of the present invention (eg, those that may exist due to the asymmetry of carbon atoms in various substituents), including enantiomeric forms and diastereomeric forms, are included in the scope of the present invention. The individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers (for example, as a pure or substantially pure optical isomer having a specific activity), or may be mixed, for example, as racemates or with all or other selected stereoisomers. The chiral centers of the compounds of the present invention may have the S or R configuration as defined in the IUPAC 1974 Recommendations.

Racemic forms can be separated by physical methods such as fractional crystallization, resolution or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. Individual optical isomers may be prepared from the racemates by any suitable method, including, without limitation, salt formation of the optically active acid followed by crystallization.

All configurational isomers of the compounds of the present invention are contemplated, either in mixture or in pure or substantially pure form. The definition of compounds of the present invention includes both cis (Z) and trans (E) alkene isomers, as well as cis and trans isomers of cyclic hydrocarbon or heterocyclic rings.

Deuterated versions of the claimed compounds may also be provided. The position at which deuteration occurs is not particularly limited, but it may be, for example, in the C _1-4 alkyl of R ⁴ and R ⁵ .

Throughout the description, groups and their components can be selected to provide stable groups and compounds.

The compounds of the present invention may be presented in the form of a diagnostic or pharmaceutical composition, which optionally includes a pharmaceutically acceptable carrier or excipient.

In accordance with the present invention, the term "diagnostic composition" refers to a composition for determining the presence of aggregated alpha-synuclein underlying a disease associated with alpha-synuclein aggregation.

In a preferred embodiment, the compound of the present invention is detectable or detectably labeled. In accordance with the present invention, it is understood that a compound is detectable or detectably labeled if its presence can be monitored using conventional methods such as NMR spectroscopy, single photon emission computed tomography (SPECT), optical detection, positron emission tomography (PET), electron microscopy, magnetic resonance imaging (MRI), spectrometry, chromatography, ELISA analysis, radiation detection, preferably PET, scintillation counting or gamma counting, more preferably PET.

When the compounds of the present invention are intended for use as probes for visualizing aggregated alpha-synuclein, they must be labeled. The exact nature of the mark will depend on the method that will be used for rendering. Positron emitting radioactive tracers (PET) having a short half-life, such as ¹⁸ F, ¹¹ C, ¹²⁵ I ^{, 123} I, ¹³¹ I, ⁷⁷ Br and ⁷⁶ Br, will generally be useful ^. Due to their short half-lives, the labeled compounds of the present invention should be prepared shortly before their use for testing. Therefore, the diagnostic composition of the present invention can also be presented 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.

One skilled in the art will be able to devise methods by which a detectable label can be attached to a compound of the present invention. The following diagrams may serve as illustrative examples.

¹⁸ F marking :

2-[ ¹⁸ F]fluoroethyl tosylate is a useful precursor for the incorporation ^{of 18} F by fluoroethylation of compounds containing nitrogen, oxygen and sulfur nucleophiles, as described in WO 2010/000372, page 32, scheme A for the preparation of compound 21 and compound 22, starting from compound 23. Compounds A and B can be prepared in the same way. Another method involves direct nucleophilic substitution of a suitable leaving group, as shown for the conversion of C to D (see J. Med. Chem . 2013 , 56 , 4568-4579, Scheme 2) or the conversion of compound 24 to compound 12 in Scheme 1.

Scheme 1

Tagging ^eleven C:

The ^11C -labeled compounds of the invention can be prepared by direct nucleophilic alkylation of a suitable [ ^11C ]MeI precursor as described in WO 2010/000372, page 32, scheme B, to obtain compound 27 and compound 28, starting with compound 23. Compound 1 of the present invention can be synthesized in a similar manner, starting with compound 2. In the same way, compound 18 and compound 19 can be synthesized, starting with compound 9, as shown in Scheme 2. Alternatively, a simple and accessible method (JM Hooker et al., Angew. Chem. Int. Ed. 2008 , 47 , 5989-5992) obtaining [ ¹¹ C]formaldehyde in the form of ^a solution in dimethylformamide by converting [ ¹¹ C]methyl iodide under mild conditions without loss of specific activity. to convert compound 2 to compound 1 via reductive amination.

Scheme 2:

The present invention provides a method for visualizing aggregated alpha-synuclein deposits, which includes the following steps:

(i) administering to the subject a detectable amount of a composition containing a detectably labeled compound of the present invention;

(ii) allowing sufficient time for the compound to bind to the aggregated alpha-synuclein; And

(iii) detection of a compound associated with aggregated alpha-synuclein.

A composition containing a detectably labeled compound can be administered to a subject by any route of administration described below, such as, for example, oral or parenteral. The labeled compound can be administered to the patient and after a period of time sufficient for the compound to bind to the aggregated alpha-synuclein, the labeled compound is detected non-invasively within the patient. Alternatively, the labeled compound may be administered to a patient, sufficient time is allowed for the compound to bind to the aggregated alpha-synuclein, and a tissue sample is then obtained from the patient and the labeled compound is detected in tissue separate from the patient. A tissue sample may also be collected from the patient before introducing the labeled compound into the tissue sample. After sufficient time for the compound to bind to the aggregated alpha-synuclein, the compound can be detected.

Visualization of aggregated alpha-synuclein can also be performed quantitatively so that the amount of aggregated alpha-synuclein can be determined.

The present invention also provides a compound of the present invention, as well as a prodrug, solvate or salt thereof, for use in the treatment or prevention of a disease associated with alpha-synuclein aggregation.

Other embodiments include the use of a compound of the present invention for the preparation of a pharmaceutical composition for the treatment or prevention of a disease associated with alpha-synuclein aggregation, as well as a method of treating or preventing a disease associated with alpha-synuclein aggregation, comprising administering a therapeutically effective amount of a compound of the present invention to a patient in need. It involves the use of the compound in the form of a "pharmaceutical composition", as described below.

The term "aggregation" in accordance with the present invention refers to the formation of oligomeric or multimeric complexes of alpha-synuclein, which may be accompanied by the integration of additional biomolecules, such as carbohydrates, nucleic acids, lipids and/or metal ions, into the complexes.

The term “alpha-synuclein aggregation disease” as used herein refers to those diseases that are characterized by the presence of aggregated alpha-synuclein. Such aggregated alpha-synuclein can form deposits in a specific tissue, more preferably in nervous tissue or brain tissue. The degree of aggregation depends on the specific disease.

In accordance with the present invention, the term "pharmaceutical composition" refers to a composition for administration to a patient, preferably a mammal, more preferably a human patient. The pharmaceutical composition of the invention contains the compounds listed above and, optionally, other molecules capable of altering the characteristics of the compounds of the invention, thereby, for example, stabilizing, modulating and/or activating their functions. The composition may be in solid, liquid or gaseous form, and may be in the form of powder(s), tablet(s), solution(s) or aerosol(s), among other things. The pharmaceutical composition of the present invention may optionally and additionally contain a pharmaceutically 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-in-water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO, etc. Compositions containing such carriers can be formulated by well-known conventional methods.

The pharmaceutical composition will be formulated and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the pharmaceutical composition, the route of administration, the schedule of administration and other factors known to practitioners. The "effective amount" of a pharmaceutical composition for purposes of the present invention is thus determined by such considerations. One skilled in the art will recognize that the effective amount of a pharmaceutical composition administered to an individual will depend, among other things, on the nature of the compound.

The pharmaceutical compositions of the invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (in the form of powders, ointments, drops or transdermal patches), buccally or as an oral or nasal spray. Preferably, they are administered intravenously when used for trace PET imaging for diagnosis, and orally when used for the treatment or prevention of disease.

By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semi-solid or liquid excipient, diluent, encapsulating material or excipient of any type.

The term "parenteral" as used herein refers to routes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.

For therapeutic purposes, the pharmaceutical composition can also be suitably administered via sustained release systems. Suitable examples of sustained release compositions include semi-permeable polymer matrices in the form of molded articles, such as films or microcapsules. Sustained release matrices include polylactides. (US Patent No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-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., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133 988). Sustained release pharmaceutical compositions may also include compounds entrapped in liposomes. Liposomes containing a pharmaceutical composition are prepared by methods known per se: DE 32 18 121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52 322; EP 36 676; EP 88 046; EP 143 949; EP 142 641; Japanese Patent Application 83-118008; US Patent Nos. 4,485,045 and 4,544,545; and EP 102324. Typically, liposomes are of the small (approximately 200-800 angstrom) single-layer type, in which the lipid content exceeds approximately 30 mol%. cholesterol, where the selected proportion is adjusted for optimal therapy.

For parenteral administration, the pharmaceutical composition is typically formulated by mixing it to the desired degree of purity in a unit injection dosage form (solution, suspension or emulsion) with a pharmaceutically acceptable carrier, i.e. a carrier that is not toxic to recipients in the dosages and concentrations used and is compatible with other ingredients of the formulation.

Typically, the formulations are prepared by bringing the components of the pharmaceutical composition into uniform and intimate contact with liquid carriers or finely divided solid carriers or both. Then, if necessary, the composition is given the desired shape. Preferably, the carrier is a parenteral vehicle, more preferably a solution isotonic with the recipient's blood. Examples of such carriers include water, saline, Ringer's solution and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also used herein, as well as liposomes. The carrier suitably contains small amounts of additives, such as substances that increase isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate, succinate, acetic acid and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than ten 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 PEGs.

The components of the pharmaceutical composition used for therapeutic administration must be sterile. Sterility is easily achieved by filtration through sterile filtration membranes (eg 0.2 µm membranes). The therapeutic components of the pharmaceutical composition are typically placed in a container having a sterile access port, such as an intravenous solution bag or vial with a stopper pierced by a hypodermic needle.

The components of the pharmaceutical composition are typically stored in single-dose or multi-dose containers, for example, in sealed ampoules or vials, as an aqueous solution or as a lyophilized reconstitution formulation. As an example of a lyophilized formulation, 10 ml vials are filled with 5 ml of a sterile filtered 1% (w/v) aqueous solution and the resulting mixture is lyophilized. A solution for infusion is prepared by reconstituting the lyophilized compound using bacteriostatic water for injection.

The present invention further provides a method of treating or preventing a disease associated with alpha-synuclein aggregation, comprising administering a therapeutically effective amount of a compound of the present invention to a patient in need thereof.

As used herein, the term “therapeutically effective amount” refers to an amount sufficient to produce the desired biological response. In the present invention, the desired biological response is inhibition of alpha-synuclein aggregation and/or reduction in the amount of aggregated alpha-synuclein present in the tissue.

The present invention also relates to the use of a compound as defined above for inhibiting alpha-synuclein aggregation in vitro or ex vivo .

The disease associated with alpha-synuclein aggregation is not particularly limited and is usually selected from Parkinson's disease, dementia with Lewy bodies and multiple system atrophy.

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

EXAMPLES

Example 1: Synthesis and testing

Chemical synthesis methods

The following methods are presented with detailed descriptions of the preparation of the compounds of the invention and illustrative examples. The compound of the invention can be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis.

All starting materials and solvents are commercial grade and are used as received unless otherwise noted.

Thin layer chromatography (TLC) is performed using Macherey-Nagel pre-coated sheets, 0.25 mm ALUGRAM® SIL G/UV254 plates, UV detection and/or carbonation reagent 10% wt. ethanol phosphomolybdic acid followed by heating at 200°C.

Flash chromatography is carried out using Merck 60 silica gel (0.063-0.100 mm).

Analytical high performance liquid chromatography (HPLC) was performed using a Waters HPLC system with a Waters 996 photodiode array sensor. All separations included a mobile phase of 0.1% trifluoroacetic acid (TFA) (v/v) in water and 0.1% TFA in acetonitrile. HPLC is carried out using a reverse phase (RP) column Eurospher RP 18, 100 Å, 5 µm, 250x4.6 mm at a flow rate of 1 ml⋅min ^-1 .

Electrospray ionization mass spectrometry (ESI-MS) and liquid chromatography/mass spectrometry (LC/MS) were performed using a Waters Micromass ZQ 4000 mass spectrometer coupled with the Waters HPLC apparatus described above.

NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer (Bruker AG, Rheinstetten, Germany) equipped with a TXI HCN z-gradient probe. All spectra were processed using TOPSPIN 3.1 (Bruker AG, Karlsruhe, Germany). ^1H NMR chemical shifts (δ) are reported in parts per million (ppm) relative to CHCl ₃ , DMSO-d5 and TFA-d1 as internal standards. The data is presented as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, qui=quintet, dd=doublet of doublets, dt=doublet of triplets, w=wide, m=multiplet), constants combinations (J, given in Hz), integration. ¹³ C NMR chemical shifts (δ) are given in parts per million (ppm) relative to CDCl ₃ , DMSO-d6 and TFA-d1 as internal standards. To record the resonances of compounds, the following experiments are used: ¹ H-1D, ¹³ C-1D NMR spectra and ¹³ C-APT (attached proton test with one J-evolution time of 1/145 s, spectra processed in such a way that quaternary and methylene groups have a positive sign, and the methyl and methine groups have a negative sign). To resolve aliasing resonances and recover undetectable resonances in the spectra of ¹ H and APT, 2D-[ ¹³ C, ¹ H]-HSQC (heteronuclear single quantum coherence), 2D-[ ¹³ C, ¹ H]-HMBC (heteronuclear multiply bond correlation) and 2D-NOESY is written for some compounds.

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

Method A: synthesis 1 N- pyrazoles

Those skilled in the art will know that compound 11 and compound 11 depicted below are two tautomeric forms of the same compound. All such tautomeric forms are considered part of the present invention. By way of illustration, all tautomeric forms of the pyrazole group, as shown, for example, for compound 11 below, are included in the present invention. Those skilled in the art will appreciate that the names of compounds contained herein are based on a nomenclature convention in which the tautomeric configuration is depicted as in relation to compound 11 below. Thus, the 1,3-benzodioxole substituent is in the third position. An alternative nomenclature convention could be based on the tautomer of compound 11 below, and in this convention the 1,3-benzodioxole substituent is in the fifth position.

Illustrative example: 2-[3-(1,3-Benzodioxol-5-yl)-1H-pyrazol-5-yl]-6-fluoropyridine, compound 11

Sodium hydride (FW 24.00, 60% in oil, 3.9 mmol, 156 mg) is added to a solution of 1-(1,3-benzodioxol-5-yl)ethanone (FW 164.16, 492 mg, 3.00 mmol) and methyl 6-fluoropyridine-2-carboxylate (FW 155.13, 605 mg, 3.9 mmol) in DMSO (7.5 ml) and THF (1.9 ml) and the reaction mixture was stirred at 20°C in for 15 hours. The reaction mixture is poured into 60 ml of ice and water containing AcOH (450 μl). The mixture was stirred for 1 hour. The resulting precipitate was filtered, washed with water (10 ml), hexane:EtOH=5:1 (10 ml), hexane (10 ml) and air dried to obtain the crude intermediate 1-(1,3 -benzodioxol-5-yl)-3-(6-fluoropyridin-2-yl)propan-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 is stirred at 70°C for 5 hours, cooled and concentrated in vacuo . The residue was suspended in MeOH (10 ml), boiled with stirring for 5 min, cooled, filtered, washed with MeOH (10 ml) and dried under high vacuum at 20°C for 15 h to give the purified product of compound 11 (471 mg, 1.66 mmol, 55% in two steps) as a white solid.

Method B: Synthesis 1 H -pyrazoles

Illustrative Example: 4-[3-(1,3-Benzodioxol-5-yl)-1H - pyrazol-5-yl]-2-bromopyridine, compound 9

A solution of potassium tert- butoxide (FW 112.21, 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 (FW 164, 16, 328 mg, 2 mmol) and methyl 2-bromopyridine-4-carboxylate (FW 216.03, 518 mg, 2.4 mmol) in dry THF (5 ml). The reaction mixture is stirred for 15 hours at 20°C. A 20 µl aliquot was removed, quenched with 1M phosphate buffer pH 7, extracted with EtOAc and analyzed by TLC. Complete conversion of the ketone is observed. The mixture is poured into 1M phosphate buffer pH 7 (15 ml) and ice water (15 ml) and stirred at 0°C for 30 minutes. The resulting yellow solid was filtered, washed with water (5x10 ml) and air dried to give 656 mg (1.88 mmol, 94%) of crude intermediate 1-(1,3-benzodioxol-5-yl)-3-(2 -bromopyridin-4-yl)propan-1,3-dione, which is used in the next step without purification. A mixture of this intermediate and hydrazine monohydrate (FW 50.06, d 1.03; 274 µl, 282 mg, 5.64 mmol) in THF (10 ml) was stirred at 50°C for 15 hours. The cooled mixture was poured into water (40 ml) and stir at 0°C for 30 minutes. The resulting precipitate is 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% in 2 steps) as a white powder.

Method C: Boc Deprotection

Illustrative example: 4-[5-(2-Bromopyridin-4-yl)-1H-pyrazol-3-yl]aniline compound 7

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 ( FW 415.28, 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 hours, and concentrated in vacuo . Add 1M phosphate buffer pH 7 (20 ml), the resulting precipitate is filtered, washed with water (2 x 10 ml) and air dried to give 543 mg (1.72 mmol, 69% in 3 steps) of the desired product as a yellow-orange solid .

Method D: Synthesis 1 H -pyrazoles

Illustrative Example: 4-[3-(1,3-Benzodioxol-5-yl)-1H - pyrazol-5-yl]-2-fluoropyridine, compound 12

i Pr ₂ NEt (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 MgBr ₂ ⋅Et ₂ O (1.56 g, 6.04 mmol) in CH ₂ Cl ₂ (35 ml). The resulting suspension was stirred for 5 minutes and then pentafluorophenyl 2-fluoropyridine-4-carboxylate (1.37 g, 4.45 mmol) in CH ₂ Cl ₂ (7 ml) was added dropwise. The reaction mixture was stirred for 48 hours. Then 1N aqueous HCl (20 ml) was added and stirring was continued for 5 minutes. The aqueous layer was extracted with CH ₂ Cl ₂ (20 ml) and the combined organic extracts were dried (MgSO ₄ ) and concentrated in vacuo . The residue is triturated with Et ₂ O (10 ml) and filtered. The solid was washed with Et ₂ O and air dried to give the crude product (1.14 g, orange solid). The crude product was recrystallized from EtOAc (8 ml) and hexane (4 ml) to give the purified intermediate 1-(1,3-benzodioxol-5-yl)-3-(2-fluoropyridin-4-yl)propan-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 is stirred at 70°C for 5 hours, cooled and concentrated in vacuo . The residue is suspended in Et ₂ O (10 ml), boiled with stirring for 5 minutes, cooled, filtered, washed with Et ₂ O (2 x 10 ml) and dried under high vacuum at 20°C for 15 hours to obtain the product, compound 12 (722 mg, 2.55 mmol, 76% in 2 steps) as a white solid.

Method E: Synthesis of pentafluorophenyl esters

Illustrative example: Pentafluorophenyl 2-fluoropyridine-4-carboxylate, compound 59

DCC (FW 206.33, 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 is continued for 15 hours, by which time a colorless precipitate has formed. The mixture is filtered through Celite® and evaporated to obtain a semi-solid. Chromatography over silica gel gave the ester, compound 59 (2.21 g, 7.2 mmol, 72%) as a clear colorless oil.

Method F: Synthesis 1 H -pyrazoles

Illustrative Example : 6-[3-(3-Bromophenyl) -1H -pyrazol-5-yl]-1,3-dioxolo[4,5- c ]pyridine, compound 20

Stage 1

To 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), Ba(OH) ₂ 8H ₂ O (5 mg) and NaOH (0.5 mg) were added and the resulting mixture was stirred at room temperature (RT) overnight. After the methanol is evaporated in vacuo, the residue is triturated with water (5 ml), the solid is collected by filtration, washed with cold methanol (0.5 ml) and dried to give the intermediate 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.

Stage 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) add an aqueous solution of H ₂ O ₂ (30%, 45 mg, 0.4 mmol), then add aqueous NaOH (10%, 16 μl, 0.04 mmol) dropwise. 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 residue is extracted with ethyl acetate (2x15 ml), the combined organic fractions are dried over Na ₂ SO ₄ , concentrated in vacuo and the residue is resuspended in toluene (0.8 ml). The toluene suspension is treated with hydrazine hydrate (35 mg, 0.7 mmol) and PTSA hydrate (5 mg) and the mixture is stirred at reflux for 1.5 h. After cooling, phosphate buffer (0.3 M, 20 ml) is added ) and the product is extracted with ethyl acetate (2x20 ml). The combined organic fractions are washed with brine (5 ml), dried over Na ₂ SO ₄ and concentrated in vacuo . The crude residue was purified by column chromatography (15 g, silica gel 63-100, CHCl ₃ /MeOH=100/1) to give a yellow solid, which was washed with Et ₂ O (1 ml) to give compound 20 (25 mg, 36%) in as a white solid.

Method G: Synthesis 1 H -pyrazoles

Illustrative Example : 4-[4-(1,3-Benzodioxol-5-yl)-1H - imidazol-2-yl]-2-bromopyridine, compound 48

A mixture of 1-(1,3-benzodioxol-5-yl)ethanone (138 mg, 0.84 mmol), MgBr ₂ ⋅Et ₂ O (542 mg, 2.1 mmol) in DCM (5 ml) was treated with DIPEA (323 mg, 426 µl, 2.5 mmol) and stir at RT for 10 minutes. Then, a crude mixture of 1H -benzotriazol-1-yl(3,6-dichloropyridazin-4-yl)methanone, which was prepared separately by stirring 3,6-dichloropyridazin-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 hours, add dropwise over 5 minutes. The resulting mixture was stirred at 25°C for 12 hours, then treated with aqueous 0.5 M HCl (2 ml) and stirred for another 10 minutes at 25°C. After adding water (20 ml), the mixture was extracted with DCM (2⋅15 ml). The combined organic fractions are washed with brine, dried over Na ₂ SO ₄ and concentrated in vacuo . The crude product was purified by column chromatography to give the intermediate 1-(1,3-benzodioxol-5-yl)-3-(3,6-dichloropyridazin-4-yl)propan-1,3-dione (190 mg, 67%) in 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 is 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% in two steps) as a light yellow solid.

Method H: Synthesis 1 H -imidazoles

Illustrative Example : 4-[4-(1,3-Benzodioxol-5-yl)-1H - imidazol-2-yl]-2-bromopyridine, compound 33

Stage 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 K ₂ CO ₃ (414 mg, 3 mmol) in DMF (6 ml) is stirred at 55-60°C for 6 hours. After cooling, the mixture is poured into water (60 ml), stirred for 10 minutes and the resulting precipitate is collected by filtration, washed with water (20 ml) on a filter and dried to give the intermediate 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.

Stage 2

A suspension of intermediate 2-(1,3-benzodioxol-5-yl)-2-oxoethyl 2-bromopyridine-4-carboxylate (364 mg, 1 mmol) and AcONH ₄ (924 mg, 12 mmol) in toluene (7 ml) heated at 100°C with vigorous stirring for 4 hours. After cooling, the reaction mixture is treated with phosphate buffer (50 ml, 0.25 M, pH 7) and extracted with ethyl acetate (2x50 ml). The combined organic fractions are washed with brine, dried over Na ₂ SO ₄ and concentrated in vacuo . The residue is purified by column chromatography (silica gel 63-100, 30 g, CHCl ₃ /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.

Method I: Synthesis 1 H -1,2,4-triazoles

Illustrative Example 4-[3-(1,3-Benzodioxol-5-yl) -1H -1,2,4-triazol-5-yl]-2-bromopyridine, compound 30

To a solution of t -BuOK (84 mg, 0.75 mmol) in n -BuOH (2 ml) add benzo[1,3]dioxole-5-carboxamidine hydrochloride (100 mg, 0.5 mmol) at 0°C and the mixture stir at RT for 20 minutes. After adding 2-bromoisonicotinic acid hydrazide (108 mg, 0.5 mmol), the yellow suspension was stirred at 85°C for 3 hours. After cooling to room temperature, ethanol (4 ml) was added and CO ₂ was bubbled in for 5 minutes. suspension. After removing the solvents in vacuo , the residue was triturated with water (10 ml), the resulting precipitate was collected by filtration, washed with water (5 ml) and dried to give compound 30 (85 mg, 49%) as a gray solid.

Method J: Synthesis 1 H -1,2,4-triazoles

Illustrative Example 4-[3-(2-Bromopyridin-4-yl) -1H- 1,2,4-triazol-5-yl]-N ,N -dimethylaniline, compound 44

A mixture of 2-bromoisonicotinic acid hydrazide (151 mg, 0.7 mmol), 4-(dimethylamino)benzonitrile (307 mg, 2.1 mmol) and K ₂ CO ₃ (48 mg, 0.5 mmol) in n -BuOH ( 2 ml) was stirred at 145°C for 8 hours. The mixture was concentrated in vacuo and the crude product was purified by two column chromatography (silica gel 63-100, 20 g, CHCl ₃ /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.

Method K: Synthesis of 4-chloro-1 H -pyrazoles

4-[3-(1,3-Benzodioxol-5-yl)-4-chloro- 1H -pyrazol-5-yl]-2-bromopyridine, compound 28

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 showed absorption of the starting material. After cooling to RT, the precipitate was collected by filtration, washed with water (2x5 ml) and recrystallized from ethanol (8 ml) to give compound 28 (52 mg, 47%) as a gray solid.

Method L: Synthesis of 1-[4-(4-R-piperazin-1-yl)phenyl]ethanones

Illustrative Example 1-{4-[4-(2-Methoxyethyl)piperazin-1-yl]phenyl}ethenone

Mixture of 1-[4-(piperazin-1-yl)phenyl]ethanone (408 mg, 2 mmol), 1-bromo-2-methoxyethane (417 mg, 3 mmol) and Cs ₂ CO ₃ (1304 mg, 4 mmol) in DMF (5 ml) stirred at 25°C overnight. After evaporation of the DMF in vacuo , the residue is partitioned between water (25 ml) and ethyl acetate (35 ml), the aqueous phase is extracted with ethyl acetate (25 ml), and the combined organic fractions are washed with brine, dried over Na ₂ SO ₄ and concentrated in vacuo . 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}ethenone (525 mg , 99%) as a light yellow solid.

2-Methoxyethyl 2,6-dibromopyridine-4-carboxylate and 2-methoxyethyl 2,6-dichloropyridine-4-carboxylate were prepared according to a published protocol (Journal of Medicinal Chemistry (2012), 55, 10564-10571)

tert- Butyl ( ₄ -acetylphenyl)methylcarbamate, tert- butyl (4-acetylphenyl) -N- ( ^2H3 )methylcarbamate , tert- butyl (4-acetylphenyl)ethylcarbamate and tert- butyl (4-acetylphenyl)(2-fluoroethyl) )carbamate was prepared according to a published protocol (Organic Letters (2020), 22, 5522-5527).

1-[4-(4-Fluoropiperidin-1-yl)phenyl]ethanone and 1-[4-(3-fluoroazetidin-1-yl)phenyl]ethanone are prepared according to a published protocol (WO 2011071570 A1).

Pentafluorophenyl 2-fluoropyridine-4-carboxylate and pentafluorophenyl 2-chloropyridine-4-carboxylate are 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 is prepared according to method L.

Exemplary compounds of the present invention are shown in Tables 1 and 2. Table 1 shows the name, structure, IUPAC name, starting materials, synthesis method, and chemical yield for a specific example compound. Table 2 shows the high-performance liquid chromatography (HPLC) retention time, molecular weight determined by low-resolution mass spectrometry coupled to HPLC, and proton nuclear magnetic resonance ( ^1H -NMR) for a specific example compound.

Table 1:

Table 2: Conn. No. HPLC retention time (min) Mass spectrum [M+H] ^{+ 1} H-NMR data (ppm) δ
(solvent, T) 1 ^4.8a 343.1 (100%), 345.2 (97%) 8.42 (d, J =5.2 Hz, 1H), 8.05 (d, J =1.4 Hz, 1H), 7.93 (d, J =8.7 Hz, 2H), 7, 87 (dd, J =5.2, 1.4 Hz, 1H), 7.66 (bd, J =7.8 Hz, 2H), 7.51 (s, 1H), 3.11 (s, 6H )
(DMSO-d6+1% DCl, 298K) 2 ^4.0a 329.1 (98%), 331.1 (100%) 8.41 (d, J =5.2 Hz, 1H), 8.04 (d, J =1.1 Hz, 1H), 7.93 (d, J =8.6 Hz, 2H), 7, 86 (dd, J =5.2, 1.4 Hz, 1H), 7.57 (d, J =8.6 Hz, 2H), 7.48 (s, 1H), 2.92 (s, 3H )
(DMSO-d6+1% DCl, 313K) 3 ^3.9a 283.2 (100%) 8.28 (d, J =5.2 Hz, 1H), 7.93 (bd, J =8.7 Hz, 2H), 7.78 (dt, J =5.2, 1.6 Hz, 1H ), 7.67 (bd, J = 8.7 Hz, 2H), 7.55 (s, 1H), 7.45 (s, 1H), 3.12 (s, 6H)
(DMSO-d6+1% DCl, 313K) 4 ^3.6a 344.1 (98%), 346.1 (100%) 8.71 (dd, J =6.2, 1.1 Hz, 1H), 8.55 (s, 1H), 8.35 (m, 2H), 8.27 (d, J =9.6 Hz , 1H), 7.41 (d, J =1.1 Hz, 1H), 7.26 (d, J =9.6 Hz, 1H), 3.38 (s, 6H)
(TFK-d1, 313K) 5 ^9.2a 344.1 (100%), 346.1 (98%) 9.07 (s, 2H), 7.82 (s, 1H), 7.60 (d, J =8.0 Hz, 2H), 7.29 (t, J =8.0 Hz, 1H), 7.26 (s, 1H), 3.37 (s, 6H)
(TFK-d1, 298K) 6 6.2 ^a 345.1 (100%), 347.3 (98%) 8.70 (s, 2H), 8.46 (d, J =6.4 Hz, 1H), 8.30 (s, 1H), 8.10 (dd, J =6.4, 1.6 Hz , 1H), 7.24 (s, 1H), 3.19 (s, 6H)
(TFK-d1, 298K) 7 ^3.3a 315.1 (98%), 317.1 (100%) 13.22 (ws, 1H), 8.39 (d, J =5.2 Hz, 1H), 8.01 (d, J =0.8 Hz, 1H), 7.83 (dd, J =5 ,2, 1.4 Hz, 1H), 7.47 (ddd, J =7.5, 1.7, 0.9 Hz, 2H), 7.19 (s, 1H), 6.64 (ddd, J =7.5, 1.7, 0.9 Hz, 2H), 5.49 (shs, 2H)
(DMSO-d6, 298K) 8 ^5.2a 285.2 (100%) 8.69 (s, 2H), 8.31 (dd, J =6.4, 2.0 Hz, 1H), 7.94 (dd, J =6.4, 1.3 Hz, 1H), 7 .76 (dd, J =3.2, 1.3 Hz, 1H), 7.22 (s, 1H), 3.17 (s, 6H)
(TFK-d1, 298K, two forms in a 10/1 ratio, main form) 9 ^10.4a 344.1 (100%), 346.2 (98%) 8.41 (d, J =5.1 Hz, 1H), 8.01 (d, J =1.2 Hz, 1H), 7.84 (dd, J =5.1, 1.5 Hz, 1H ), 7.38 (d, J =1.7 Hz, 1H), 7.34 (s, 1H), 7.33 (dd, J =8.1, 1.7 Hz, 1H), 7.00 (d, J =8.1 Hz, 1H), 6.06 (s, 2H)
(DMSO-d6+1% DCl, 313K) 10 ^9.8a 344.1 (100%), 346.2 (98%) 9.05 (d, J =1.2 Hz, 1H), 8.67 (d, J =2.0 Hz, 1H), 8.46 (shs, 1H), 7.37 (d, J =2 .0 Hz, 1H), 7.32 (dd, J =8.1, 1.5 Hz, 1H), 7.27 (s, 1H), 6.99 (d, J =8.1 Hz, 1H ), 6.06 (s, 2H)
(DMSO-d6+1% DCl, 313K) eleven ^8.7a 284.2 8.04 (kv, J =8.0 Hz, 1H), 7.89 (dd, J =7.5, 2.4 Hz, 1H), 7.44 (d, J =1.7 Hz, 1H ), 7.38 (dd, J =8.1, 1.7 Hz, 1H), 7.22 (s, 1H), 7.09 (dd, J =8.0, 2.4 Hz, 1H) , 6.98 (d, J =8.1 Hz, 1H), 6.05 (s, 2H)
(DMSO-d6+1% DCl, 298K) 12 ^8.4a 284.2 (100%) 8.26 (d, J =5.2 Hz, 1H), 7.76 (dt, J =5.2, 1.7 Hz, 1H), 7.53 (s, 1H), 7.39 (d , J =1.7 Hz, 1H), 7.37 (s, 1H), 7.33 (dd, J =8.1, 1.8 Hz, 1H), 7.01 (d, J =8, 1 Hz, 1H), 6.06 (s, 2H)
(DMSO-d6+1% DCl) 13 ^7.4a 284.2 8.98 (t, J =1.7 Hz, 1H), 8.62 (d, J =2.7 Hz, 1H), 8.21 (ddd, J =8.1, 2.7, 1, 7 Hz, 1H), 7.37 (d, J =1.7 Hz, 1H), 7.32 (dd, J =8.1, 1.8 Hz, 1H), 7.32 (s, 1H) , 7.01 (d, J =8.1 Hz, 1H), 6.07 (s, 2H)
(DMSO-d6+1% DCl, 298K) 14 ^9.8a 300.1 (100%) 8.43 (d, J =5.2 Hz, 1H), 7.87 (s, 1H), 7.81 (dd, J =5.2, 1.4 Hz, 1H), 7.39 (d , J =1.6 Hz, 1H), 7.34 (s, 1H), 7.34 (dd, J =8.2, 1.6 Hz, 1H), 7.00 (d, J =8, 1 Hz, 1H), 6.06 (s, 2H)
(DMSO-d6+1% DCl, 313K) 15 11.2 ^a 344.2 (100%), 346.2 (97%) 7.97 (d, J =7.8 Hz, 1H), 7.80 (t, J =7.8 Hz, 1H), 7.55 (d, J =7.8 Hz, 1H), 7. 45 (d, J =1.6 Hz, 1H), 7.39 (dd, J =8.1, 1.6 Hz, 1H), 7.20 (s, 1H), 6.97 (d, J =8.1 Hz, 1H), 6.04 (s, 2H)
(DMSO-d6+1% DCl, 298K) 16 ^7.3a 344.0 (100%), 346.0 (97%) 9.47 (d, J =1.7 Hz, 1H), 9.44 (d, J =6.4 Hz, 1H), 9.04 (dd, J =6.4, 1.7 Hz, 1H ), 8.21 (s, 1H), 8.12 (dd, J =8.1, 1.7 Hz, 1H), 8.04 (d, J =1.7 Hz, 1H), 7.80 (d, J =8.1 Hz, 1H), 6.86 (s, 2H)
(TFK-d1, 298K) 17 ^7.5a 355.2 (100%), 357.2 (98%) 8.40 (d, J =5.2 Hz, 1H), 8.02 (d, J =1.4 Hz, 1H), 7.84 (dd, J =5.2, 1.4 Hz, 1H ), 7.74 (d, J =8.6 Hz, 2H), 7.31 (s, 1H), 7.11 (shd, J =8.4 Hz, 2H), 3.76 (t, J =7.0 Hz, 2H), 3.31 (t, J =7.0 Hz, 2H), 2.10 (qui, J =7.0 Hz, 2H)
(DMSO-d6+1% DCl, 313K) 18 ^12.6a 358.2 (100%), 360.2 (98%) 8.51 (d, J =5.1 Hz, 1H), 7.89 (d, J =0.7 Hz, 1H), 7.69 (dd, J =5.1, 1.3 Hz, 1H ), 7.35 (s, 1H), 7.33 (d, J =9.3 Hz, 1H), 7.12 (s, 1H), 6.96 (d, J =7.9 Hz, 1H ), 6.05 (s, 2H), 3.97 (s, 3H)
(DMSO-d6, 298K) 19 ^13.7a 358.1 (100%), 360.2 (98%) 8.36 (d, J =5.0 Hz, 1H), 7.90 (s, 1H), 7.64 (d, J =4.7 Hz, 1H), 6.92 (s, 1H), 6.91 (d, J =8.9 Hz, 2H), 6.61 (s, 1H), 6.06 (s, 2H), 3.92 (s, 3H)
(CDCl ₃ , 298K) 20 17.3 ^b 344.2 (97%),
346.2 (100%) 8.37 (s, 1H), 8.07 (t, J =1.8 Hz, 1H), 8.03 (s, 1H), 7.89-7.82 (m, 2H), 7.61 (ddd, J =8.0, 2.0, 0.9 Hz, 1H), 7.47 (t, J =7.9 Hz, 1H), 6.52 (s, 2H)
(DMSO-d6+1% DCl, 298K) 21 17.7 ^b 329.1 (98%),
331.1 (100%) 8.46 (d, J =6.4 Hz, 1H), 8.31 (s, 1H), 8.12 (d, J =6.4 Hz, 1H), 7.70 (d, J =8 .6 Hz, 2H), 7.43 (d, J =8.6 Hz, 2H), 7.22 (s, 1H)
(TFK-d1, 298K) 22 15.2 ^b 285.2 (100%)
287.2 (34%) 8.46 (d, J =5.2 Hz, 1H), 7.97 (d, J =8.6 Hz, 2H), 7.94 (s, 1H), 7.85 (dd, J =5 ,2, 1.4 Hz, 1H), 7.64 (d, J =8.6 Hz, 2H), 7.56 (s, 1H), 2.92 (s, 3H)
(DMSO-d6+1% DCl, 298K) 23 15.9 ^b 343.2 (99%)
345.2 (100%) 8.43 (d, J =5.2 Hz, 1H), 8.07 (d, J =1.2 Hz, 1H), 7.98 (d, J =8.6 Hz, 2H), 7, 88 (dd, J =5.2, 1.5 Hz, 1H), 7.69 (d, J =8.6 Hz, 2H), 7.57 (s, 1H), 3.34 (sq, J =7.3 Hz, 2H), 1.27 (t, J =7.3 Hz, 3H)
(DMSO-d6+1% DCl, 298K) 24 14.3 ^b 269.2 (100%) 8.30 (d, J =5.3 Hz, 1H), 7.98 (d, J =8.6 Hz, 2H), 7.80 (m, 1H), 7.66 (d, J =8 .6 Hz, 2H), 7.59 (s, 1H), 7.54 (s, 1H), 2.92 (s, 3H)
(DMSO-d6+1% DCl, 298K) 25 21.2 ^b 361.3 (100%)
363.2 (100%) 8.42 (d, J =5.2 Hz, 1H), 8.07 (d, J =0.8 Hz, 1H), 7.92-7.84 (m, 3H), 7.49 (s , 1H), 7.41 (d, J =8.6 Hz, 2H), 4.73 (dt, J =47.3, 4.8 Hz, 2H), 3.61 (dt, J =27, 2, 4.8 Hz, 2H)
(DMSO-d6+1% DCl, 298K) 26 15.9 ^b 398.3 (99%)
400.4 (100%) 8.40 (d, J =5.2 Hz, 1H), 8.05 (s, 1H), 7.87 (d, J =5.2 Hz, 1H), 7.74 (d, J =8 .7 Hz, 2H), 7.39 (s, 1H), 7.11 (d, J =8.7 Hz, 2H), 3.97-3.87 (m, 2H), 3.52-3 .43 (m, 2H), 3.27-3.10 (m, 4H), 2.80 (s, 3H)
(DMSO-d6+1% DCl, 298K) 27 19.5 ^b 325.3 (100%) 8.28 (d, J =5.3 Hz, 1H), 7.88 (d, J =8.8 Hz, 2H), 7.82-7.77 (m, 1H), 7.60-7 .52 (m, 3H), 7.47 (s, 1H), 3.44 (m, 4H), 3.96 (m, 4H)
(DMSO-d6+1% DCl, 298K) 28 25.8 ^b 378.0 (76%)
380.1 (100%) 8.49 (dd, J =5.3, 0.6 Hz, 1H), 8.05 (dd, J =1.5, 0.6 Hz, 1H), 7.93 (dd, J =5, 3, 1.4 Hz, 1H), 7.33-7.28 (m, 2H), 7.08 (d, J =8.6 Hz, 1H), 6.10 (s, 2H)
(DMSO-d6+1% DCl, 298K) 29 15.0 ^b 344.1 (100%)
346.1 (98%) 8.52 (s, 1H), 8.51 (d, J =5.3 Hz, 1H), 8.18 (s, 1H), 7.92 (d, J =5.2, 1.3 Hz , 1H), 7.68-7.60 (m, 2H), 7.21 (d, J =8.1 Hz, 1H), 6.19 (s, 2H)
(DMSO-d6+1% DCl, 298K) thirty 22.6 ^b 345.2 (100%)
347.2 (93%) 8.50 (d, J =5.1 Hz, 1H), 8.14 (s, 1H), 8.01 (d, J =5.1 Hz, 1H), 7.66 (dd, J =8 ,1, 1.6 Hz, 1H), 7.61 (d, J =1.6 Hz, 1H), 7.08 (d, J =8.1 Hz, 1H), 6.12 (s, 2H )
(DMSO-d6+1% DCl, 298K) 31 13.3 ^b 343.2 (100%)
345.1 (98%) 8.59 (dd, J =5.2, 0.6 Hz, 1H), 8.28 (dd, J =1.6, 0.6 Hz, 1H), 8.07 (s, 1H), 8 .03 (dd, J =5.3, 1.6 Hz, 1H), 7.75 (d, J =8.9 Hz, 2H), 6.94 (d, J =8.9 Hz, 2H) , 3.00 (s, 6H)
(DMSO-d6+1% TFA-d1, 298K) 32 20.4 ^b 345.1 (99%)
347.1 (100%) 8.43 (d, J =5.3 Hz, 1H), 8.12 (d, J =1.9 Hz, 1H), 8.03 (s, 1H), 7.84 (dd, J =5 ,2, 1.4 Hz, 1H), 7.66 (d, J =1.8 Hz, 1H), 7.49 (s, 1H), 6.20 (s, 2H)
(DMSO-d6+1% DCl, 298K) 33 16.2 ^b 344.2 (100%)
346.2 (96%) 8.60 (dd, J =5.2, 0.6 Hz, 1H), 8.27 (dd, J =1.5, 0.6 Hz, 1H), 8.12 (s, 1H), 8 .02 (dd, J =5.2, 1.5 Hz, 1H), 7.46 (d, J =1.8 Hz, 1H), 7.41 (dd, J =8.1, 1.8 Hz, 1H), 7.06 (d, J =8.1 Hz, 1H), 6.09 (s, 2H)
(DMSO-d6+1% TFA-d1, 298K) 34 24.1 ^b 344.1 (96%)
346.1 (100%) 8.12 (d, J =1.8 Hz, 1H), 8.03 (t, J =1.8 Hz, 1H), 7.83 (d, J =7.7 Hz, 1H), 7, 66 (d, J =1.8 Hz, 1H), 7.53 (m, 1H), 7.42 (t, J =7.9 Hz, 1H), 7.30 (s, 1H), 6, 19 (s, 2H)
(DMSO-d6+1% DCl, 298K) 35 17.8 ^b 325.3 (100%)
327.4 (35%) 8.71 (dd, J =5.3, 0.6 Hz, 1H), 8.21 (dd, J =1.6, 0.6 Hz, 1H), 8.18 (s, 1H), 8 .04 (dd, J =5.3, 1.6 Hz, 1H), 7.71 (d, J =8.9 Hz, 2H), 6.69 (d, J =8.9 Hz, 2H) , 3.30 (m, 4H), 1.98 (m, 4H)
(DMSO-d6+1% TFA-d1, 298K) 36 15.0 ^b 286.2 (100%)
288.2 (33%) 9.20 (s, 1H), 8.72 (s, 1H), 8.04 (d, J =8.7 Hz, 2H), 7.64 (d, J =8.7 Hz, 2H), 7.49 (s, 1H), 2.92 (s, 3H)
(DMSO-d6+1% DCl, 298K) 37 19.3 ^b 407.1 (52%)
409.1 (100%)
411.1 (50%) 8.13 (s, 2H), 7.96 (d, J =8.6 Hz, 2H), 7.66 (d, J =8.6 Hz, 2H), 7.65 (s, 1H), 2.91 (s, 3H)
(DMSO-d6+1% DCl, 298K) 38 16.3 ^b 343.2 (100%)
345.1 (98%) 7.94 (d, J =8.7 Hz, 2H), 7.86 (s, 1H), 7.76 (s, 1H), 7.58 (d, J =8.7 Hz, 2H), 7.50 (s, 1H), 2.91 (s, 3H), 2.49 (s, 3H)
(DMSO-d6+1% DCl, 298K) 39 13.4 ^b 286.2 (100%)
288.2 (34%) 9.71 (d, J =1.8 Hz, 1H), 8.26 (d, J =1.8 Hz, 1H), 7.94 (d, J =8.6 Hz, 2H), 7, 67 (s, 1H), 7.61 (d, J =8.6 Hz, 2H), 2.92 (s, 3H)
(DMSO-d6+1% DCl, 298K) 40 14.2 ^b 286.2 (100%)
288.2 (30%) 8.80 (d, J =5.2 Hz, 1H), 8.09-8.01 (m, 3H), 7.64 (d, J =8.6 Hz, 2H), 7.54 (s , 1H), 2.92 (s, 3H)
(DMSO-d6+1% DCl, 298K) 41 15.2 ^b 285.2 (100%)
287.2 (34%) 8.02 (d, J =8.7 Hz, 2H), 7.99-7.92 (m, 2H), 7.59 (d, J =8.7 Hz, 2H), 7.46 (dd , J =6.7, 2.0 Hz, 1H), 7.37 (s, 1H), 2.92 (s, 3H)
(DMSO-d6+1% DCl, 298K) 42 19.6 ^b 333.1 (100%)
334.1 (61%)
335.1 (61%) 8.01-7.96 (m, 4H), 7.89 (d, J =8.8 Hz, 2H), 7.67 (s, 1H), 3.14 (s, 6H)
(DMSO-d6+1% DCl, 298K) 43 15.4 ^b 300.2 (100%)
302.2 (33%) 9.02 (s, 1H), 8.05 (s, 1H), 7.65 (d, J =8.6 Hz, 2H), 7.24 (s, 1H), 6.78 (d, J =8.6 Hz, 2H), 2.94 (s, 6H)
(DMSO-d6+2% acetic acid-d4, 298K) 44 19.2 ^b 344.2 (100%)
346.3 (100%) 8.53 (d, J =5.1 Hz, 1H), 8.27 (d, J =8.8 Hz, 2H), 8.24 (s, 1H), 8.08 (dd, J =5 ,1, 1.3 Hz, 1H), 7.76 (d, J =8.6 Hz, 2H), 3.13 (s, 6H)
(DMSO-d6+1% DCl, 298K) 45 16.9 ^b 343.2 (100%)
345.2 (98%) 8.50 (d, J =5.3 Hz, 1H), 8.48 (s, 1H), 8.20 (d, J =1.0 Hz, 1H), 7.97-7.91 (m , 3H), 6.89 (d, J =9.1 Hz, 2H), 3.04 (s, 6H)
(DMSO-d6+1% TFA-d1, 298K) 46 26.5 ^b 422.1 (52%)
424.1 (100%)
426.1 (49%) 8.08 (s, 2H), 7.49 (s, 1H), 7.38 (d, J =1.6 Hz, 1H), 7.33 (dd, J =8.1, 1.6 Hz , 1H), 7.03 (d, J =8.1 Hz, 1H), 6.07 (s, 2H)
(DMSO-d6+1% DCl, 298K) 47 24.2 ^b 358.1 (100%)
360.1 (98%) 7.84 (s, 1H), 7.75 (s, 1H), 7.39 (d, J =1.7 Hz, 1H), 7.37 (s, 1H), 7.33 (dd, J =8.1, 1.7 Hz, 1H), 7.01 (d, J =8.1 Hz, 1H), 6.06 (s, 2H), 2.49 (s, 3H)
(DMSO-d6+1% DCl, 298K) 48 22.5 ^b 335.1 (100%)
337.2 (66%) 8.31 (s, 1H), 7.48-7.45 (m, 2H), 7.38 (dd, J = 8.1, 1.8 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H), 6.08 (s, 2H)
(DMSO-d6+1% DCl, 298K) 49 14.9 ^b 324.3 (100%) 9.02-8.84 (m, 1H), 8.28 (d, J =5.2 Hz, 1H), 7.78 (d, J =5.1 Hz, 1H), 7.71 (d , J =8.7 Hz, 2H), 7.54 (s, 1H), 7.34 (s, 1H), 7.11 (d, J =8.7 Hz, 2H), 3.48-3 .40 (m, 4H), 3.30-3.21 (m, 4H)
(DMSO-d6+1% TFA-d1, 298K) 50 16.3 ^b 386.3 (100%)
388.3 (37%) 8.43 (d, J =5.2 Hz, 1H), 7.91 (s, 1H), 7.84 (dd, J =5.2, 1.3 Hz, 1H), 7.74 (d , J =8.8 Hz, 2H), 7.38 (s, 1H), 7.11 (d, J =8.8 Hz, 2H), 4.96 (dt, J =47.2, 4, 3 Hz, 2H), 4.00-3.88 (m, 2H), 3.67-3.50 (m, 4H), 3.34-3.20 (m, 4H)
(DMSO-d6+1% DCl, 298K) 51 17.1 ^b 442.4 (93%)
444.2 (100%) 8.41 (d, J =5.2 Hz, 1H), 8.04 (s, 1H), 7.87 (dd, J =5.2, 1.2 Hz, 1H), 7.73 (d , J =8.8 Hz, 2H), 7.38 (s, 1H), 7.11 (d, J =8.8 Hz, 2H), 3.96-3.87 (m, 2H), 3 .78 (t, J =4.6 Hz, 2H), 3.61-3.52 (m, 2H), 3.36 (t, J =4.6 Hz, 2H), 3.31 (s, 3H), 3.29-3.15 (m, 4H)
(DMSO-d6+1% DCl, 298K) 52 19.1 ^b 401.2 (100%)
403.2 (98%) 8.43 (d, J =5.2 Hz, 1H), 8.07 (s, 1H), 8.00 (s, 4H), 7.88 (dd, J =5.3, 1.3 Hz , 1H), 7.60 (s, 1H), 5.17-4.96 (m, 1H), 3.79-3.53 (m, 4H), 2.57-2.36 (m, 2H ), 2.34-2.17 (m, 2H)
(DMSO-d6+1% DCl, 298K) 53 ^4.7a 299.3 (100%) 8.46 (dd, J =5.2, 0.2 Hz, 1H), 8.00 (dd, J =8.8, 2.2, 2.2 Hz, 2H), 7.94 (d, J =0.9 Hz, 1H), 7.89-7.79 (m, 3H), 7.58 (s, 1H), 3.14 (s, 6H)
(DMSO-d6+1% DCl, 298K) 54 17.0 ^b 284.2 (100%) 8.79 (t, J =6.5 Hz, 1H), 8.19 (dd, J =9.3, 2.5 Hz, 1H), 7.67 (ddd, J =7.8, 6, 5, 2.5, Hz, 1H), 7.60 (s, 1H), 7.42 (d, J =1.8 Hz, 1H), 7.35 (dd, J =8.1, 1, 8 Hz, 1H), 7.04 (d, J =8.1 Hz, 1H), 6.08 (s, 2H)
(DMSO-d6+1% DCl, 298K) 55 12.2 ^a 329.2 (100%)
331.1 (33%) 8.42 (d, J =5.2 Hz, 1H), 7.88 (s, 1H), 7.82 (dd, J =5.2, 1.4, Hz, 1H), 7.64 ( ddd, J =8.6, 2.3, 2.3 Hz, 2H), 7.30 (shs, 1H), 6.58 (ddd, J =8.6, 2.3, 2.3 Hz, 2H), 5.58 and 5.43 (dtt, J =57.6, 5.8, 3.1 Hz, 2×0.5 H), 4.20 (dddd, J =20.7, 9, 4, 5.7, 1.0 Hz, 2H), 3.93 (dddd, J =24.3, 9.4, 3.1, 1.2 Hz, 2H)
(DMSO-d6, 298K) 56 15.3 ^b 329.2 (100%)
331.1 (98%) 9.08 (d, J =1.8 Hz, 1H), 8.71 (d, J =2.2 Hz, 1H), 8.53 (t, J =2.0 Hz, 1H), 7, 91 (bd, J =8.6 Hz, 2H), 7.50 (bd, J =8.6 Hz, 2H), 7.44 (s, 1H), 2.90 (s, 3H)
(DMSO-d6+1% DCl, 298K) 57 14.6 ^b 329.2 (100%)
331.1 (98%) 8.48 (d, J =5.3 Hz, 1H), 8.15 (d, J =1.8 Hz, 1H), 7.57 (d, J =8.6 Hz, 2H), 7, 56 (m, 1H), 7.08 (s, 1H), 6.60 (bd, J =8.6 Hz, 2H), 2.71 (s, 3H)
(DMSO-d6, 298K) 58 16.4 ^b 329.2 (100%)
331.1 (98%) 8.02-7.98 (m, 3H), 7.83 (t, J =7.8 Hz, 1H), 7.60-7.57 (m, 3H), 7.35 (s, 1H) , 2.92 (s, 3H)
(DMSO-d6+1% DCl, 298K)

^a gradient 50% CH ₃ CN/50% H ₂ O → 100% CH ₃ CN in 30 min

^b gradient 5% CH ₃ CN /100% H ₂ O → 100% CH ₃ CN in 30 min

Example 2: Binding Studies to Aggregated Alpha-Synuclein, Tau and A Beta

Two types of in vitro fibril binding assays are used to analyze the binding affinity and target selectivity of compounds: saturation assay and competition assay.

1) Preparation of fibrils

Alpha-synuclein and tau46 are purified from E. coli according to established protocols (Nuscher B, et al., J. Biol. Chem., 2004; 279(21):21966-75), Aβ _1-42 is obtained from rPeptide , Watkinsville, GA, USA. Fibrils are obtained by aggregation of recombinant protein with constant stirring. Specifically, 70 μM alpha-synuclein mixed with 100 mM NaCl in 50 mM Tris, pH 7.0+0.02% NaN ₃ is incubated at 1400 rpm, 37°C for 96 hours. 10 μM tau46 is incubated with 0.03 mg/ml heparin in 50 mM Tris, pH=7.0 at 1000 rpm, 37°C for 72 hours. Aβ _1-42 is dissolved in 20 mM _NaPi , pH=8.0+0, 2 mM EDTA + 0.02% NaN ₃ (Deeg AA, et al., Biochim. Biophys. Acta, 2015; 1850(9): 1884-90; Goedert M, et al. Nature, 1996; 383(6600): 550-3).

2a) Binding saturation analysis

Sonicated fibrils of αSYN (0.04 μM) or tau46 (0.4 μM) or Aβ _1-42 (6 μM), diluted in PBS, are incubated with decreasing concentrations of [ ³ H]-labeled compound (48 nM or 24 nM - 23 pM) in 50 mM Tris base, 10% ethanol, 0.05% Tween20, pH 7.4. To determine nonspecific binding, the appropriate unlabeled compound (400 nM) is added to a duplicate set of binding reactions.

Assay plates are incubated with stirring at 37°C for 2 h and covered with plastic film (Resealable Tape, PerkinElmer, Waltham, MA, USA). incubated with 5 mg/ml polyethylenimine (PEI, poly(ethylenimine solution), Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) at 4°C for 30 min. After incubation, bound and free ligands are separated by vacuum filtration using a cell harvester (Filtermate harvester, PerkinElmer, Waltham, MA, USA). The filter is washed three times with buffer cooled to 4°C and then dried in a microwave oven for 2 minutes at medium power. Fused scintillator plates (MeltiLex™ B/HS, PerkinElmer, Waltham, MA, USA) are fused into the filter using a heating plate set at 120°C. After solidification at room temperature, the filter is sealed in a plastic bag (MicroBeta® Sample Bag, PerkinElmer, Waltham, MA, USA). Tritium accumulation was immediately counted in a liquid scintillation counter (Wallac MicroBeta® TriLux, 1450 LSC & Luminescence Counter, PerkinElmer, Waltham, MA, USA). Radioactivity is plotted against increasing concentrations of the tritium-labeled compound or the cold compound. Data were fit using nonlinear regression analysis in GraphPad Prism (GraphPad Software, Inc., version 7.03, La Jolla, CA, USA).

2b) Modified binding saturation assay

For saturation binding assay, a fixed concentration of sonicated recombinant αSYN (15 nM/well) or tau46 (250 nM/well) or Aβ _1-42 (1 μM/well) human fibrils diluted in phosphate-buffered saline (PBS) is incubated in low-binding plates (96-well microplate test, Ratiolab GmbH, Dreieich, Germany) with [ ³ H]-compound 1 or [ ³ H]-compound 2 in increasing concentrations (from 0.05 nM to 12 nM/24 nM) in 30 mM Tris HCl, 10% ethanol, 0.05% Tween20, pH 7.4 (hereinafter called incubation buffer) in a total volume of 200 μl/well. Non-specific labeled binding was determined by co-incubation with 400 nM unlabeled Compound 1 or Compound 2. Optimal fibril concentrations were determined using a concentration determination assay.

Plates covered with removable sealing tape (PerkinElmer) are incubated on a shaker (MaxQ™ 6000, 1.9 cm orbital diameter, Thermo Fisher Scientific Inc., Marietta, OH, USA) at 45 rpm for two hours at 37°C . After incubation, bound and free radioligands are separated by vacuum filtration through a glass fiber flat filter B (PerkinElmer) using a flat filter cell harvester (PerkinElmer). To collect cells from plates containing αSYN and Aβ _1-42 fibrils, the flat filter is further incubated with 5 mg/ml polyethylenimine for 30 minutes at 4C before collecting cells. The filter is washed three times with 100 ml (approximately 1 ml/well) of ice-cold incubation buffer and then dried in the microwave for 2.5 minutes at medium power. Melt scintillation sheets (MeltiLex™ B/HS, PerkinElmer) are fused into the filter using a heating plate set at 120°C. After solidification at room temperature, the filter was sealed in a plastic sample bag (PerkinElmer). Tritium accumulation was immediately counted on a Wallac MicroBeta® TriLux liquid scintillation counter (PerkinElmer). Radioactivity is plotted against the concentration of ³ H-labeled compound. Data were fit using nonlinear regression analysis in GraphPad Prism (GraphPad Software, Inc., version 7.03, La Jolla (CA), USA).

3a) Competitive binding assay

Sonicated recombinant αSYN (200 nM/well) or tau46 (208 nM/well) human fibrils at a fixed concentration, diluted in phosphate-buffered saline (PBS), are incubated in low-binding plates (96-well microplate assay, Ratiolab GmbH , Dreieich, Germany) together with 1 nM [ ^3H ] compound 1 and serial 1:4 dilutions of the cold compound of interest, starting with 1 µM diluted in 50 mM Tris base, 10% EtOH, 0.05% Tween20, pH 7.4. To calculate K _i values, the K _D value of compound 1 relative to sonicated recombinant human αSYN fibrils was set to 3 nM.

Assay plates are incubated with agitation at 37°C for 2 h, covered with plastic film (Resealable Tape, PerkinElmer, Waltham, MA, USA). Before cell collection, a filter (printed flat filter B, PerkinElmer, Waltham, MA, USA) is incubated with 5 mg/ml polyethylenimine (PEI, poly(ethylenimine solution), Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) at 4°C for 30 min. After incubation, bound and free ligands are separated by vacuum filtration using a cell harvester (Plant Filter Cell Collector, PerkinElmer, Waltham, MA, USA). The filter is washed three times with buffer cooled to 4°C and then dried in a microwave oven for 2 minutes at medium power. The filter was sealed in a plastic bag (MicroBeta® sample bag, PerkinElmer, Waltham, MA, USA) along with added scintillator (BETAPLATE SCINT, PerkinElmer, Waltham, MA, USA). Tritium accumulation was immediately counted in a liquid scintillation counter (Wallac MicroBeta® TriLux, 1450 LSC & Luminescence Counter, PerkinElmer, Waltham, MA, USA). Radioactivity is plotted against increasing concentrations of the tritium-labeled compound or the cold compound. Data points were fit using nonlinear regression analysis in GraphPad Prism (GraphPad Software, Inc., version 7.03, La Jolla, CA, USA).

3b) Modified Competitive Binding Assay

For competitive binding experiments, a fixed concentration of recombinant αSYN fibrils (sonicated, 15 nM/well) is incubated with 1 nM [ ³ H]-compound 1 or [ ³ H]-compound 2 and decreasing concentrations of serial dilution of unlabeled competitor (serial dilution 1:4 or 1:3.5 or 1:3 indicating concentration ranges of unlabeled competitor 1 µM-1 pM or 1 µM-4 nM or 1 µM-17 pM, respectively) in 30 mM Tris-HCl, 10% ethanol , 0.05% Tween20, pH 7.4, in a total volume of 200 µl/well. The plates are incubated for 4.5 hours at room temperature, covered with removable sealing tape (PerkinElmer, Waltham, MA, USA).

Filtration and readings were performed as described for saturation binding assays. [ ³ H]-labeled ligand binding [in CMP] is plotted against increasing competitor concentrations and the data are fitted using nonlinear regression analysis to calculate IC ₅₀ and K _i values (GraphPad Software, Inc., version 7.03, La Jolla (CA ), USA). In the competitive binding assay with sonicated recombinant αSYN fibrils, the calculation of K _i values is based on K _D values of 0.6 nM and 0.2 nM for compound 1 and compound 2, respectively.

Saturation analysis of fibril binding yields K _D values. This assay is performed with directly radiolabeled compounds (eg ³ H labeling). The K _D values are shown in Tables 3a and 3b. Saturation binding assays are performed using fibril aggregates derived from recombinant human alpha-synuclein, Aβ _1-42 and tau46, as target structures, respectively.

The results show high affinity for alpha-synuclein fibrils. Connection 1, connection 2 and connection 12 showed very high affinity at K values_D below 10 nM. Compound 1 and compound 2 was also tested for binding to A beta and tau46 fibrils and showed good to excellent selectivity, demonstrating the utility of these compounds for the diagnostic detection of aggregated alpha-synuclein.

Table 3a: Saturation analysis using protocol 2a Conn. No. K _D [nM] for aggregated alpha-synuclein K _D [nM] for aggregated tau46 K _D [nM] for aggregated A beta 1 < 2 > 5 > 100 2 < 1 > 15 > 100 12 7 ND ND 18 126 ND ND

Table 3b: Saturation analysis using protocol 2b Conn. No. K _D [nM] for aggregated alpha-synuclein 1 0.6 2 0.2

Fibril competition analysis provides K _i values. K _i is a quantitative measure that represents the concentration of a non-radioactive test compound (competitor ligand) required to replace 50% of the reference compound, in this case [ ³ H] compound 1 (1 nM) or [ ³ H] compound 2 (1 nM), which is associated with target structures (1 nM), in our case, recombinant alpha-synuclein and tau46 fibrils. This assay is suitable for screening non-radioactive ligands. The _Ki values obtained for the test compounds are given in Tables 4a, 4b and 5.

Table 4a: Competitive assay using protocol 3a (reference ligand [ ^3H ]-compound 1 (1 nM)) Conn. No. Experiment A Experiment B K _i (nM) alpha-synuclein (fibrils) K _i (nM) alpha-synuclein (sonicated fibrils) K _i (nM) tau46 (fibrils) 2 ND < 2 9 3 <0.1 3 4 4 8 13 31 5 27 >60 ND 6 24 >300 ND 8 43 ND ND 9 2 < 4 28 10 34 25 >100 12 47 9 ND 12* 42 ND ND 17 ND 2 1

* repetition

Table 4b: Competitive assay protocol 3b (reference ligand [ ^3H ]-compound 1 (1 nM)) Conn. No. K _i (nM) alpha-synuclein (sonicated fibrils) 2 0.3 1 0.4 9 2.1 56 2.6 20 3.1 57 3.3 58 5.1 16 5.2 15 6.7

Table 5: Competition assay for protocol 3b (reference ligand [ ^3H ]-compound 2 (1 nM))

The results of various competition assays indicate high binding affinity to alpha-synuclein fibrils for the tested compounds (lower K _i values indicate higher binding affinity). In the case of the competition assay against compound 1 (Table 4a), similar K _i values for alpha-synuclein fibrils and tau46 fibrils indicate similar selectivity as compound 1 for compounds with higher K _i values for tau46 fibrils rather than alpha fibrils -synuclein, where data suggest further improvement in selectivity.

In addition, compounds 63, 64 and 65 were tested in direct comparison with the corresponding compounds containing a nitrogen atom in one of the phenyl rings. While Table 6 gave a _Ki value of 0.4 nM for compound 1, the _Ki value for compound 65 (which lacks a nitrogen atom on the phenyl ring) was much higher (1.9 nM), indicating significantly improved binding affinity for compound 1. Analysis of binding properties based on K _i values showed significantly improved binding affinity for N-containing compounds. The K _i values obtained for the test compounds in the competitive binding assay with the reference ligand [ ³ H] compound 1 (1 nM) are shown in Table 6.

Example 3: Therapeutic effect in cell culture

To identify potential therapeutically beneficial effects, the compounds are tested in two cellular models of aggregated alpha-synuclein-dependent toxicity in H4 cells. These cell models allow for inducible overexpression of alpha-synuclein half-Venus (V1S+SV2) or full-length alpha-synuclein fusion constructs (model detailed in: Bartels M, Weckbecker D, Kuhn PH, Ryazanov S, Leonov A, Griesinger C, Lichtenthaler SF , Bötzel K, Giese A: Iron-mediated aggregation and toxicity in a novel neuronal cell culture model with inducible alpha-synuclein expression , Sci 2019 Jun 24; In addition, the addition of DMSO and FeCl ₃ promotes the aggregation of alpha-synuclein, which is associated with increased cytotoxicity. Cell culture assays exploit these effects by detecting changes in cell number and changes in the proportion of condensed nuclei, indicative of apoptosis, to provide information about which compounds show the most promising effects and thus may be therapeutically useful.

The data are summarized below in Table 7. In these experiments, cells were incubated with 100 μM FeCl ₃ and 0.75% DMSO in the presence of 10 μM anle 138c as a positive control, compounds 1-19 (10 μM) or DMSO as a negative control. After 48 h, cells were imaged by the OPERA high-throughput imaging system and analyzed using Acapella software (Perkin Elmer). The table shows the change in the number of cells and the change in the proportion of cells with condensed nuclei. (i.e. apoptotic cells) compared to DMSO control. A decrease in the proportion of condensed nuclei (in the absence of a strong decrease in the number of cells) indicates a favorable therapeutic effect.

Table 7: Therapeutic effects of the compound in cell models V1S+SV2 (H4 cells) Alpha-synuclein (H4 cells) Conn. No. Change in cell number (relative to DMSO control) [%] Change in the proportion of condensed nuclei (relative to DMSO control) [%] Change in cell number (relative to DMSO control) [%] Change in the proportion of condensed nuclei (relative to DMSO control) [%] 1 -21.2 -11.7 -27.3 -65.4 2 -25.8 -49.4 -44.2 -79.0 3 -11.3 -31.9 -34.8 -79.1 4 -36.9 33.7 -48.6 -30.1 5 -12.4 -27.3 -14.6 -34.2 6 -34.0 53.6 -31.0 5.4 7 -30.8 -46.1 -37.8 -62.5 8 -4.5 14.2 -5.7 10.7 9 -22.5 -31.8 -35.3 -23.8 10 -32.8 -50.3 -35.3 -60.2 eleven -17.4 123.2 -20.0 13.7 12 -22.2 24.9 -9.8 28.4 13 -6.1 46.0 -18.1 -55.5 14 -21.9 61.0 -32.0 -47.3 15 -11.3 23.1 -11.2 20.3 16 -40.2 56.8 -47.1 0.8 17 -8.2 -38.3 -28.9 -55.0 18 -11.8 -19.9 -21.3 -18.2 19 -5.7 3.2 -30.3 -13.2

Example 4: In vivo biodistribution of [ ¹¹ C]-labeled compound 1 and compound 2

[ ¹¹ C]-labeled compound 1 and compound 2, respectively, were administered intravenously into the tail vein of mice. The mice are then imaged using a small animal PET scanner. Good penetration of the blood-brain barrier with SUV values >1.5 was observed for both compounds. In addition, rapid washout from the brain was found. The half-lives for [ ¹¹ C]-labeled compound 1 and compound are 12 minutes and 9 minutes, respectively.

Example 5: Autoradiography Using Human Brain Tissue

Autoradiography is performed on histological sections of frozen brain tissue using tritiated compound 1 and brain tissue obtained from the cingulate gyrus of a patient with dementia with Lewy bodies. When using [ ³ H]-compound 1 at a concentration of 3 nM for tissue incubation followed by washing steps, preferential binding to gray matter can be observed, which is a pattern identical to the known distribution of aggregated alpha-synuclein (Figure 1, left panel). When specific binding is blocked by an excess of non-tritiated (ie, cold) Compound 1, no or very weak nonspecific binding of Compound 1 to brain tissue can be seen (Figure 1, right panel).

These data demonstrate that Compound 1 binds specifically and with high affinity to pathologically aggregated alpha-synuclein present in a human patient with synucleinopathy and allows the diagnostic detection of aggregated alpha-synuclein.

Claims (61)

1. A compound represented by the general formula Ia, Ib, IIa or IIb: or its salt, Where selected from And And selected from , And ; Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ are independently selected from CR ³ and N provided that at least one of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represents N, and provided that in formulas Ia or Ib at least one of Y ¹ , Y ³ , Y ⁴ and Y ⁶ represents N; R ¹ is independently selected from hydrogen, C _1-4 alkyl and -(CH ₂ )-OP(=O)(OR)(OR), wherein C _1-4 alkyl may be optionally substituted with one or more halogens; R ² is independently selected from hydrogen, halogen and C _1-4 alkyl, wherein C _1-4 alkyl may be optionally substituted with one or more halogens; R represents hydrogen or a cation; R ³ represents hydrogen; R ⁴ and R ⁵ are independently selected from H and C _1-4 alkyl, wherein the C _1-4 alkyl may be optionally substituted with one or more halogens or wherein R ⁴ and R ⁵ together with the nitrogen atom to which they are bonded form a 4- A 6-membered saturated heterocyclic ring selected from azetidinyl, piperidinyl, piperazinyl and morpholinyl, wherein the 4-6 membered saturated heterocyclic ring may be optionally substituted with one or more R ⁶ ; R ⁶ is independently selected from halogen and C _1-4 alkyl, wherein C _1-4 alkyl may be optionally substituted with one or more halogens; And Hal is a halogen, or the connection is provided that the connection does not constitute . 2. The connection according to claim 1, characterized in that represents or represents or , characterized in that R ¹ and R ² are as defined in paragraph 1. 3. The connection according to claim 2, characterized in that represents or represents , characterized in that R ¹ and R ² are as defined in paragraph 1. 4. Connection according to any one of paragraphs. 1-3, characterized in that one, or two, or three of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represent N. 5. Connection according to any one of paragraphs. 1-4, characterized in that in formulas Ia or Ib, Y ¹ represents N and at least one of Y ³ , Y ⁴ and Y ⁶ represents N; or in formulas 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. 6. Connection according to any one of paragraphs. 1-5, characterized in that Y ¹ represents N and Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represent CR ³ , preferably Y ¹ represents N and Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represent CH. 7. Connection according to any one of paragraphs. 1-6, characterized in that R ² represents H. 8. Connection according to any one of paragraphs. 1-7, characterized in that at least one of R ⁴ and R ⁵ represents C _1-4 alkyl, wherein C _1-4 alkyl may be optionally substituted by one or more halogens. 9. A compound represented by the general formula Ia, Ib, IIa or IIb: or its salt, Where selected from And And selected from , And ; Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ are independently selected from CR ³ and N provided that at least one of Y ¹ , Y ² , Y ³ , Y ⁴ , Y ⁵ , Y ⁶ , Y ⁷ and Y ⁸ represents N, and provided that in formulas Ia or Ib at least one of Y ¹ , Y ³ , Y ⁴ and Y ⁶ represents N; R ¹ is independently selected from hydrogen, C _1-4 alkyl and -(CH ₂ )-OP(=O)(OR)(OR), wherein C _1-4 alkyl may be optionally substituted with one or more halogens; R ² is independently selected from hydrogen, halogen and C _1-4 alkyl, wherein C _1-4 alkyl may be optionally substituted with one or more halogens; R represents hydrogen or a cation; R ³ represents hydrogen; R ⁴ and R ⁵ are independently selected from H and C _1-4 alkyl, wherein the C _1-4 alkyl may be optionally substituted with one or more halogens or wherein R ⁴ and R ⁵ together with the nitrogen atom to which they are bonded form a 4- 6-membered saturated heterocyclic ring selected from azetidinyl, piperidinyl and morpholinyl; And Hal is a halogen, characterized in that the compound is detectably labeled with ¹⁸ F, ¹¹ C, ¹²⁵ I, ¹²³ I, ¹³¹ I, ⁷⁷ Br and ⁷⁶ Br. 10. The compound according to claim 9, characterized in that the compound is detectably labeled with ¹⁸ F or ¹¹ C. 11. Diagnostic composition for diagnosing a disease associated with alpha-synuclein aggregation, containing a diagnostically effective amount of a compound defined in any one of paragraphs. 1-9, or salts thereof, and optionally a pharmaceutically acceptable carrier. 12. A pharmaceutical composition for the treatment of a disease associated with alpha-synuclein aggregation, containing a therapeutically effective amount of a compound defined in any one of paragraphs. 1-8, or salts thereof, and optionally a pharmaceutically acceptable carrier. 13. Connection according to any one of paragraphs. 1-9 or a salt thereof for use in the diagnosis of a disease associated with alpha-synuclein aggregation. 14. Connection according to any one of paragraphs. 1-8 or a salt thereof for use in the treatment of a disease associated with alpha-synuclein aggregation. 15. The compound for use according to claim 13 or 14, characterized in that the disease associated with alpha-synuclein aggregation is selected from Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. 16. Use of the compound according to any one of paragraphs. 1-9 or salts thereof for inhibiting alpha-synuclein aggregation in vitro . 17. Connection according to any one of paragraphs. 1-9 or a salt thereof for use in visualizing alpha-synuclein aggregates. 18. A method for visualizing deposits of aggregated alpha-synuclein, where the method includes the following steps: (i) administering to the subject a detectable amount of the detectably labeled compound of claim 9 to the subject; (ii) allowing sufficient time for the compound to bind to the aggregated alpha-synuclein; And (iii) detection of a compound associated with aggregated alpha-synuclein. 19. A method for producing a detectably labeled compound according to claim 9 or a salt thereof, where the method includes the interaction of two precursor compounds, where the first precursor compound is a compound according to any one of claims. 1-8 or a salt thereof, and the second precursor compound is 2-[ ¹⁸ F]fluoroethyl tosylate, TBA[ ¹⁸ F], [ ¹¹ C]MeI, [ ¹¹ C]CH ₂ O or 99% tritium gas/Kerr catalyst. 20. A method for producing a detectably labeled compound according to claim 9 or a salt thereof, where the method includes the interaction of two precursor compounds, where the first precursor compound is compound 24 or a salt thereof, and the second precursor compound is ¹⁸ FK.