Novel tracer for the in vivo detection of the aggregation of alpha-synuclein

Abstract

The present invention discloses a compound represented by Formula (I): (I) or a radioactively labelled compound or pharmaceutically acceptable salt thereof for use as radioctive tracer compounds in monitoring a disease therapy in an individual. The present invention also discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof or a composition for use in in vivo monitoring of an early stage of diseases, preferably Parkinson's disease, Multiple System Atrophy, Dementia with Lewy Bodies.

Description

TITLE: Novel Tracer For The in vivo Detection Of The Aggregation Of Alpha-SynucleinFIELD OF INVENTION

[0001] The present invention relates to a compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof for use as radioactive tracer compounds in monitoring a disease therapy such as Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies in an individual. The present invention also relates to a composition for use in aforementioned diseases in an individual. The present invention further relates to a method for making a radioactively labelled compound.BACKGROUND OF THE INVENTION

[0002] Parkinson’s disease (PD) is a progressive disorder of degeneration of nerve cells in the substantia nigra, which controls movement. These nerve cells die or become impaired, losing the ability to produce an important chemical called dopamine. Parkinson's is the second-most common neurodegenerative disease after Alzheimer's disease. More than 10 million people worldwide are living with PD out of which approx., one million patients are only from the USA. These 1 million patients ae expected to be 1.2 million by 2030.

[0003] At present, in Europe alone, the proportion of people suffering from Parkinson’s disease (PD) is 1.6% for people of >65 years and up to 3% for people > 80 years. As the average age of the population in the EU continues to rise, the overall number of PD patients also increases, placing a significant burden on society due to associated healthcare costs.

[0004] Neurodegenerative diseases such as Parkinson’s disease (PD) are manifested by inclusion bodies of alpha- sy nuclein (a-syn) also called a-synucleinopathies.

[0005] McCann, H.; Stevens, C.; Cartwright, H ; Halliday, G. a-Synucleinopathy phenotypes. Park. Relat. Disord. 2014, 20, S62-S67 discloses that abnormal alpha-synuclein (a-syn) depositions (a- synucleinopathies) in neurons, nerve fibers, or glia cells are the hallmark for neurodegenerative diseases such as Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA).

[0006] Spela Korat et. Al. Alpha-Synuclein PET Tracer Development — An Overview about Current Efforts, Pharmaceuticals 2021, 14, 847, (https: / / doi.org / 10.3390 / phl4090847) mentions, the clinical diagnosis of PD is not straightforward in the early phases of the illness which seriously complicates the identification of targets for novel therapies and the interpretation of treatment outcomes. It is imperative to correctly diagnose the illness as early as possible since early intervention is key to limiting the neurodegenerative process. The pathological characteristic of PD is deposition of a-syn protein in the brain followed by dopaminergic neuronal loss.

[0007] Currently, a-syn deposition cannot be assessed accurately in vivo. Only histological examination of post-mortem brain tissue can detect these inclusions so far. The possibility of non-invasively detecting a- syn therefore provides valuable insights into the disease progression of a-synucleinopathies. In particular, a-syn imaging can quantify changes in monomeric, oligomeric, and fibrillic a-syn over time and improve early diagnosis of various a-synucleinopathies or monitor treatment progress.

[0008] Spela Korat et. al. indicates, positron emission tomography (PET) is a non-invasive in vivo imaging technique that can quantify target expression and drug occupancies when a suitable tracer exists. Positron emission tomography (PET) molecular imaging has the potential to reveal the pathogenesis of brain disorders when suitable tracers are available. PET can localize and quantify drug targets, monitor treatment effect, or image disease pathophysiology on a molecular basis. Various studies have indicated toward the importance of imaging with respect to patient inclusion in clinical trials, especially for PD.

[0009] Although, there is no clinical positron emission tomography (PET) tracer available and therefore a- synucleinopathies (e.g., Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies) cannot be diagnosed in vivo. Developing a first PET tracer for a-synucleinopathies would revolutionize the field because we could diagnose the disease at an early stage and consequently create a potential market for monitoring the efficacy of therapy.

[0010] As such, novel a-syn PET tracers are highly sought after but the development thereof is reasonably challenging. For instance, the low abundance of a-syn within the brain necessitates the development of a high-affinity ligand. Moreover, a-syn depositions are, in contrast to amyloid proteins, predominantly localized intracellularly, limiting their accessibility. Furthermore, another challenge is the ligandselectivity over structurally similar amyloids such as amyloid-beta or tau, which are often co-localized with a-syn pathology. The lack of a defined crystal structure of a-syn has also hindered rational drug and tracer design efforts.[Oil] Therefore, to overcome the above-mentioned challenges, there was a need to develop a PET tracer which could be used as a diagnostic imaging agent for in vitro and in vivo imaging of alpha-synuclein to identify a-synucleinopathies and can be utilized for early diagnosis of neurogenerative diseases such as Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies.OBJECTIVE OF THE INVENTION

[0012] Main objective of the present invention is to provide compounds, preferably radioactively labelled compounds.

[0013] Another objective of the present invention is to provide compounds, preferably radioactively labelled compound for monitoring a disease therapy such as Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies in an individual.

[0014] Yet another objective of the present invention is to provide a composition for use in Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies in an individual.

[0015] Still another objective of the present invention is to provide a method for synthesizing the radioactively labelled compound for monitoring a disease therapy such as Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies in an individual.

[0016] Yet another objective of the present invention is to provide a compound, preferably radioactively labelled compound to diagnose the disease such as Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies at an early stage and consequently create a potential market for monitoring the efficacy of therapy.

[0017] Still another objective of the present invention is to provide compounds, preferably radioactively labelled compounds to detect alpha synuclein in vitro and in vivo in humans.SUMMARY OF THE INVENTION

[0018] Main aspect of the present invention provides a compound represented by Formula (I):Formula (I) or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein:Xi is C or N,B is C or N,Y or Z is independently selected from O, N or C,Wherein Zi represents phenyl, pyridyl, pyridone, N-methyl-2-pyridone, optionally substituted with one or more substituents selected from C1-3 alkyl, hydroxy, C1-3 alkoxy, halogen, a mono, bi or tri-fluorinated methoxy group; andRi is selected from

[0019] Another aspect of the present invention provides a compound represented by Formula (I):Formula (I) or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein:Xi is C or N,B is C or N,Y or Z is independently selected from O, N or C,Wherein Zi represents phenyl, pyridyl, pyridone, N-methyl-2-pyridone, optionally substituted with one or more substituents selected from C1-3 alkyl, hydroxy, C1.3 alkoxy, halogen, a mono, bi or trifluorinated methoxy group; andRi is selected from

[0020] Another aspect of the present invention provides a compound represented by Formula (II):10 Formula II or a radioactively labelled compound or pharmaceutically acceptable salt thereof,Wherein:Xi or X2 each independently selected from C or N,Y or Z or B is independently selected from N or C,R9is H or CH3Rio is H or FR11 is H, CH3 or a mono, bi or tri-fluorinated methyl group.

[0021] Another aspect of the present invention provides a compound represented by Formula (II):Formula II or a radioactively labelled compound or pharmaceutically acceptable salt thereof,Wherein:Xi or X2 each independently selected from C or N,Y or Z or B is independently selected from N or C,R9is H or CH3RIO is H or FRn is H, CH3or a mono, bi or tri-fluorinated methyl group; provided that if Y = N, Z=O, B=C, and X1=C, then R8 is not

[0022] Yet another aspect of the present invention provides a compound represented by Formula (III):Formula IIIR13 is H, CH3 or a mono, bi or tri-fluoro methyl group.

[0023] Still another aspect of the present invention provides compounds, preferably radioactively labelled compound for monitoring a disease therapy such as Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies in an individual.

[0024] Yet another aspect of the present invention provides a composition for use in Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies in an individual.

[0025] Still another aspect of the present invention provides a method for synthesizing the radioactively labelled compound for monitoring a disease therapy such as Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies in an individual.

[0026] Yet another aspect of the present invention provides a compound, preferably radioactively labelled compound to diagnose the disease such as Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies at an early stage and consequently create a potential market for monitoring the efficacy of therapy.

[0027] Still another aspect of the present invention provides compounds, preferably radioactively labelled compounds to detect alpha synuclein in vitro and in vivo in humans.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Fig 1 : Represents immunohistochemistry of MSA (Putamen region) (Human tissue) with pSerl259 a-syn antibody. Top part of the tissue represents presence of a-syn pathology (“rich a-syn region”). Bottom part of the tissue shows that there is no a-syn pathology (“poor a-syn region”).

[0029] Fig 2: Represents MSA (Putamen region) (Human tissue), ARG Small molecule SKP-02, demonstrating increased binding of SKP02 to alpha-synuclein rich regions compared to alpha-synuclein poor regions.

[0030] Fig 3: Represents MSA (Putamen region) (Human tissue), ARG Small molecule SKP-03, demonstrating increased binding of SKP03 to alpha-synuclein rich regions compared to alpha-synuclein poor regions.

[0031] Fig 4: Represents MSA (Putamen region) (Human tissue), ARG Small molecule SKP-05, demonstrating increased binding of SKP05 to alpha-synuclein rich regions compared to alpha-synuclein poor regions.

[0032] Fig 5: Represents MSA (Putamen region) (Human tissue), ARG Small molecule SKP-07, demonstrating increased binding of SKP07 to alpha-synuclein rich regions compared to alpha-synuclein poor regions.

[0033] Fig 6: Represents MSA (Putamen region) (Human tissue), ARG Small molecule SKP-08, demonstrating increased binding of SKP08 to alpha-synuclein rich regions compared to alpha-synuclein poor regions.

[0034] Fig 7: Left: overview of autoradiography of small molecules SKP-02, SKP-03, SKP-05, SKP-07, SKP-08 on PD (Substantia Nigra region) (Human tissue). Right: Represents immunohistochemistry ofPD (Substantia Nigra region) (Human tissue) with pSerl259 a-syn antibody. Middle part of the tissue (‘Mid- High pathology) represents presence of a-syn pathology (“rich a-syn region”). Side parts of the tissue (‘No pathology") shows that there is no a-syn pathology (“poor a-syn region”).

[0035] Fig 8: Represents PD (Substantia Nigra region) (Human tissue), ARG Small molecule SKP-02, demonstrating increased binding of SKP02 to alpha-synuclein rich regions compared to alpha-synuclein poor regions.

[0036] Fig 9: Represents PD (Substantia Nigra region) (Human tissue), ARG Small molecule SKP-03, demonstrating increased binding of SKP03 to alpha-synuclein rich regions compared to alpha-synuclein poor regions. A relatively high nonspecific binding is present.

[0037] Fig 10: Represents PD (Substantia Nigra region) (Human tissue), ARG Small molecule SKP-05, demonstrating increased binding of SKP05 to alpha-synuclein rich regions compared to alpha-synuclein poor regions.

[0038] Fig 11 : Represents PD (Substantia Nigra region) (Human tissue), ARG Small molecule SKP-07, demonstrating marginally increased binding of SKP07 to alpha-synuclein rich regions compared to alpha- synuclein poor regions A relatively high nonspecific binding is present.

[0039] Fig 12: Represents PD (Substantia Nigra region) (Human tissue), ARG Small molecule SKP-08, demonstrating increased binding of SKP08 to alpha-synuclein rich regions compared to alpha-synuclein poor regions A relatively high nonspecific binding is present.

[0040] Fig 13: Top: overview of autoradiography of small molecules SKP-02, SKP-03, SKP-05, SKP-07, SKP-08 on PSP (Substantia Nigra) (Human tissue). Bottom: Represents immunohistochemistry of PSP (Substantia Nigra) (Human tissue) with AT8. The top part of the tissue (‘Mid-High pathology) represents presence of tau pathology (“rich tau region”). Bottom part of the tissue (‘No pathology") shows that there is no tau pathology (“poor tau region”).

[0041] Fig 14: Represents PSP (Substantia Nigra) (Human tissue), ARG Small molecule SKP-02, demonstrating binding of SKP02 to Tau-protein aggregates rich regions compared to Tau-protein aggregates poor regions.

[0042] Fig 15: Represents PSP (Substantia Nigra) (Human tissue), ARG Small molecule SKP-03, demonstrating binding of SKP03 to Tau-protein aggregates rich regions compared to Tau-protein aggregates poor regions.

[0043] Fig 16: Represents PSP (Substantia Nigra) (Human tissue), ARG Small molecule SKP-05, demonstrating low binding of SKP05 to Tau-protein aggregates rich regions compared to Tau-protein aggregates poor regions.

[0044] Fig 17: Represents PSP (Substantia Nigra) (Human tissue), ARG Small molecule SKP-07, demonstrating binding of SKP07 to Tau-protein aggregates rich regions compared to Tau-protein aggregates poor regions A relatively high nonspecific binding is present.

[0045] Fig 18: Represents PSP (Substantia Nigra) (Human tissue), ARG Small molecule SKP-08, demonstrating binding of SKP08 to Tau-protein aggregates rich regions compared to Tau-protein aggregates poor regions.

[0046] Fig 19: Top: overview of autoradiography of small molecules SKP-02, SKP-03, SKP-05, SKP-07, SKP-08 on AD (Putamen) (Human tissue). Bottom: represents immunohistochemistry of AD (Putamen) (Human tissue) with 4G8 antibody. There are areas with high pathology of A0 (‘ A[3 +”) and low pathology of Ap (‘A -”).

[0047] Fig 20: Represents overview of autoradiography of small molecules SKP-02, SKP-03, SKP-05, SKP-07, SKP-08 on LBV (Putamen) (Human tissue).

[0048] Fig 21: Represents in vivo study - WT C57B16 / J, Dynamic Reconstruction. Time activity curves of Brain uptake of1^-labelled SKP02, SKP03, SKP05, SKP07, SKP08 and SKP12, Demonstrating high brain uptake and fast washout for1^-labelled SKP02 and SKP-05, moderate wash-out fornC-labelled SKP08 and SKP03. FornC-labelled SKP07 no washout was observed.nC-labelled SKP12 had limited uptake in the brain.

[0049] Fig 22: In vitro binding assay results on alpha-synuclein and amyloid-beta recombinant fibrils. Binding affinities of new compounds against [3H]alpha synuclein reference. Other benchmark compounds (e.g. SIL26, MODAGOOl and PIB) were tested in the assay.

[0050] Fig 23: in vitro saturation binding curves for [3H]SKP-08 on amyloid beta fibrils.

[0051] Fig 24: Represents in vitro autoradiography using3H-21 and fibril injected mouse model

[0052] Fig 25: Represents in vitro autoradiography using3H-SKP02,03,05,7, 08 and 18F-ACI12589.

[0053] Fig 26: Represents in vitro autoradiography in PD tissue

[0054] Fig 27: Represents in vitro autoradiography in MSA tissue.

[0055] Fig 28: Represents in vitro autoradiography in PSP tissue.

[0056] Fig 29: Represents in vitro autoradiography in AD tissue.DETAIL DESCRIPTION OF THE INVENTION

[0057] The term "pharmaceutically acceptable" is used to specify that an object (for example a salt, dosage form, excipient) is suitable for use in patients.

[0058] The term “Radioactively labelled compound” includes any compound that has been joined with a radioactive substance.

[0059] The term “Phenyl” includes a univalent hydrocarbon radical formally derived from benzene by the removal of a hydrogen atom.

[0060] The term “C1.3 alkyl” includes Cl alkyl (methyl), C2 alkyl (ethyl) and C3 alkyl (propyl as n-propyl and isopropyl). In one embodiment, the C1-3 alkyl is methyl.

[0061] The term “C1.3 alkoxy” includes Cl alkoxy (methoxy), C2 alkoxy (ethoxy) and C3 alkoxy (propoxy as n-propoxy and isopropoxy). In one embodiment, the C1-3 alkoxy is methoxy.

[0062] The term “Halogen” includes a group in the periodic table consisting of six chemically related elements: fluorine (F), chlorine (Cl), bromine (Br), iodine (I), astatine (At), and tennessine (Ts).

[0063] The term “Radioactive tracer compounds” are chemical compounds in which one or more atoms have been replaced by a radionuclide.

[0064] The term "therapy" is intended to have its normal meaning of dealing with a disease in order to entirely or partially relieve one, some or all of its symptoms, or to correct or compensate for the underlying pathology. The term "therapy" also includes "prophylaxis" unless there are specific indications to the contrary. The terms "therapeutic" and "therapeutically" should be interpreted in a corresponding manner.

[0065] The term "treatment" is used synonymously with "therapy". Similarly, the term "treat" can be regarded as "applying therapy" where "therapy" is as defined herein.

[0066] Main embodiment of the present invention discloses a compound represented by Formula (I):Formula (I) or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein:Xi is C orN,B is C or N,Y or Z is independently selected from O, N or C,Wherein Zi represents phenyl, pyridyl, pyridone, N-methyl-2-pyridone, optionally substituted with one or more substituents selected from C1-3 alkyl, hydroxy, C1-3 alkoxy, halogen, a mono, bi or tri -fluorinated methoxy group; and

[0067] Another embodiment of the present invention discloses a compound represented by Formula (I):Formula (I) or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein:Xi is C or N,B is C or N,Y or Z is independently selected from O, N or C,Wherein Zi represents phenyl, pyridyl, pyridone, N-methyl-2-pyridone, optionally substituted with one or more substituents selected from C1-3 alkyl, hydroxy, C1.3 alkoxy, halogen, a mono, bi or trifluorinated methoxy group; andor

[0068] The compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein Zi represents phenyl, pyridyl, pyridone, N-methyl-2-pyridone optionally substituted with one or more substituents selected from methyl, methoxy, hydroxy, flourine, iodine.

[0069] Another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula la:laWhereinXi or X2 each independently selected from C or N,R2is H or CH3R3 is H or FR4 is H or CH3 or a mono, bi or tri -fluorinated methyl group.

[0070] Yet another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the compound is represented by Formula la:WhereinXi or X2 each independently selected from C or N,R2 is H or CH3R3 is H or FR4 is H or CH3 or a mono, bi or tri-fluorinated methyl group, provided that if X1=C, then R1 is not

[0071] Yet another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein the compound is represented by FormulalbWherein R5 or Re each independently selected from I, OCH3 or a mono, bi or tri-fluorinated methoxy group.

[0072] Still another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula IcIcWhereinY or Z each independently selected from O, N or C R7 is H, CH3 or a mono, bi or tri-fluorinated methyl group.

[0073] Yet another embodiment of the present invention discloses a compound represented by Formula (II):Formula II or a radioactively labelled compound or pharmaceutically acceptable salt thereof,Wherein: Xi or X2 each independently selected from C or N,Y or Z or B is independently selected from N or C,R9is H or CH3Rio is H or FRu is H, CH3 or a mono, bi or tri-fluorinated methyl group.

[0074] Yet another embodiment of the present invention discloses a compound represented by Formula (II):Formula II or a radioactively labelled compound or pharmaceutically acceptable salt thereof, Wherein: Xi or X2 each independently selected from C or N,Y or Z or B is independently selected from N or C,R9is H or CH3Rio is H or FR11 is H, CH3 or a mono, bi or tri-fluorinated methyl group; provided thatif Y = N, Z=O, B=C, and X1=C, then R8 is not

[0075] Yet another embodiment of the present invention discloses a compound represented by Formula (III):R13 is H, CH3 or a mono, bi or tri-fluoro methyl group.

[0076] Still another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein the compound is selected from the group comprising of:

[0077] Still another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein the compound is selected from the group comprising of:

[0078] Yet another embodiment of the present invention discloses a compound represented by Formula (I):Formula (I) or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein:Xi is C or N,B is C or N,Y or Z is independently selected from O, N or C,Wherein Zi represents phenyl, pyridyl, pyridone, N-methyl-2-pyridone, optionally substituted with one or more substituents selected from C1-3 alkyl, hydroxy, C1.3 alkoxy, halogen, a mono, bi or trifluorinated methoxy group; and

[0079] Yet another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein Zi represents phenyl, pyridyl, pyridone, N-methyl-2-pyridone optionally substituted with one or more substituents selected from methyl, methoxy, hydroxy, flourine, iodine.

[0080] Y et another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula la:WhereinXi or X2each independently selected from C or N,R2is H or CH3Rj is H or FR4 is H or CH3 or a mono, bi or tri-fluorinated methyl group.

[0081] Yet another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula lb:Wherein R5 or Re each independently selected from OCH3 or a mono, bi or tri-fluorinated methoxy group.

[0082] Yet another embodiment of the present invention discloses a compound represented by Formula (II):Formula II or a radioactively labelled compound or pharmaceutically acceptable salt thereof, Wherein:Xi or X2 each independently selected from C or N, Y or Z or B is independently selected from N or C,R9is H or CH3RIO is H or F Rn is H, CH3or a mono, bi or tri-fluorinated methyl group;

[0083] Yet another embodiment of the present invention discloses a compound represented by Formula (III):R13 is H, CH3 or a mono, bi or tri-fluoro methyl group.

[0084] Yet another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein the compound is selected from the group comprising of:

[0085] Yet another embodiment of the present invention discloses the compound or pharmaceutically acceptable salt thereof, wherein the radioactively labelled compounds are radiolabeled with Carbon- 11 (nC), Fluorine-18 (18F) or Tritium (3H) or a radionuclide of Iodine.

[0086] Still another embodiment of the present invention discloses a composition comprising a compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.

[0087] Yet another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof or a composition, for use as radioctive tracer compounds in monitoring a disease therapy in an individual.

[0088] Still another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof or a composition, for use in the monitoring of an early stage of diseases, preferably Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies.

[0089] Yet another embodiment of the present invention discloses the compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof or a composition, for use in in vivo monitoring of an early stage of diseases, preferably Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies.

[0090] Still another embodiment of the present invention discloses a method for making a radioactively labelled compound, wherein radioactively unlabelled variant of compounds of claims 1 to 8 are radiolabelled with Carbon- 11 (nC), Fluorine- 18 (18F), or Tritium (3H), or a radionuclide of Iodine.

[0091] Yet another embodiment of the present invention discloses a method for detection of the aggregation of alpha-synuclein protein, comprising contacting said protein with an amount of radioactively labelled compound which is sufficient to be detected by Positron Emission Tomography (PET) by forming at least one PET image and determining the aggregation of alpha-synuclein protein by observing the image.

[0092] Still another embodiment of the present invention discloses a compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof or a composition, for use in method of treating a disease selected from Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies.EXPERIMENTAL SECTION;Materials, Instruments and Sources

[0093] Chemicals and solvents were purchased from commercial suppliers and used as received. When necessary, the reaction was performed in oven-dried glassware. Thin-layer chromatography (TLC) was performed on 5 x 20 cm plates with a layer thickness of 0.25 mm (silica gel 60 F254), were developed with ninhydrin. All the compounds used for the binding assays were >95% pure as determined by nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography-mass spectrometry (LC-MS) and electrospray ionization-high resolution mass spectrometry (HR-MS (ESI)), 'll NMR (500 MHz) and13C NMR (MHz) were performed on NMR spectrometers with chemical shifts (8) reported as parts per million (ppm) relative to the solvent CDCh: 'H 7.26ppm,13C 77.16ppm, (CDa^SO: 1H 2.50ppm, MeOH, ACN.

[0094] Splitting patterns are indicated as following s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublet), and br (broad singlet). Coupling constant is reported in hertz (Hz). Spectrums were acquired with MestReNova, version 14.1.1-24571. Liquid chromatography-mass spectrometry (LC / MS) results were obtained using the Shimadzu LCMS-2010 EV equipped with a LC- 20 AD pump system, SPD-m20A diode array detector and a Xbridge C18 5pM column (100 mm x 4.6 mm). The mobile phases involved were H2O with 0.1% TFA (Solvent A) and ACN with 0.1% TFA (Solvent B). The following method of 8 minutes was used for measurements: A linear gradient of 5% solvent B : 95% solvent A to 90% solvent B : 10% solvent A over 4.5 minutes, then 1.5 minutes isocratic flow with 90% solvent B : 10% solvent A, followed by a linear gradient of 90% solvent B : 10% solvent A to 5% solvent B : 95% solvent A for 0.5 minutes and at last 1.5 minutes isocratic flow with 5% solvent B : 95% solvent A. The chromatograms were detected on UV wavelengths 254 nm. The microwave reaction, Electrospray ionization-high resolution mass spectrometry (HR-MS (ESI)) were carried out using a Bruker micro TOF-Q instrument in a positive ion mode (capillary potential of 4500V) and interpreted with Bruker Compass Data Analysis 4.0 software. Flash chromatography separation was performed onBtichi Switzerland (Flawil, Switzerland), Sepacore system (comprising a C-620 control unit, a C-660 fraction collector, two C-601 pumps and a C-640 UV detector) equipped with Buchi Sepacore pre-packed flash columns. Chromatograms were acquired with SepacoreControl 1.2 4000.0 Standard Edition. Yields refer to purified and spectroscopically pure compounds. Methods used in flash column chromatography were based on linear gradient. Preparative HPLC was performed on Jasco PU-2089 / PU- 2087 Plus station (Easton, Maryland, USA) accompanied with Jasco UV-2075 Plus UV detector (254nm) and Nal radioactivity detector (Raytest, Straubenhardt, Germany) with an Alltima Cl 8 10pm (250 x 10mm) column and 254nm. Chromatograms were acquired with Chrom NAV CFR-FES software. Method used in preparative HPLC system was XX.General procedure for the synthesis of nitriles (2, 6, 10, 14, 17)Procedure a:1, 8, 0, 13 2, e, 10, 14, 17

[0095] This general procedure was based on Damont A. et al. [https: / / doi.org / 10.1021 / acs.jmedchem.5b00932]. To anhydrous THF cooled at -60 °C, 1.6 M n- butyllithium solution was added in hexane (100 mmol) and then, cautiously, a solution of anhydrous acetonitrile (100 mmol) in anhydrous tetrahydrofuran was added over a period of 10 min while maintaining the temperature below -50 °C. The mixture was then stirred for 30 min at -60 °C. Then, methyl 4-iodobenzoate (45.8 mmol) dissolved in anhydrous THF was added dropwise to the mixture while maintaining the temperature below -50 °C. Once the addition finished, the reaction mixture was stirred for 1 h at -60 °C and 1 h at -45 °C and monitored by TLC. When no starting material was detected on TLC, the reaction was quenched with water at -40 °C under vigorous stirring. Then, a IM HC1 solution was added to acidify the aqueous layer to pH 2. An inorganic precipitate was filtered off, and the filtrate was extracted three times with ethyl acetate. The combined organic layers were washed with water and brine, dried overNa2SO4, filtered, and concentrated to dryness.General procedure for the synthesis of 3-isoxazol-5-amines (3, 7, 11, 15, 18)Procedure b :2, 6, 10, 14, 17 3, 7, 11, 15, 18

[0096] This general procedure was based on Ge, Y. et rz / .[https: / / doi.org / 10.1021 / acs.orglett.8b00971], NH2OH HCl (3.0 mmol) and NaOAc (3.0 mmol) were stirred in MeOH at room temperature for 1 hour and then / Lketonitrile (1.0 mmol) was added to the mixture. The reaction mixture was continued to react until the starting material was consumed completely, shown by TLC detection. Then water was added toquench the reaction and product was extracted three times with ethyl acetate. The combined the organic phases were washed with brine, dried over NasSCh, filtered, and concentrated to dryness. If needed, the crude reaction mixture was purified by column chromatography on silica gel to get product.General procedure for the synthesis of A-aryl amides (4, 8. 12. 16. 19)Procedure c:3, 7, 11, 15, 18 4, 8, 12, 16, 19

[0097] This general procedure was based on [US2009 / 0069288 Al], 3-(4-iodophenyl)isoxazol-5-amine and anhydrous pyridine were stirred for 15 - 20 min. After that, 4-m ethoxybenzoyl chloride was added. The mixture was heated at 150 °C and monitored by TLC (ninhydrin was used as visualization reagent). Upon completion, reaction was stopped, and solvent was removed under reduced pressure. Then saturated bicarbonate solution was added to quench the reaction and product was extracted with three times with ethyl acetate. The combined organic phases were washed with water, brine, dried over Na2SO4, filtered and concentrated. Flash column chromatography was performed to achieve pure product.General procedure for the synthesis of acetyl chlorides (21, 24, 39, 47, 53, 61)Procedure d:

[0098] In an oven dried flask carboxylic acid was dissolved in anhydrous DCM under argon and a catalytic amount of anhydrous DMF (3-4 drops) were added. To the resulting solution was slowly added oxalyl chloride at 0°C and the reaction mixture was stirred for several hours at room temperature. Solvent was removed under vacuum and the crude product was used directly for the next-step synthesis.

[0099] EXAMPLE 1: Synthesis of compound N-(3-(4-iodophenyl)isoxazol-5-yl)-4-methoxybenzamide (4):a. Synthesis of 3-(4-iodophenyl)-3-oxopropanenitrile (2): The title compound was prepared according to the procedure a. Starting compound 1 (methyl 4-iodobenzoate) (6.0 g, 23.0 mmol), anhydrous THF (60 ml, 0.74 mol), anhydrous ACN (2.6 ml, 50.0 mmol), 1.6 M n-butyllithium solution in hexanes (15 ml, 24.0 mmol) were taken. The product was obtained as an orange solid (6.2 g, yield 86 %).1H NMR (500 MHz, DMSO) 5 7.99 (d, J= 8.5 Hz, 2H), 7.70 (d, J= 8.5 Hz, 2H), 4.75 (s, 2H). 13C NMR (126 MHz, DMSO) 5 189.37 [C], 137.84 [2 X CH], 133.95 [C], 129.97 [2 X CH], 115.72 [C], 103.21 [C], 29.98 [CH2] ppm. HRMS (ESI) m / z calculated for C9H6INO : 270.9494; found 271.9548 [M+H+], b. Synthesis of 3-(4-iodophenyl)isoxazol-5-amine (3): The title compound was prepared according to the procedure b. Starting compound 2 (1 g, 3.7 mmol), NH2OH HCI (0.77 g, 11.0 mmol), NaOAc (0.91 g, 11.0 mmol), MeOH (11.1 ml) were taken. The product was obtained as an orange solid (1.0 g, yield 95%).1H NMR (500 MHz, DMSO 57.81 (d, J= 8.4 Hz, 2H), 7.52 (d, J= 8.4 Hz, 2H), 6.82 (s, 2H), 5.40 (s, 1H). 13C NMR (126 MHz, DMSO) 6 170.97 [C], 161.54 [C], 137.43 [2 X CH], 129.28 [C], 128.01 [2 X CH], 95.93 [C], 74.75 [CH], HRMS (ESI) m / z: calculated for C9H7IN2O: 285.9603; found 286.9663 [M+H+], c. Synthesis of N-(3-(4-iodophenyl)isoxazol-5-yl)-4-methoxybenzamide (4): The title compound was prepared according to the adjusted procedure c. Starting compound 3 (200 mg, 0.70 mmol), anhydrous pyridine (3.5 ml, 43.3 mmol), 4-methoxybenzoyl chloride (189 pL, 1.4 mmol). The mixture was heated at 150 °C for 10 min using microwave. The crude material was purified by flash column chromatography using ethyl acetate / heptane (0% - 100%) as eluent to yield 4 as a beige solid (151.3 mg, yield 52 %).1H NMR (500 MHz, DMSO) 8 11.94 (s, 1H), 8.05 (d, J= 8.9 Hz, 2H), 7.89 (d, J= 8.4 Hz, 2H), 7.69 (d, J= 8.4 Hz, 2H), 7.09 (d, J= 9.0 Hz, 2H), 6.92 (s, 1H), 3.85 (s, 3H). 13C NMR (126 MHz,DMS0- 6) 5 163.00 [C], 162.98 [C], 162.74 [C], 161.90 [C], 137.89 [2 X CH], 130.19 [2 X CH], 128.41 [2 X CH], 128.32 [C], 124.42 [C], 113.90 [2 X CH], 97.03 [C], 86.56 [CH], 55.53 [CH3], HRMS (ESI) m / z : calculated for C17H13IN2O3 : 419.9971; found 421.0049 [M+H+],

[0100] EXAMPLE 2: Synthesis of compound 4-methoxy-N-(3-(6-(pyrrolidin-l-yl)pyridin-3-yl)isoxazol- 5-yl)benzamide (8): a. Synthesis of 3-oxo-3-(6-(pyrrolidin-l-yl)pyridin-3-yl)propanenitrile (6): The title compound was prepared according to the procedure a. Starting compound 5 (methyl 6-(pyrrolidin-l-yl)nicotinate ) (0.9 mg, 4.36 mmol), anhydrous IHF (16.2 ml), anhydrous ACN (0.5 ml, 9.53 mmol), 1.6 M n- butyllithium solution in hexanes (6.0 ml, 9.53 mmol). The crude material was purified by column chromatography using ethyl acetate / n-hexane (1 / 1) as eluents to yield 6 as a beige solid (377 mg, yield 40 %).1H NMR (500 MHz, CDC13) 5 8.68 (d, J= 2.4 Hz, 1H), 7.96 (dd, J= 9.2, 2.4 Hz, 1H), 6.39 (d, J = 9.9 Hz, 1H), 3.93 (s, 2H), 3.56 (d, J = 114.7 Hz, 4H), 2.06 (s, 4H). 13C NMR (126 MHz, CDC13) 8 183.76, 158.98, 151.58, 136.70, 118.94, 114.20, 106.56, 47.18, 28.44. HRMS (ESI) m / z. calculated for C12H13N3O: 215.1059; found 216.1138 [M+H+]; found 238.0956 [M+Na+], b. Synthesis of 3-(6-(pyrrolidin-l-yl)pyridin-3-yl)isoxazol-5-amine (7): The title compound was prepared according to the procedure b. Starting compound 6 (200 mg, 0.93 mmol), NH2OH HC1 (193.7 mg, 2.7 mmol), NaOAc (228.7 mg, 2.7 mmol), MeOH (2.8 ml). The product was obtained as a beige solid (217 mg, yield 94 %). 1H NMR (500 MHz, DMSO) 8 8.40 (d, J = 2.4 Hz, 1H), 7.80 - 7.73 (m, 1H), 6.66 (s, 2H), 6.48 (d, J= 8.1 Hz, 1H), 5.30 (s, 1H), 3.46 - 3.35 (m, 4H), 2.01 - 1.85 (m, 4H). 13C NMR (126 MHz, DMSO) 8 170.45 [C], 160.72 [C], 157.15 [C], 146.18 [2 X CH], 134.40 [2 X CH], 113.22 [C], 106.09 [2 X CH], 74.21 [2 X CH], 46.38 [CH2], 24.93 [CH2], HRMS (ESI) m / z. calculated for C12H14N4O: 230.1168; found 231.1281 [M+H+]; found 253.1099 [M+Na+], c. Synthesis of compound 4-methoxy-N-(3-(6-(pyrrolidin-l-yl)pyridin-3-yl)isoxazol-5-yl)benzamide (8): The title compound was prepared according to the procedure c. Starting compound 7 (20 mg,0.087 mmol), anhydrous pyridine (0.44 ml), 4-methoxybenzoyl chloride (24 pL, 0.174 mmol). The mixture was heated at 150 °C for 10 min. The crude material was purified by flash column chromatography using ethyl acetate / heptane (3 / 1) as eluents to yield 8 as a white solid (30 mg, yield 95 %).1HNMR (500 MHz, DMSO) 5 11.84 (s, 1H), 8.55 (d, J= 2.4 Hz, 1H), 8.05 (d, J= 9.0 Hz, 2H), 7.93 (dd, J= 8.8, 2.4 Hz, 1H), 7.09 (d, J= 9.0 Hz, 2H), 6.82 (s, 1H), 6.54 (d, J= 8.7 Hz, 1H), 3.85 (s, 3H), 3.50 - 3.39 (m, 4H), 2.01 - 1.92 (m, 4H). 13C NMR (126 MHz, DMSO) 8 162.91 [C], 162.69 [C], 162.21 [C], 160.89 [C], 157.40 [C], 146.54 [CH], 134.64 [CH], 130.14 [2 X CH], 124.52 [C], 113.87 [2 X CH], 112.15 [C], 106.39 [CH], 85.86 [CH], 55.52 [CH3], 46.45 [CH2], 24.94 [CH2], HRMS (ESI) m / . calculated for C20H20N403 : 364.1535; found 365.1636 [M+H+]; found 387.1459 [M+Na+],

[0101] EXAMPLE 3: Synthesis of compound 4-methoxy-N-(3-(thiazol-2-yl)isoxazol-5-yl)benzamide ri2 - a. Synthesis of compound 3-oxo-3-(thiazol-2-yl)propanenitrile (10): The title compound was prepared according to the procedure a. Starting compound 9 (methyl thiazole-2-carboxylate)(0.95 mg, 6.64 mmol), anhydrous THF (25 ml), anhydrous ACN (0.76 ml, 14.5 mmol), 1.6 M n- butyllithium solution in hexanes (9.1 ml, 14.5 mmol). The crude material was purified by flash column chromatography using MeOH / DMC (1 / 99) as eluents to yield 10 as an orange solid (0.357 mg, yield 86 %).1H NMR (500 MHz, CDC13) 5 8.06 (d, J= 2.9 Hz, 1H), 7.83 (d, J= 2.9 Hz, 1H), 4.32 (s, 2H). 13C NMR (126 MHz, CDC13) 5 181.29 [C], 163.49 [C], 145.50 [CH], 128.49 [CH], 113.35 [C], 29.11 [CH2], ESI (+)-MS: m / z 153 [M+H+], b. Synthesis of compound 3-(thiazol-2-yl)isoxazol-5-amine (11): The title compound was prepared according to the procedure b. Starting compound 10 (152.2 mg, 1 mmol), NH2OH HC1 (208.5 mg, 3.0 mmol), NaO Ac (246.1 mg, 3.0 mmol), MeOH (3.1 ml). The reaction was monitored by LCMS . The product was obtained as an orange solid (147 mg, yield 88 %).1H NMR (500 MHz, DMSO) 5 8.00 (d, J= 3.2 Hz, 1H), 7.89 (d, J= 3.2 Hz, 1H), 7.03 (s, 2H), 5.43 (s, 1H). 13C NMR (126 MHz, DMSO) 8 171.46 [C], 158.60 [C], 156.83 [C], 143.58 [CH], 121.58 [CH], 74.98 [CH], HRMS (ESI)m / z: calculated for C6H5N3OS: 167.0153; found 190.0056 [M+Na+], c. Synthesis of compound 4-methoxy-N-(3-(thiazol-2-yl)isoxazol-5-yl)benzamide (12): The title compound was prepared according to the procedure c. Starting compound 11 (80 mg, 0.478 mmol), anhydrous pyridine (2.4 ml), 4-methoxybenzoyl chloride (130 pL , 0.957 mmol). The mixture was heated at 150 °C for 10 min. The crude material was purified by flash column chromatography using ethyl acetate / heptane (linear gradient; 2 / 3) as eluents to yield 12 as an orange solid (59 mg, yield 41 %).1H NMR (500 MHz, DMSO) 6 12.11 (s, 1H), 8.10 (d, J = 3.1 Hz, 1H), 8.06 (d, J= 9.0 Hz, 2H), 8.00 (d, J= 3.2 Hz, 1H), 7.10 (d, J= 8.9 Hz, 2H), 6.87 (s, 1H), 3.86 (s, 3H). 13C NMR (126 MHz, DMSO- 6) 8 163.53 [C], 163.10 [C], 162.84 [C], 158.68 [C], 155.59 [C], 144.11 [CH], 130.29 [2 X CH], 124.24 [C], 122.58 [CH], 113.93 [2 X CH], 86.42 [CH], 55.54 [CH3], HRMS (ESI) m / . calculated for C14H11N3O3S: 301.0521; found 324.0470 [M+Na+],

[0102] EXAMPLE 4: Synthesis of compound N-(3-(5-iodopyridin-2-yl)isoxazol-5-yl)-4- methoxybenzamide (16): a. Synthesis of compound 3-(5-iodopyridin-2-yl)-3-oxopropanenitrile (14): The title compound was prepared according to the procedure a. Starting compound 13 (methyl 5-iodopicolinate) (1.05 g, 4.0 mmol), anhydrous THF (14.8 ml), anhydrous ACN (0.46 ml, 8.73 mmol), 1.6 Mn-butyllithium solution in hexanes (5.4 ml, 8.73 mmol). The product was obtained as a brown solid (348 mg, yield 32 %).1H NMR (500 MHz, CDC13) 8 8.91 (d, J= 2.9 Hz, 1H), 8.25 (dd, J= 8.2, 2.0 Hz, 1H), 7.85 (d, J = 9.0Hz, lH), 4.32 (s, 2H). 13C NMR(126 MHz, CDC13) 8 189.12 [C], 155.73 [CH], 149.57[CH],146.15 [C], 123.84 [CH], 114.19 [C], 100.82 [C], 28.62 [CH2], HRMS (ESI) m / r. calculated for C8H5IN2O: 271.9447; found 294.9351 [M+Na+], b. Synthesis of compound 3-(5-iodopyridin-2-yl)isoxazol-5-amine (15): The title compound was prepared according to the procedure b. Starting compound 14 (272.0 mg, 1.0 mmol), NH2OH HC1 (208.5 mg, 3.0 mmol), NaOAc (246.1 mg, 3.0 mmol), MeOH (3.1 mmol). The crude material was purified by flash column chromatography using ethyl acetate / n-hexane (2 / 3) as eluents to yield 15 as xx solid (175 mg, yield 61 %).1H NMR (500 MHz, DMSO) 5 8.89 (d, J= 3.2 Hz, 1H), 8.26 (dd, J= 8.2, 2.1 Hz, 1H), 7.68 (d, J = 8.2 Hz, 1H), 6.87 (s, 2H), 5.43 (s, 1H). 13C NMR (126 MHz, DMSO) 8 171.31 [C], 162.74 [C], 155.28 [CH], 147.68 [C], 145.15 [CH], 122.35 [CH], 94.99 [C], 75.45 [CH], HRMS (ESI) m / z'. calculated for C8H6IN3O: 286.9556; found 309.9486 [M+Na+], c. Synthesis of compound N-(3-(5-iodopyridin-2-yl)isoxazol-5-yl)-4-methoxybenzamide (16): The title compound was prepared according to the procedure c. Starting compound 15 (50 mg, 0.174 mmol), anhydrous pyridine (0.92 ml), 4-methoxybenzoyl chloride (47 pL, 0.347 mmol). The mixture was heated at 150 °C for 10 min. The crude material was purified by flash column chromatography using ethyl acetate / toluene (linear gradient; 1 / 4) as eluents to yield 16 as solid (59 mg, yield 80 %).1H NMR (500 MHz, DMSO) 5 12.01 (s, 1H), 8.98 (d, J= 1.2 Hz, 1H), 8.34 (dd, J= 8.3, 2.2 Hz, 1H), 8.05 (d, .7= 8.9 Hz, 2H), 7.84 (d, = 9.2 Hz, 1H), 7.09 (d, J= 9.0 Hz, 2H), 6.91 (s, 1H), 3.85 (s, 3H). 13C NMR (126 MHz, DMSO) 163.19 [C], 163.03 [C], 162.87 [C], 162.76 [C], 155.67 [CH], 146.59 [C], 145.52 [CH], 130.23 [2 X CH], 124.38 [C], 122.72 [CH], 113.88 [2 X CH], 95.90 [C], 86.94 [CH], 55.52 [CH3], HRMS (ESI) m / z: calculated for C16H12IN3O3: 420.9923; found 443.9852 [M+Na+],

[0103] EXAMPLE 5: Synthesis of compound 4-methoxy-N-(3-phenylisoxazol-5-yl)benzamide (19):a. Synthesis of compound 3-phenylisoxazol-5 -amine (18): The title compound was prepared according to the procedure b. Starting compound 17 (3-oxo-3-phenylpropanenitrile) (1000 mg, 7.0 mmol), NH2OH HC1 (1436.1 mg, 21.0 mmol), NaOAc (1695.4 mg, 21.0 mmol), methanol (20.7 ml). The product was obtained as an orange solid (1.077 mg, yield 98 %).1HNMR (500 MHz, DMSO) 6 7.72 (dd, J= 7.7, 1.9 Hz, 2H), 7.49 - 7.39 (m, 3H), 6.77 (s, 2H), 5.39 (s, 1H). 13C NMR (126 MHz, DMSO-d6): 5 170.97 [C], 162.45 [C], 129.96 [C], 129.50 [CH], 128.76 [2 X CH], 126.19 [2 X CH], 75.04 [CH], HRMS (ESI) m / z: calculated for C9H8N2O: 160.0637; found 183.0532 [M+Na+], b. Synthesis of compound 4-methoxy-N-(3-phenylisoxazol-5-yl)benzamide (19):The title compound was prepared according to the procedure c. Starting compound 18 (90 mg, 0.562 mmol), anhydrous pyridine (3.5 ml), 4-methoxybenzoyl chloride (86 pL, 0.633 mmol). The mixture was heated at 150 °C for 10 min. The crude material was purified by flash column chromatography using ethyl acetate / toluene (linear gradient; 1 / 3) as eluents to yield 19 as a white solid (50 mg, yield 28 %).1H NMR (600 MHz, DMSO) 8 11.93 (s, 1H), 8.06 (d, J= 9.0 Hz, 2H), 7.92 - 7.86 (m, 2H), 7.52 (dd, J= 5.1, 2.0 Hz, 3H), 7.10 (d, J= 9.0 Hz, 2H), 6.90 (s, 1H), 3.86 (s, 3H). 13C NMR (151 MHz, DMSO- 6) 8 163.01 [C], 162.82 [C], 162.75 [C], 162.57 [C], 130.21 [2 X CH], 129.08 [2 X CH], 128.84 [C], 126.49 [3 X CH], 124.48 [C], 113.92 [2 X CH], 86.66 [CH], 55.55 [CH3], HRMS (ESI) m / z : calculated for C17H14N2O3 : 294.1004; found 295.1065 [M+H+]; found 317.0915 [M+Na+],

[0104] EXAMPLE 6: Synthesis of compound 2-fluoro-N-(3-(4-iodophenyl)isoxazol-5-yl)-4- methoxybenzamide (22):Rl= F and X= CH a. Synthesis of compound 2-fluoro-4-methoxybenzoyl chloride (21): The title compound was prepared according to the procedure d. Starting compound 20 (2-fluoro-4-methoxybenzoic acid) (500 mg, 2.939 mmol), anhydrous DCM (2.6 ml), anhydrous DMF (3-4 drops), oxalyl chloride (0.756 mL, 8.816 mmol). The reaction mixture was stirred for 6h at room temperature. Solvent was removed under vacuum and the crude product was used directly for the next-step synthesis. b. Synthesis of compound 2-fluoro-N-(3-(4-iodophenyl)isoxazol-5-yl)-4-methoxybenzamide (22): Starting compound 3 (350 mg, 1.223 mmol) was dissolved in anhydrous pyridine (6.1 ml) and left to stir for 10 - 15 min under argon. Afterwards, it was slowly added to the 21 and the reaction mixture was stirred for 10 min at 150 °C. Solvent was removed under reduced pressure. Then saturated bicarbonate solution was added to quench the reaction and product was extracted three times with ethyl acetate. The combined organic layers were washed with water, brine, dried over Na2SC>4, filtered, and concentrated to dryness. The crude reaction mixture was purified by flash column chromatography using ethyl acetate / toluene (linear gradient; 1 / 10) as eluents to yield 22 as a yellow-white solid (324 mg, yield 62 %).1H NMR (600 MHz, DMSO) 5 11.97 (s, 1H), 7.89 (d, J= 8.4 Hz, 2H), 7.73 (t, J= 8.6 Hz, 1H), 7.69 (d, J= 8.5 Hz, 2H), 7.00 (dd, J= 12.7, 2.4 Hz, 1H), 6.93 (dd, J= 8.7, 2.4 Hz, 1H), 6.91 (s, 1H), 3.86 (s, 3H). 13C NMR (XX): 5 XX ppm. HRMS (ESI) m / z calculated for C17H12FIN2O3: 437.9877; found 438.9938 [M+H+]; found 460.9760 [M+Na+],

[0105] EXAMPLE 7: Synthesis of compound N-(3-(4-iodophenyl)isoxazol-5-yl)-6-methoxynicotinamide(25):Rl= H and X= N a. Synthesis of compound 6-methoxynicotinoyl chloride (24): The title compound was prepared according to the procedure d. Starting compound 23 (6-methoxynicotinic acid) (122.5 mg, 0.8 mmol), anhydrous DCM (0.65 ml), catalytic amount of anhydrous DMF (3-4 drops), oxalyl chloride (0.206 ml, 2.4 mmol). The reaction mixture was stirred for 2h at room temperature. Solvent was removed under vacuum and the crude product was used directly for the next-step synthesis. b. Synthesis of compound N-(3-(4-iodophenyl)isoxazol-5-yl)-6-methoxynicotinamide (25): Starting compound 3 (114.4 mg, 0.4 mmol) was dissolved in anhydrous pyridine (2.0 ml) and left to stir for 10 - 15 min under argon. Afterwards, it was slowly added to the 24 and the reaction mixture was stirred for 10 min at 150 °C. Solvent was removed under reduced pressure. Then saturated bicarbonate solution was added to quench the reaction and product was extracted three times with ethyl acetate, the organic layers were washed with water, brine, dried over ISfeSCU, filtered, and concentrated to dryness. The crude reaction mixture was purified by flash column chromatography using ethyl acetate / n-hexane (linear gradient; 2 / 3) as eluents to yield 25 as a white solid (30 mg, yield 18 %).1HNMR (600 MHz, DMSO) 5 12.15 (s, 1H), 8.88 (d, J= 2.6 Hz, 1H), 8.31 (dd, J= 8.7, 2.6 Hz, 1H), 7.90 (d, J= 8.5 Hz, 2H), 7.70 (d, 8.4 Hz, 2H), 6.99 (d, J= 8.7 Hz, 1H), 6.94 (s, 1H), 3.96(s, 3H). 13C NMR (151 MHz, DMSO) 8 165.95 [C], 162.68 [C], 162.00 [C], 161.98 [C], 148.30 [CH], 138.94 [CH], 137.93 [2 X CH], 128.45 [2 X CH], 128.24 [C], 122.06 [C], 110.51 [CH], 97.18 [C], 86.83 [CH], 53.91 [CH3], HRMS (ESI) m / r. calculated for C16H12IN3O3: 420.9923; found 421.9978 [M+H+],

[0106] EXAMPLE 8: Synthesis of compound 4-iodo-N-(5-(4-methoxyphenyl)-l,2,4-oxadiazol-3- yl)benzamide (30a) and 2-(5-( 4-iodophenyl)-l, 2, 4-oxadiazol-3-yl)-l-( 4-methoxyphenyl)ethan-l-one (30b): a. Synthesis of compound 4-iodobenzoyl chloride (27). The title compound was prepared according to the procedure d. Starting compound 26 (4-iodobenzoic acid) (0.992 g, 4 mmol), anhydrous DCM (10 ml), catalytic amount of anhydrous DMF (3-4 drops), oxalyl chloride (0.926 mL). The reaction mixture was stirred for 2h at room temperature and then refluxed for 3h. After cooling, solvent was removed under vacuum and the crude product was used directly for the next-step synthesis. b. Synthesis of compound N-carbamimidoyl-4-iodobenzamide (28): Starting material 27 was dissolved in anhydrous THF (10 ml) and was slowly added dropwise to a solution of guanidine hydrochloride (18 mmol) in 12 ml of NaOH solution (c = 2 M). The reaction mixture was stirred for Ih at room temperature. When no starting material was detected on TLC, the reaction was quenched with water and the product was extracted three times with ethyl acetate. The combined organic phases were washed with NaOH (c = 1 M) and subsequently with water. The combined organic layers were dried over Na2SO4, fdtered, and concentrated to dryness. The crude reaction mixture was purified by flash column chromatography using MeOH / DCM (1 / 9) as eluents to give 28 as white solid (311 mg, yield 32 %).IH NMR (500 MHz, DMSO) 5 8.00 (s, IH), 7.84 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.5 Hz, 2H), 6.75 (s, 2H). 13C NMR (126 MHz, DMSO) 5 174.88 [C], 162.97 [C], 138.66 [C], 136.56 [2 X CH], 130.56 [2 X CH], 98.14 [C], HRMS (ESI) m / r. calculated for C8H8IN3O: 288.9712; found 289.9795 [M+H+], c. Synthesis of compound 5-(4-iodophenyl)-l,2,4-oxadiazol-3-amine (29): To a stirring solution of 28 (90 mg, 0.311 mmol) in anhydrous DMF (1.6 mL), PIDA (150 mg, 0.467 mmol) was added at 0°C. The reaction mixture continued to react at room temperature until no starting material was detected on TLC (typically around 6 h). When no starting material was detected on TLC, solvent was removed under reduced pressure. Then saturated bicarbonate solution was added, and theproduct was extracted three times with ethyl acetate. The combined organic phases were washed with water, brine, dried over Na2SO4, filtered and concentrated. The crude reaction mixture was purified by flash column chromatography using ethyl acetate / n-hexane / TEA (2 / 3 / 0.1) as eluents to yield 29 as a white solid (42 mg, yield 47 %).1H NMR (600 MHz, DMSO) 5 7.98 (d, J= 8.5 Hz, 2H), 7.74 (d, J= 8.4 Hz, 2H), 6.43 (s, 2H). 13C NMR (151 MHz, DM SO-6 / 6) 5 172.50 [C], 169.02 [C], 138.30 [2 X CH], 128.96 [2 X CH], 123.45 [C], 100.63 [C], HRMS (ESI) m / z : calculated for C8H6IN3O: 286.9556; found 287.9750 [M+H+], d. Synthesis of compound 4-iodo-N-(5-(4-methoxyphenyl)-l,2,4-oxadiazol-3-yl)benzamide (30a) and 2-(5-(4-iodophenyl)-l ,2, 4-oxadiazol-3-yl)-l-(4-methoxyphenyl)ethan-l-one (30b):I. The title compound was prepared according to the adjusted procedure c with the addition of DMAP. Starting compound 29 (30 mg, 0.105 mmol), anhydrous pyridine (0.525 ml), 4- methoxybenzoyl chloride (28 pL, 0.209 mmol), DMAP (5.4 mg, 0.044 mmol). The mixture was heated at 150 °C for 3h. Afterwards, solvent was removed under reduced pressure and the crude material was purified by flash column chromatography using ethyl acetate / n-hexane (0% - 100%) as eluent to yield 30a, 30b as a beige solid (8 mg, yield 18 %). Signals are referred to both isomers. rH NMR (600 MHz, DMSO) 5 11.67 (s, 1H), 11.49 (s, 1H), 8.09 - 8.02 (m, 4H), 7.94 (d, J = 8.6 Hz, 4H), 7.86 (d, J= 8.5 Hz, 2H), 7.80 (d, J= 8.5 Hz, 2H), 7.20 (d, J= 9.0 Hz, 2H), 7.08 (d, J = 9.0 Hz, 2H), 3.89 (s, 3H), 3.86 (s, 3H).13C NMR (151 MHz, DMSO) 5 173.91, 173.45, 164.26, 164.04, 163.66, 163.15, 162.67, 138.55, 137.45, 132.18, 130.35, 130.06, 129.74, 129.25, 124.71, 122.83, 115.67, 115.07, 113.82, 101.57, 100.71, 55.68, 55.53. ESI (+)-MS: m / z 422 [M+H+],II. Starting material 29 (100 mg, 0.348 mmol) was dissolved in anhydrous benzene (3.5 ml) and anhydrous pyridine (35 pl) was added, under argon. Afterwards, 4-methoxybenzoyl chloride (59 pl, 0.435 mmol) was added and reaction mixture was allowed to stand at room temperature (with occasional shaking) for 17 days, monitored by TLC. The reaction was stopped, and solvent was removed under reduced pressure. Then water was added to quench the reaction and product was extracted three times with ethyl acetate. The combined organic phases were washed with brine, dried over Na2SO4, filtered and concentrated. The crude reaction mixture was purified by flash column chromatography twice using MeOH / DCM (1% - 5%) and ethyl acetate / n-hexane (1 / 3) as eluents to to yield 30a, 30b as a beige solid (17 mg, yield 12 %). Signals are referred to both isomers. rH NMR (600 MHz, DMSO) 5 11.67 (s, 1H), 11.49 (s, 1H), 8.09 - 8.02 (m, 4H), 7.94 (d, J = 8.6 Hz, 4H), 7.86 (d, J= 8.5 Hz, 2H), 7.80 (d, J= 8.5 Hz, 2H), 7.20 (d, J= 9.0 Hz, 2H), 7.08 (d, J = 9.0 Hz, 2H), 3.89 (s, 3H), 3.86 (s, 3H).13C NMR (151 MHz, DMSO) 5 173.91, 173.45, 164.26, 164.04, 163.66, 163.15, 162.67, 138.55, 137.45, 132.18, 130.35, 130.06, 129.74, 129.25, 124.71, 122.83, 115.67, 115.07, 113.82, 101.57, 100.71, 55.68, 55.53. HRMS (ESI) m / r. calculated for C16H12IN3O3 : 420.9923; found 422.0006 [M+H+], 443.9825 [M+Na+],

[0107] EXAMPLE 9: Synthesis of compound N-(3-(4-iodophenyl)-l,2,4-oxadiazol-5-yl)-4- methoxybenzamide (37):a. Synthesis of compound (E)-4-iodobenzaldehyde oxime (32): Starting material 31 (4- iodobenzaldehyde) (2.4 g, 10.344 mmol) and NH2OH HC1 (0.863 g, 12.413 mmol) were ground together with a pestle in a mortar. Then, NaOH (0.496 g, 12.413 mmol) was added to the reaction mixture, and everything was ground together with MeOH (0.3 ml), for 2 min at room temperature. The reaction mixture was left for 5 min, after which it was grounded for another 5 minutes with MeOH (0.33 ml) and monitored by TLC. Then water was added to quench the reaction and the product was extracted three times with ethyl acetate, the organic layers were washed with water, brine, dried over Na2SO4, filtered, and concentrated to dryness. The crude reaction mixture was purified by flash column chromatography using ethyl acetate / n-hexane (linear gradient; 1 / 3) as eluents to yield 32 as a white solid (1.19 g, yield 47 %).1HNMR (500 MHz, DMSO) 8 11.35 (s, 1H), 8.10 (s, 1H), 7.77 (d, J= 8.5 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H). 13C NMR (126 MHz, DMSO-c / 6) 8 147.44 [CH], 137.53 [2 X CH], 132.65 [C], 128.29 [2 X CH], 95.71 [C], ESI (+)-MS: m / z 248 [M+H+], b. Synthesis of compound (Z)-N-hydroxy-4-iodobenzimidoyl chloride (33): Starting material 32 (1.19 g, 4.817 mmol) was dissolved in anhydrous DMF (19 ml) under argon. To the solution, 7V-chlorosuccinimide (0.779 g, 5.831 mmol) was slowly added. The reaction mixture continued to react at room temperature until no starting material was detected on TLC (typically around 12 h). The reaction was stopped, and solvent was removed under reduced pressure. Then water was added to quench the reaction and the product was extracted with ethyl acetate, the combined organic phases were washed with brine, dried over ISfeSCU, filtered and concentrated and the crude product was used directly for the next-step synthesis. c. Synthesis of compound (lZ,2Z)-N-(tert-butyl)-N'-hydroxy-2-(hydroxyimino)-2-(4- iodophenyl)acetimidamide (34): This procedure was based on Mercalli V., et al. [https: / / doi.org / 10.1021 / acs.joc.5b01676]. Firstly, hydroxyl solution was prepared using NH20H HC1 (1.04g, 15 mmol) in MeOH (10 mL) that was added to a stirred solution of KOH (841.65 mg, 15 mmol) in MeOH (4 ml) at 0 °C using ice bath. The mixture was stirred for 30 min at room temperature, and the precipitate potassium chloride was removed, and the filtratewas used as such. Then, starting material 33 (1 g, 3.553 mmol) was dissolved in anhydrous DCM (12 ml). Isocyanide (0.4 ml, 3.553 mmol), hydroxylamine (solution 1 M in MeOH, 1.2 equiv), andNaHCO3 (298.4 mg, 3.553 mmol) were added, and the reaction was stirred at room temperature under argon until all of the starting material was consumed (typically 16 h). Upon completion, the reaction mixture was concentrated under reduced pressure and the crude reaction mixture was purified by flash column chromatography using ethyl acetate / n-hexane (linear gradient; 1 / 1) as eluents to yield 34 as solid (235 mg, yield 38%). Signals are referred to both isomers (ratio between 50: 50).1H NMR (500 MHz, DMSO) 5 11.85 (s, 1H), 11.26 (s, 1H), 9.84 (s, 1H), 8.53 (s, 1H), 7.76 (dd, J= 13.6, 8.5 Hz, 4H), 7.40 (d, J= 8.5 Hz, 2H), 7.31 (d, J= 8.7 Hz, 2H), 5.35 (s, 1H), 5.12 (s, 1H), 1.31 (s, 9H), 1.07 (s, 9H). 13C NMR (126 MHz, DMSO-t / 6) 8 149.85 [C], 149.32 [C], 148.88 [C], 145.34 [C], 137.25 [2 X CH], 137.09 [3 X CH], 134.84 [C], 133.15 [C], 127.94 [2 X CH], 127.73 [2 X CH], 95.65 [C], 94.92[C], 50.79 [C], 50.73 [C], 30.66 [3 X CH3], 28.50 [3 X CH3], HRMS (ESI) m / z : calculated for C12H16IN3O2: 361.0287; found [M+H+] 362.0395. d. Synthesis of compound N-(tert-butyl)-3-(4-iodophenyl)-l,2,4-oxadiazol-5-amine (35): Starting material 34 (470 mg, 1.301 mmol) was dissolved in anhydrous toluene (6.0 ml) under argon, cooled to 0 °C in an ice bath. Then, triphenylphosphine (682.6 mg, 2.603 mmol) was added. Afterwards, diethyl azodicarboxylate (0.408 ml, 2.603 mmol) was added dropwise and the resulting solution was heated at reflux under argon and reaction was monitored by TLC. When no starting material was detected on TLC (typically after 21 h), the reaction mixture was concentrated under reduced pressure and the crude reaction was purified by flash column chromatography using ethyl acetate / n-hexane (linear gradient; 1 / 3) as eluents to yield 35 as solid (301 mg, yield 68 %).1HNMR (500 MHz, DMSO) 8 8.36 (s, 1H), 7.89 (d, J= 8.5 Hz, 2H), 7.67 (d, J= 8.5 Hz, 2H), 1.39 (s, 9H). 13C NMR (126 MHz, DMSO) 8 170.25 [C], 166.60 [C], 137.78 [2 X CH], 128.75 [2 X CH], 127.06 [C], 97.86 [C], 51.58 [C], 28.33 [3 X CH3], HRMS (ESI) m / r. calculated for C12H14IN3O: 343.0182; found 366.0097 [M+Na+],e. Synthesis of compound 3-(4-iodophenyl)-l,2,4-oxadiazol-5-amine (36): In a 10 ml flask staring material 35 (250 mg, 0.729 mmol) was dissolved in neat TFA (7.3 mL) under argon. The reaction was heated at reflux and monitored periodically by TLC. When no starting material was detected on TLC (typically after 48 h), the reaction mixture was quenched with saturated bicarbonate solution and product was extracted three times with ethyl acetate, the organic layers were washed with water, brine, dried over Na2SO4, filtered, and concentrated to dryness. The crude reaction mixture was purified by flash column chromatography using ethyl acetate / n-hexane / TEA (linear gradient; 1 / 1 / 0.1) as eluents to yield 36 as an orange solid (121 mg, yield 58 %).1HNMR (500 MHz, DMSO) 8 7.96 (s, 2H), 7.88 (d, J= 8.5 Hz, 2H), 7.64 (d, J= 8.5 Hz, 2H. 13C NMR (126 MHz, DMSO) 5 172.16 [C], 166.96 [C], 137.77 [2 X CH], 128.39 [2 X CH], 127.07 [C], 97.81 [C], HRMS (ESI) m / z: calculated for C8H6IN3O : 286.9556; found 287.9628 [M+H+], f. Synthesi s of compound N-( 3-( 4-iodophenyl)-l , 2, 4-oxadiazol-5-yl)-4-methoxybenzamide (37) : Starting material 36 (50 mg, 0.174 mmol), anhydrous pyridine (869 pL) and DMAP (63.8 mg, 0.523 mmol) were stirred for 45 min in a flask. Afterwards, 4-methoxybenzoyl chloride (94 ul, 0.697 mmol) was added. The mixture was heated at 150 °C for 10 min and monitored by TLC. Upon completion, solvent was removed under reduced pressure. Then saturated bicarbonate solution was added to quench the reaction and product was extracted with ethyl acetate, the combined organic phases were washed with water, brine, dried over Na2SO4, filtered and concentrated. The crude reaction was purified twice by flash column chromatography using toluene / ethyl acetate (linear gradient; 6 / 1) as eluents to yield 37 as a white solid (20 mg, yield 27 %).1H NMR (500 MHz, DMSO) 12.50 (s, 1H), 8.05 (d, J= 8.9 Hz, 2H), 7.96 (d, J= 8.5 Hz, 2H), 7.77 (d, J = 8.4 Hz, 2H), 7.10 (d, J= 9.0 Hz, 2H), 3.86 (s, 3H). 13C NMR (126 MHz, DMSO) 5 167.82 [C], 166.84 [C], 163.53 [C], 163.12 [C], 138.12 [2 X CH], 130.71 [2 X CH], 128.61[2 X CH], 125.96 [C], 124.11 [C], 113.94 [2 X CH], 98.80 [C], 55.58 [CH3], HRMS (ESI) m / z calculated for C16H12IN3O3 : 420.9923; found 422.0009 [M+H+],

[0108] EXAMPLE 10: Synthesis of compounds (E)-5-(4-iodophenyl)-3-(4-methoxystyryl)iso &z.o\Q (41a) and (E)-3-(4-iodophenyl)-5-(4-methoxystyryl)isoxazole (41b):a. Synthesis of compound (E)-3-(4-methoxyphenyl)acryloyl chloride (39): The title compound was prepared according to the procedure d. Starting compound 38 ((E)-3-(4- methoxyphenyl)acrylic acid) (890 mg, 5 mmol), anhydrous DCM (10 ml), catalytic amount of anhydrous DMF (3-4 drops), oxalyl chloride (0.515 mL, 6 mmol). The reaction mixture was stirred for 6h at room temperature. Solvent was removed under vacuum and the crude product was used directly for the next-step synthesis. b. Synthesis of compound (2Z,4E)-3-hydroxy-l-(4-iodophenyl)-5-(4-methoxyphenyl)penta-2,4- dien-l-one (40): A solution of 4’ -iodoacetophenone (861 mg, 3 mmol) in anhydrous THF (10 mL) was cooled to -78 °C, and 1 M lithium bis(trimethylsilyl)amide in THF (LiHMDS, 3.9 mL, 3.858 mmol) was added dropwise and stirred at -78 °C for 1 h.To the reaction mixture was added 39 in anhydrous THF (4 mL) dropwise at the same temperature and stirred at the room temperature until no starting material was detected on TLC (typically 18h). After the completion, the reaction mixture was diluted with saturated NH4C1 (aq) and extracted three times with ethyl acetate. The combined organic layer was washed with water, dried overNa2SO4, and concentrated. The crude reaction mixture was purified by flash column chromatography twice using ethyl acetate / n-hexane (linear gradient; 2 / 3) and ethyl acetate / n-hexane (1 / 4) as eluents to yield 40 as a yellow solid (90 mg, yield 7 %).1HNMR (500 MHz, DMSO) 5 7.95 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 8.5 Hz, 2H), 7.73 - 7.66 (m, 3H), 7.03 (d, J= 9.0 Hz, 2H), 6.83 (d, J= 16.0 Hz, 1H), 6.77 (s, 1H), 3.82 (s, 3H). 8 13C NMR (126 MHz, DMSO-t / 6) 8 186.35 [C], 181.24 [C], 161.18 [C], 140.34 [CH], 137.81 [2 X CH], 134.82 [C], 130.07 [2 X CH], 128.82 [2 X CH], 127.28 [C], 121.03 [CH], 114.58 [2 X CH], 101.12 [C], 96.89 [CH], 55.37 [CH3], HRMS (ESI) m / z. calculated for: C18H15IO3 ; found 428.9979 [M+Na+], c. Synthesis of compounds (E)-5-(4-iodophenyl)-3-(4-methoxystyryl)\'^o^azo\e (41a) and (E)-3- ( 4-iodophenyl)-5-(4-methoxystyryl)isoxazole ( 1b):A solution of 40 (40 mg, 0.098 mmol) in EtOH (1.3 ml) was added NH2OH HC1 (13.7 mg, 0.197 mmol) and stirred at 80 °C until no starting material was detected on TLC (typically around 24 h). Then, solvent was removed under reduced pressure. The crude reaction mixture was purified twice by flash column chromatography using DCM / MeOH / NH4OH (100 / 1 / 1 - 100 / 2 / 1) and DCM (100%) as eluents to yield 41a, 41b as a yellow solid (24 mg, yield 60 %). %). Signals are referred to both isomers.1HNMR (600 MHz, DMSO) 8 7.94 (d, J= 8.5 Hz, 2H), 7.91 (d, J= 8.5 Hz, 2H), 7.71 - 7.61 (m, 8H), 7.49 (s, 1H), 7.41 (d, J = 1.3 Hz, 1H), 7.38 (d, J= 2.5 Hz, 1H), 7.17 (s, 1H), 7.14 (d, J= 1.5 Hz, 1H), 7.12 (s, 1H), 6.99 (dd, J = 8.9, 2.4 Hz, 4H), 3.80 (d, J = 2.1 Hz, 6H). 13C NMR (151 MHz, DMSO) 8 170.15, 168.27, 163.18, 162.03, 160.71, 160.48, 138.63, 138.45, 136.82, 135.10, 129.44, 129.17, 128.94, 128.67, 128.61, 128.38, 127.77, 126.79, 114.87, 114.84, 113.60, 111.35, 99.50, 98.59, 97.87, 97.55, 55.77, 55.72. HRMS (ESI) m / z. calculated for C18H14INO2: 403.0069; found 404.0147 [M+H+],

[0109] EXAMPLE 11: Synthesis of compound N-(3-(4-iodophenyl)isoxazol-5-yl)-4-methoxy-N- methylbenzamide (42):Starting material 3 (62.8 mg, 0.149 mmol) was dissolved in anhydrous DMF (0.382 mmL) under argon, cooled to 0 °C in an ice bath, and NaH (60% dispersion in mineral oil; 17.9 mg, 0.747 mmol) was added. After 1 h, iodomethane (32.7 uL, 0.523 mmol) was added. The ice bath was removed, and the reaction mixture was stirred at room temperature for 24 h and monitored by TLC. When no starting material was present anymore, water was added to quench the reaction and product was extracted three times with ethyl acetate, the organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to dryness. The crude reaction mixture was purified by flash column chromatography (ethyl acetate / n-hexane; 0% to 100%) to give the product as a white solid (35.9 mg, yield 55 %).1H NMR (500 MHz, DMSO) 8 7.87 (d, J= 8.5 Hz, 2H), 7.59 (d, J= 8.5 Hz, 2H), 7.49 (d, J= 8.9 Hz, 2H), 6.97 (d, J= 8.9 Hz, 2H), 6.79 (s, 1H), 3.78 (s, 3H), 3.43 (s, 3H). 13C NMR (126 MHz, DMSO) 8 168.91 [C], 166.70 [C], 162.68 [C], 161.96 [C], 138.46 [2 X CH], 130.50 [2 X C], 128.72 [2 X CH], 128.46 [C], 126.69 [C], 114.20 [2 X CH], 97.82 [C], 91.98 [C], 55.86 [CH3], 36.96 [CH3], HRMS (ESI) m / z calculated for C18H15IN2O3: 434.0127; found 435.0204 [M+H+]; found 457.0014 [M+Na+],

[0110] EXAMPLE 12: Synthesis of compound 6-hydroxy-N-(3-(4-iodophenyl)isoxazol-5- yl)nicotinamide (49):a. Synthesis of compound Methyl 6-(methoxymethoxy)nicotinate (45): To a solution of 44 (methyl 6-hydroxynicotinate) (1.57g, 10.252 mmol) in anhydrous DMF (96 mb) was slowly added NaH (60% dispersion in mineral oil; 0.277 g, 11.534 mmol), and the resulting mixture was stirred at room temperature for 40 min. After cooling the reaction mixture to 0 °C,chloromethyl methyl ether (3.2 ml, 42.291 mmol) was added. Next, K2CO3 (8 eq) was added to the reaction to adjust pH to 9. The reaction mixture was stirred for 4h at room temperature and monitored by TLC. Upon completion, the reaction mixture was quenched with water and the product was extracted three times with ethyl acetate. The collected organic layer was washed with brine and dried over Na2SO4, filtered and evaporated under reduced pressure. The crude reaction mixture was purified by flash column chromatography (ethyl acetate / n- hexane = 5 / 1) to give product as yellow oil (944 mg, 47%).1H NMR (500 MHz, CDC13) 8 8.26 (d, J= 2.6 Hz, 1H), 7.86 (dd, J= 9.5, 2.5 Hz, 1H), 6.55 (d, J= 9.6 Hz, 1H), 5.33 (s, 2H), 3.87 (s, 3H), 3.42 (s, 3H). 13C NMR (126 MHz, Chloroformed) 8 164.73 [C], 162.78 [C], 141.56 [CH], 139.13 [CH], 120.43 [CH], 110.34 [C], 79.03 [CH2], 57.64 [CH3], 52.28 [CH3], HRMS (ESI) m / r. calculated for C9H11NO4 : 197.0688; found 198.0775 [M+H+], b. Synthesis of compound 6-(methoxymethoxy)nicotinic acid (46): To a solution of 45 (934 mg mmol, 4.737 mmol) in THF (13.1 ml) and miliQ water (3.9 ml) was added NaOH (473.6 mg, 11.841 mmol) at room temperature. Then, the reaction was heated at 50 °C and stirred until all of the starting material was consumed (typically 19 h as judged by TLC). Upon completion, the reaction mixture was cooled down and concentrated under reduced pressure. To the residue was added water and IM HC1 to acidify the aqueous layer to pH to 3. The solution was left stirring for 2h (at room temperature) and the yellow precipitate was formed and collected by filtration. Precipitate was washed with water and dried under reduced pressure to give product as yellow solid (668.3mg, 77%).1H NMR (500 MHz, DMSO) 8 12.92 (s, 1H), 8.40 (d, J= 2.3 Hz, 1H), 7.86 - 7.76 (m, 1H), 6.46 (d, J= 9.6 Hz, 1H), 5.30 (s, 2H), 3.29 (s, 3H). 13C NMR (126 MHz, DMSO-t76) 8 165.19 [C], 161.63 [C], 143.23 [CH], 139.33 [CH], 119.40 [CH], 109.39 [C], 78.48 [CH2], 56.40 [CH3], HRMS (ESI) m / z : calculated for C8H9NO4: 183.0532; found 184.0617 [M+H+], c. Synthesis of compound 6-(methoxymethoxy)nicotinoyl chloride (47): The title compound was prepared according to the procedure d. Starting compound 46 (170 mg, 0.928 mmol),anhydrous DCM (0.8 ml), catalytic amount of anhydrous DMF (3-4 drops), oxalyl chloride (0.24 mL, 2.784 mmol) at 0 °C using ice bath. The reaction mixture was stirred for 2h at room temperature. Solvent was removed under vacuum and the crude product was used directly for the next-step synthesis. d. Synthesis of compound N-(3-(4-iodophenyl)isoxazol-5-yl)-6-(methoxymethoxy)nicotinamide (48): The solution of 3 and anhydrous pyridine (1ml) was left stirring for 15-20 min, under argon, at room temperature. Afterwards, the solution of 47 in anhydrous pyridine (1ml) was added to the solution of 3. The reaction mixture was heated to 150°C for lOmin. Solvent was removed under reduced pressure. Then saturated bicarbonate solution was added to quench the reaction and product was extracted three times with ethyl acetate, the organic layers were washed with water, brine, dried over Na2SO4, filtered, and concentrated to dryness. The crude reaction mixture was purified by flash column chromatography (linear gradient; ethyl acetate / n-hexane = 5 / 1) to give the product as beige solid (60 mg, yield 32 %).1H NMR (500 MHz, DMSO) 5 11.97 (s, 1H), 8.64 (d, J= 2.7 Hz, 1H), 8.01 (dd, J= 9.6, 2.7 Hz, 1H), 7.88 (d, J= 8.5 Hz, 2H), 7.68 (d, J= 8.5 Hz, 2H), 6.88 (s, 1H), 6.54 (d, J= 9.6 Hz, 1H), 5.31 (s, 2H), 3.31 (s, 3H). 13C NMR (126 MHz, DMSO-c / 6) 5 162.65 [C], 161.94 [C], 161.40 [C], 160.67 [C], 142.30 [CH], 138.57 [CH], 137.89 [2 X CH], 128.41 [2 X CH], 128.22 [C], 119.48 [CH], 110.98 [C], 97.11 [C], 86.57 [CH], 78.89 [C], 56.67 [CH3], HRMS (ESI) m / z : calculated for C17H14IN3O4 : 451.0029; found 452.0114 [M+H+], e. Synthesis of compound 6-hydroxy-N-(3-(4-iodophenyl)isoxazol-5-yl)nicotinamide (49): To 48 (53.0 mg, 0.117 mmol) was added 4N HC1 in 1,4 dioxane (8ml). Then the reaction was heated at 50 °C and stirred until all of the starting material was consumed. Upon completion, the reaction mixture was cooled down and concentrated under reduced pressure. Then, water was added to quench the reaction and the product was extracted three times with ethyl acetate. The combined organic phases were brined, dried over Na2SO4, filtered and concentrated. The crude reaction was purified by flash column chromatography (ethyl acetate 100%) to give the product as beige solid (30 mg, yield 63 %).1HNMR (500 MHz, DMSO) 5 12.21 (s, 1H), 11.84 (s, 1H), 8.32 (d, J= 2.9 Hz, 1H), 7.97 (dd, J= 9.7, 2.8 Hz, 1H), 7.88 (d, J= 8.5 Hz, 2H), 7.68 (d, J= 8.4 Hz, 2H), 6.86 (s, 1H), 6.43 (d, J = 9.6 Hz, 1H). 13C NMR (151 MHz, DMSO-<76) 8 162.79 [C], 162.30 [C], 161.97 [C], 160.93 [C], 139.54 [CH], 139.11 [CH], 137.96 [2 X CH], 128.47 [2 X CH], 128.28 [C], 119.54 [CH], 110.60 [C], 97.16 [C], 86.48 [CH]. HRMS (ESI) m / z: calculated for Cl 5H10IN303: 406.9767; found 407.9849 [M+H+],

[0111] EXAMPLE 13: Synthesis of compound 4-hydroxy-N-(3-phenylisoxazol-5-yl)benzamide (50):Starting material 19 was dissolved in anhydrous DCM. To the resulting solution a solution of IM BBr3 in DCM was added dropwise at room temperature, under argon atmosphere. The mixture was stirred for xx h and monitored by TLC. Upon completion, the solution was quenched with water and the product was extracted three times with ethyl acetate, the organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to dryness. The crude reaction mixture was purified by flash column chromatography to give product as solid.1H NMR (500 MHz, DMSO) 8 11.82 (s, 1H), 10.32 (s, 1H), 7.95 (d, J= 8.9 Hz, 2H), 7.90 - 7.85 (m, 2H), 7.51 (dd, J= 5.0, 2.0 Hz, 3H), 6.89 (d, J= 7.6 Hz, 3H). 13C NMR (126 MHz, DMSO-76) 8 163.20 [C], 163.07 [C], 162.54 [C], 161.53 [C], 130.38 [2 X CH], 130.20 [CH], 129.08 [2 X CH], 128.91 [C], 126.48 [2 X CH], 123.01 [C], 115.20 [2 X CH], 86.47 [CH], HRMS (ESI) m / z: calculated for C16H12N2O3: 280.0848; found 281.0922 [M+H+], 303.0743 [M+Na+],

[0112] EXAMPLE 14: Synthesis of compound 2-fluoro-4-hydroxy-N-(3-(4-iodophenyl)isoxazol- 5-yl)benzamide (51):Starting material 22 was dissolved in anhydrous DCM. To the resulting solution a solution of IM BBr3 in DCM was added dropwise at room temperature, under argon atmosphere. The mixture was stirred and monitored by TLC. Upon completion, the solution was quenched with water and the product was extracted three times with ethyl acetate. The organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to dryness. The crude reaction mixture was purified by flash column chromatography to give product as beige solid.1H NMR (500 MHz, DMSO) 8 11.84 (s, 1H), 10.69 (s, 1H), 7.88 (d, J= 8.5 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 7.63 (t, J = 8.6 Hz, 1H), 6.89 (s, 1H), 6.76 - 6.65 (m, 2H). 13C NMR (126 MHz, DMSO) . HRMS (ESI) m / z: calculated for C16H10FIN2O3: 423.9720; found 424.9804 [M+H+],

[0113] EXAMPLE 15: Synthesis of compound 4-hydroxy-N-(3-(5-iodopyridin-2-yl)isoxazol-5- yl)benzamide (55):a. Synthesis of compound 4-(chlorocarbonyl)phenyl acetate (53): The title compound was prepared according to the procedure d. Starting compound 52 (4-acetoxybenzoic acid), anhydrous DCM, catalytic amount of anhydrous DMF (3-4 drops), oxalyl chloride at 0 °C using ice bath. The reaction mixture was stirred at room temperature. Solvent and excess of oxalyl chloride was removed under vacuum and the crude product was used directly for the next-step synthesis without further purification or characterization.b. Synthesis of compound 4-((3-(5-iodopyridin-2-yl)isoxazol-5-yl)carbamoyl)phenyl acetate (54): Starting compound 15 was dissolved in anhydrous pyridine and left to stir for 10 - 15 min under argon. Afterwards, it was slowly added to the 53 and the reaction mixture was stirred for 10 min at 150 °C. Solvent was removed under reduced pressure. Then saturated bicarbonate solution was added to quench the reaction and product was extracted three times with ethyl acetate. The combined organic layers were washed with water, brine, dried over Na2SO4, filtered, and concentrated to dryness. The crude reaction mixture was purified by flash column chromatography using ethyl acetate / toluene (1 / 3) as eluents to yield 54 as a solid (32 mg, yield 14 %).1HNMR (500 MHz, DMSO) 8 12.24 (s, 1H), 9.00 (d, J= 2.3 Hz, 1H), 8.37 (dd, J= 8.3, 2.2 Hz, 1H), 8.12 (d, J= 8.7 Hz, 2H), 7.87 (d, J= 8.2 Hz, 1H), 7.35 (d, J= 8.7 Hz, 2H), 6.96 (s, 1H), 2.33 (s, 3H). 13C NMR (126 MHz, DMSO). ESI (+)-MS: m / z 248 calculated for: xx; c. Synthesis of compound 4-hydroxy-N-(3-(5-iodopyridin-2-yl)isoxazol-5-yl)benzamide (55): To the suspension of 54 in MeOH, was added IM aqueous solution of NaOH followed by water. After addition, a clear solution was obtained, and the reaction mixture was stirred at room temperature and monitored by TLC. Upon completion, the reaction mixture concentrated in vacuo and the remaining solution was quenched with water. Then, IM HC1 solution was added to acidify the aqueous layer. A beige precipitate was obtained which was diluted with water and extracted three times with ethyl acetate. The collected organic layer was washed with brine and dried over Na2SO4, filtered and evaporated under reduced pressure. The crude reaction mixture was purified by preparative HPLC to give product as white solid (12 mg, yield 60 %).1H NMR (500 MHz, DMSO) 6 11.90 (s, 1H), 10.33 (s, 1H), 8.98 (d, .7= 3.1 Hz, 1H), 8.34 (dd, J = 8.2, 2.1 Hz, 1H), 7.95 (d, J= 8.7 Hz, 2H), 7.84 (d, J= 9.2 Hz, 1H), 6.91 - 6.85 (m, 3H). 13C NMR (126 MHz, DMSO-t / 6) 8 163.31 [C], 163.18 [C], 162.85 [C], 161.57 [C], 155.66 [CH], 146.63 [C], 145.52 [CH], 130.40 [2 X CH], 122.82 [C], 122.72 [CH], 115.18 [2 X CH], 95.89 [C], 86.77 [CH], HRMS (ESI) m z calculated for C15H10IN3O3: 406.9767; found 407.9847 [M+H+],

[0114] EXAMPLE 16: Synthesis of compound 4-hydroxy-N-(3-(6-(pyrrolidin-l-yl)pyridin-3- yl)isoxazol-5-yl)benzamide (59):59a. Synthesis of compound 4-(chlorocarbonyl)phenyl acetate (57): The title compound was prepared according to the procedure d. Starting compound 56 (4-acetoxybenzoic acid), anhydrous DCM, catalytic amount of anhydrous DMF (3-4 drops), oxalyl chloride at 0 °C using ice bath. The reaction mixture was stirred at room temperature. Solvent was removed under vacuum and the crude product was used directly for the next-step synthesis. b. Synthesis of compound 4-((3-(6-(pyrrolidin-l-yl)pyridin-3-yl)isoxazol-5-yl)carbamoyl)phenyl acetate (58): Starting compound 7 (138 mg, 0.695 mmol) was dissolved in anhydrous pyridine (1.7 ml) and left to stir for 10 - 15 min under argon. Afterwards, it was slowly added to the 57 and the reaction mixture was stirred for 10 min at 150 °C. Solvent was removed under reduced pressure. Then saturated bicarbonate solution was added to quench the reaction and product was extracted three times with ethyl acetate. The combined organic layers were washed with water, brine, dried over Na2SO4, filtered, and concentrated to dryness. The crude reaction mixture was purified by flash column chromatography using ethyl acetate / n-hexane (5 / 1) as eluents to yield 54 as white solid (33 mg, yield 22 %).1H NMR (500 MHz, DMSO) 5 12.06 (s, 1H), 8.58 (d, J = 2.3 Hz, 1H), 8.11 (d, J = 8.7 Hz, 2H), 7.96 (dd, J= 8.9, 2.4 Hz, 1H), 7.35 (d, J= 8.7 Hz, 2H), 6.88 (s, 1H), 6.57 (d, J= 8.9 Hz, 1H), 3.46 (s, 4H), 2.33 (s, 3H), 1.98 (s, 4H). 13C NMR. ESI (+)-MS: m / z 248 calculated for: xx;c. Synthesis of compound 4-hydroxy-N-(3-(6-(pyrrolidin-l-yl)pyridin-3-yl)isoxazol-5- yl)benzamide (59): To the suspension of 58 (33 mg, 0.084 mmol) in MeOH (0.54 ml), was added IM aqueous solution of NaOH (0.43 ml, 0.433 mmol) followed by water (54 pl). After addition, clear solution was obtained, and the reaction mixture was stirred at room temperature and monitored by TLC. Upon completion, the reaction mixture concentrated in vacuo and the remaining solution was quenched with water. Then, IM HC1 solution was added to acidify the aqueous layer to pH 6. A beige precipitate was obtained which was diluted with water and extracted three times with ethyl acetate. The collected organic layer was washed with brine and dried over Na2SO4, filtered and evaporated under reduced pressure. The crude reaction mixture was purified by preparative HPLC to give product as a beige solid (13 mg, yield 45 %). lHNMR (500 MHz, DMSO) 5 11.79 (s, 1H), 10.33 (s, 1H), 8.51 (d, J= 2.4 Hz, 1H), 8.13 (d, J= 8.7 Hz, 1H), 7.95 (d, J= 8.9 Hz, 2H), 6.93 (s, 1H), 6.89 (d, J= 8.9 Hz, 2H), 6.81 (s, 1H), 3.52 (s, 4H), 2.00 (s, 4H). 13C NMR. HRMS (ESI) m / z'. calculated for C19H18N4O3: 350.1379; found 351.1463 [M+H+],

[0115] EXAMPLE 17: Synthesis of compound N-(3-(4-iodophenyl)isoxazol-5-yl)-l-methyl-6-oxo-l,6- dihydropyridine-3-carboxamide (62) :a. Synthesis of compound l-methyl-6-oxo-l,6-dihydropyridine-3-carbonyl chloride (61): The title compound was prepared according to the procedure d. Starting compound 60 (l-methyl-6-oxo- l,6-dihydropyridine-3-carboxylic acid) (120mg, 0.784 mmol), anhydrous DCM (0.64 ml), catalytic amount of anhydrous DMF (3-4 drops), oxalyl chloride (0.202 mL, 2.651mmol) at 0 °C using ice bath. The reaction mixture was stirred for 3 h at room temperature. Solvent was removed under vacuum and the crude product was used directly for the next-step synthesis.b. Synthesis of compound N-(3-(4-iodophenyl)isoxazol-5-yl)-l-methyl-6-oxo-l, 6-dihydropyridine-3- carboxamide (62): Starting compound 3 (100 mg, 0.350 mmol) was dissolved in anhydrous pyridine (1.7 ml) and left to stir for 10 - 15 min under argon. Afterwards, it was slowly added to the 61 and the reaction mixture was stirred for 10 min at 150 °C. Solvent was removed under reduced pressure. Then saturated bicarbonate solution was added to quench the reaction and product was extracted three times with ethyl acetate. The combined organic layers were washed with water, brine, dried over Na2SO4, filtered, and concentrated to dryness. The crude reaction mixture was purified by flash column chromatography using ethyl acetate / n-hexane (85 / 15) as eluents to yield 62 as white solid (50 mg, yield 34 %).'HNMR (500 MHz, DMSO) 5 11.84 (s, 1H), 8.66 (d, J= 2.7 Hz, 1H), 7.99 (dd, J= 9.5, 2.7 Hz, 1H), 7.88 (d, J= 8.5 Hz, 2H), 7.68 (d, J= 8.5 Hz, 2H), 6.86 (s, 1H), 6.48 (d, J= 9.6 Hz, 1H), 3.52 (s, 3H).RADIOCHEMISTRY

[0116] For optimization reactions, the stock solution of [3H] methyl nosylate / methyl nosylate was prepared. The stock solution of the [3H] methyl nosylate has activity of 1.0 GBq / ml and Amof 2.98 TBq / mmol. The molar mass of [3H] methyl nosylate is 223.029 g / mol. activity 1.0 . , concentation = - - molar mass = - * 223.029 = 74.84 ug / miAm 2.981

[0117] A stock solution of methyl nosylate was prepared with a concentration of 7.5 mg / mL 10 pL of this stock solution was diluted with 980 pL of ACN and 10 pL of the stock solution of [3H] methyl nosylate (1 GBq / mL). This resulted in a concentration of 10 MBq / mL of activity with the same molarity as methyl nosylate. This was used to perform the optimization reactions.Radiosynthesis of [3H]SKP02 ([3H]8)

[0118] Manual labeling of [3H]SKP-02 was carried out by preparing a solution of 59 (1.2 pmol), anhydrous CS2CO3 (15.3 pmol) dissolved in anhydrous DMSO (300 pl). Solution was left stirring for 30 min at 60°C. In the meantime, 55 pL of [3H]methyl nosylate (92.5 GBq / ml in ACN, Vitrax Co., 2.84 GBq / pmol) was added to the 2 ml HPLC vial and solvent was evaporated by cautiously introducing helium gas to the reaction vial. Afterwards, solution of 59 was cooled down and added to the reaction vial with dry [3H]methyl nosylate and was left to react for few seconds at room temperature. Reaction was monitored by analytical HPLC method (isocratic conditions, Luna, 5pm, C18(2), 100A, 250 x 4.6 mm; Iml / min; ACN : water = 50 : 50, 25 min; 254 nm; tr ([3H]SKP02) = 16.0 min, tr ([3H]methyl nosylate)= 9.06 min). When no [3H]methyl nosylate was present, the reaction mixture was quenched with 1ml solution (ACN : water = 50 : 50) and was injected on semi -preparative HPLC (isocratic conditions, Alltima, ACN : miliQ water = 50 : 50, 25 min; 254 nm; 3ml / min; retention time ([3H]SKP01) around 35min) and fractions (30 sec) were collected. Afterwards, fractions with retention time between 30 min and 40 min were studied by using analytical HPLC method (Luna, 5pm, C18(2), 100A, 250 x 4.6 mm; Iml / min; ACN : miliQ water = 50 : 50; 254 nm). Only fractions that were pure were combined and added to 5 mL of miliQ water. This was transferred over a conditioned plus short tC18 SPE column which was rinsed with miliQ water (5 mL) and air (20 mL). Product was eluted with 1.42 ml EtOH (accurately measured with Hamilton syringe). [3H]SKP02 (25.94 MBq) was obtained with 51 % RCY, > 99% RCP and chemical purity, as determined on analytical HPLC. Successful labelling was confirmed with coinjection with standard reference.Radiosynthesis of [3H]SKP03 ([3H]25)

[0119] Manual labeling of [3H]SKP-03 was carried out by preparing a solution of 49 (0.5 mg, 1.2 pmol), anhydrous CS2CO3 (2.8 pmol) dissolved in anhydrous DMSO (300 pl). Solution was left stirring for 30 min at 60°C. In the meantime, 120 L of [3H]methyl nosylate (92.5 GBq / ml in ACN, Vitrax Co., 2.84 GBq / pmol) was added to the 2 ml HPLC vial and solvent was evaporated by cautiously introducing helium gas to the reaction vial. Afterwards, solution of 49 was cooled down and added to the reaction vial with dry [3H] methyl nosylate and was left to react for few seconds at room temperature. Reaction was monitored by analytical HPLC method (isocratic conditions, Luna, 5pm, C18(2), 100A, 250 x 4.6 mm; Iml / min; ACN : water = 50 : 50, 254 nm; tr ([3H]methyl nosylate) = 8.48 min, [3H]SKP-12) = 10.15 min; and isocratic conditions, Luna, 5pm, C18(2), 100A, 250 x 4.6 mm; Iml / min; ACN : water = 65 : 35, 254 nm; tr ([3H]SKP-12) = 6 min, [3H]SKP-03 = 11.21 min). When no [3H] methyl nosylate was present, the reaction mixture was quenched with 1 ml solution (ACN : water = 50 : 50) and was injected on semipreparative HPLC (isocratic conditions, Alltima, ACN : miliQ water = 60 : 40, 25 min; 254 nm; 3ml / min; retention time ([3H]SKP12) around 14 min and [3H]SKP03) around 23) and fractions (30 sec) were collected. Afterwards, fractions with retention time between 18 min and 28 min were studied by using analytical HPLC method (isocratic conditions, Luna, 5pm, C18(2), 100A, 250 x 4 6 mm; Iml / min; ACN : water = 65 : 35, 254 nm). Only fractions that were pure were combined and added to x mL of miliQ water. This was transferred over a conditioned plus short tC18 SPE column which was rinsed with miliQwater (5 mL) and air (20 mL). Product was eluted with 1.49 ml EtOH (accurately measured with Hamilton syringe). [3H]SKP03 (2.4 MBq) was obtained with 2 % RCY, > 99% RCP, chemical purity, as determined on analytical HPLC. Successful labeling was confirmed with co-inj ection with standard reference. [3H]SKP12 (9.4 MBq) was obtained with 8 % RCY, > 99% RCP, chemical purity, as determined on analytical HPLC. Successful labeling was confirmed with co-inj ection with standard reference.Radiosynthesis of [3H]SKP05 ([3H]19)

[0120] Manual labeling of [3H]SKP05 was carried out by adding approximately 50 pL of [3H]methyl nosylate ( 92.5 GBq / ml in ACN, Vitrax Co., 2.84 GBq / pmo) to the 2 mL HPLC vial. Solvent (ACN) was evaporated by cautiously introducing helium gas to the reaction vial (inlet helium gas, outlet porapak column). Solution of precursorcompound 50 (0.34 mg, 1.2 pmol) dissolved in anhydrous DMSO (0.3 ml) and anhydrous CS2CO3 (1.6 mg, 4.8 pmol) was left stirring for 30 min at 60°C. Afterwards, vial with precursor was cooled down and added to the reaction vial with dry [3H|methyl nosylate and was left to react for few seconds at room temperature. Reaction was monitored by analytical HPLC method (isocratic conditions, Luna, 5pm, C18(2), 100A, 250 x 4.6 mm; Iml / min; ACN : miliQ water = 55 : 45, 25 min; 254 nm). When no [3H]methyl nosylate was present, the reaction mixture was quenched with 1ml solution (ACN : water = 50 : 50) and was injected on semi-preparative HPLC (isocratic conditions, Alltima, ACN : miliQ water = 50 : 50, 30 min; 254 nm; 3ml / min; retention time ([3H]SKP05) around 33 min) and fractions (30 sec) were collected. Afterwards, fractions with retention time between 30 min and 36 min were studied by using analytical HPLC method. Only fractions that were pure were combined and added to 10 mL of miliQ water. This was transferred over a conditioned plus short tC18 SPE column which was rinsed with miliQ water (5 mL) and air (20 mL). Product was eluted with 1.43 ml EtOH (accurately measured with Hamilton syringe). [3H]SKP05 (14.1 MBq) was obtained with 31% RCY, > 99% RCP and chemical purity, as determined on analytical HPLC. Successful labeling was confirmed with co-inj ection with standard reference.Radiosynthesis of [3H]SKP07 ([3H]22)

[0121] Manual labeling of [3H] SKP07 was carried out by adding approximately 55 pL of [3H]methyl nosylate ( 92.5 GBq / ml in ACN, Vitrax Co., 2.84 GBq / pmol) to the 2 mL HPLC vial. Solvent (ACN) was evaporated by cautiously introducing helium gas to the reaction vial (inlet helium gas, outlet porapak column). Solution of compound 51 (0.51 mg, 1.2 pmol) dissolved in anhydrous DMSO (0.3 ml) and anhydrous CS2CO3 (1.6 mg, 4.8 pmol) was left stirring for 30 min at 60°C. Afterwards, vial with precursor was cooled down and added to the reaction vial with dry [3H]methyl nosylate and was left to react for few seconds at room temperature. Reaction was monitored by analytical HPLC method (isocratic conditions, Luna, 5pm, C18(2), 100A, 250 x 4.6 mm; Iml / min; ACN : miliQ water = 65 : 35 , 25 min; 254 nm). When no [3H]methyl nosylate was present, the reaction mixture was quenched with 1ml solution (ACN : water = 50 : 50) and was injected on semi-preparative HPLC (isocratic conditions, Alltima, ACN : miliQ water = 65 : 35, 25 min; 254 nm; 3ml / min; retention time ([3H]SKP07) around 25 min) and fractions (30 sec) were collected. Afterwards, fractions with retention time between 20 min and 30 min were studied by using analytical HPLC method. Only fractions that were pure were combined and added to 10 mL of miliQ water. This was transferred over a conditioned plus short tC18 SPE column which was rinsed with miliQ water (5 mL) and air (20 mL). Product was eluted with 1.42 ml EtOH (accurately measured with Hamilton syringe). [3H]SKP07 (14.2 MBq) was obtained with 27 % RCY, > 99% RCP and chemical purity, as determined on analytical HPLC successful labelling was confirmed with co-inj ection with standard reference.Radiosynthesis of [3H] SKP08 ([3H] 16)

[0122] Manual labeling of [3H]SKP08 was carried out by adding approximately 70 pL of [3H]methyl nosylate ( 92.5 GBq / ml in ACN, Vitrax Co., 2.84 GBq / pmol) to the 2 mb HPLC vial. Solvent (ACN) was evaporated by cautiously introducing helium gas to the reaction vial (inlet helium gas, outlet porapak column). Solution of compound 55 (0.50 mg, 1.2 pmol) dissolved in anhydrous DMSO (0.3 ml) and anhydrous CS2CO3 (5 mg, 15.34 pmol) was left stirring for 30 min at 60°C. Afterwards, vial with precursor was cooled down and added to the reaction vial with dry [3H]methyl nosylate and was left to react for few seconds at room temperature. Reaction was monitored by analytical HPLC method (isocratic conditions, Luna, 5pm, C18(2), 100A, 250 x 4.6 mm; Iml / min; ACN : miliQ water = 60 : 40; 254 nm). When no [3H]methyl nosylate was present, the reaction mixture was quenched with 1ml solution (ACN : water = 50 : 50) and was injected on semi-preparative HPLC (isocratic conditions, Alltima, ACN : miliQ water = 55 : 45, 25 min; 254 nm; 3ml / min; retention time ([3H]SKP08) around 20 min) and fractions (30 sec) were collected. Afterwards, fractions with retention time between 15 min and 25 min were studied by using analytical HPLC method. Only fractions that were pure were combined and added to 10 mL of miliQ water. This was transferred over a conditioned plus short tC18 SPE column which was rinsed with miliQ water (5 mL) and air (20 mL). Product was eluted with 1.44 ml EtOH (accurately measured with Hamilton syringe). [3H]SKP08 (33.16 MBq) was obtained with 51 % RCY, > 98% RCP and chemical purity, as determined on analytical HPLC. successful labeling was confirmed with co-inj ection with standard reference.Radiosynthesis of carbon-11 labelled compounds

[0123] [nC] CO2 was trapped in a solution of LiAlH4 in THF (0.1 ml) at room temperature by helium flow(10 ml / min). Afterwards, the solution was heated at 130 °C and the helium flow was increased to 100 ml / ml. After a few minutes, helium flow was adjusted to 20 ml / min and HI (55% solution, 0.2ml) was added. [nC]CH3lwas trapped into a second reaction vial with solution of precursor (1.2 pmol) and DMSO (300 pL) and anhydrous CS2CO3 (15.34 pmol). After complete trapping of [11C]CH3l, the reaction was stirred at 40 °C for 2 min. Afterwards, it was quenched with the 2 ml of ACN / water (1 / 1) and injected on preparative HPLC. When the correct product was eluted, it was collected and diluted with water (40 ml). The solution was concentrated on SepPak, rinsed with water (10ml) and subsequently eluted with ethanol (96%) and diluted with saline solution to give final solution of ethanol < 10%. The molar activity was calculated against a calibration curve using the same HPLC system. Successful labeling was confirmed with co-inj ection with standard reference.COMPARATIVE STUDY

[0124] Comparison of SKP radioligands with125I-BJ1094 (125I-21) (compound of prior art)

[0125] In vitro binding determined by in vitro autoradiography:Following the manuscript on125I-21 (https: / / doi.org / 10.1007 / sl l307-023-01814-9), in vitro autoradiography was not performed. It is the gold standard for novel radioligand characterization. The homogenate assay is not sufficient to be used, scientifically, to prove binding of radioligand to alpha synuclein. Recombinant fibrils are not representative to human pathology, therefore cannot be used for accurate characterization of binding (https: / / doi.org / 10.3390 / phl4090847). Following thorough discussion on the need of in vitro autoradiography vs homogenate assay (https: / / doi.org / 10.1021 / acs.jmedchem.3c00779), it was concluded that the homogenate assay needs support by in vitro autoradiography data. Therefore, it is challenging to draw any conclusion whether 125I-21 binds to human pathology in MSA, PD or DLB postmortem tissue. Nonetheless,125I-21 was radiolabeled using tritium (3H-21) and the ligand in fibril injected mouse model was studied. Comparison with some of ligands of the present invention is provided below.Present radioligands bind to alpha synuclein and this is supported not only by recombinant fibrils but also by detailed in vitro autoradiography and immunostaining, which is exemplified in Figures. This is the result of structure activity relationship study, which was executed based on rational drug design, ligand-based design. With the chemical changes (left ring, right ring and core structure) of125I-21 , the change of binding was exemplified which is supported by in vitro autoradiography. Some chemical changes were detrimental for binding while others show increased binding to either MSA or PD tissue.

[0126] Comparative study of present radioligands with125I-21 using fibril injected mouse model is provided as follows:o CNS MPO calculation shows that ligands of the present invention have higher prediction to enter the brain and have better physiochemical properties. This is based on the calculation of CNS MPO (Table 1) that is > 3.8 for all radioligands of the present invention. CNS PET MPO value is either in a similar range or above 3.0. o Calculation of in vitro contrast (Table 1) for all our radioligands is > 7.1, except for SKP07. This indicates that some of our radioligands have better interaction with alpha synuclein fibrils or lower nonspecific binding.

[0127] Some of the radioligands of the present invention show selectivity over amyloid beta, which is one of the main prerequisite for alpha synuclein tracer development due to co-existence and colocalization with other amyloid proteins (e.g. amyloid beta and tau). The selectivity profile of some of the tracers of the present invention is in the similar range to recently published PET tracers (https: / / doi.Org / 10.21203 / rs.3.rs-2189800 / yl ). For125I-21 , it is difficult to know whether the ligand binds to amyloid beta or tau because of the lack of in vitro autoradiography experiments.

[0128] In vivo pharmacokinetic profile: some of the PET tracers of the present invention show superior properties (higher brain uptake, better time-activity curve which shows clear brain uptake and fast clearance, because of the use of healthy animals - no biological target in the brain; and lower nonspecific binding). These properties are essential for further characterization of tracer in human neuroimaging.Table 1. I / / silico parameters, CNS MPO, CNS PET MPO, in vitro contrast and in vivo brain uptake.

[0129] Comparison of SKP radioligands with TG-1-90B (compound of prior art)Present alpha synuclein compounds are not similar to TG-1-90B. The only similarity is with SKP04 where the change is in amide bond, used as a linker. This is a common bioisosterism and is widely used in the rational modification of lead compounds [https: / / doi.org / 10.1021 / acs.jmedchem.0c00530]. Additionally, following the reference (https: / / doi.org / 10.1021 / acsomega.7b01897) some of radioligand of the present invention show superior binding properties to alpha synuclein.

[0130] Comparison of present radioligands with the3H-ACI12589 (R-isomer) benchmark compoundHead to head comparison with3H-ACI12589 revealed the following: - Radioligands of the present invention can recognize alpha synuclein in MSA tissue (exemplified inFigure 27). Medium to strong autoradiographic signal is detected for3H-SKP compounds. On the other hand, no / low signal is detected from3H-ACI12589.

Claims

CLAIMS1. A compound represented by Formula (I):Formula (I) or a radioactively labelled compound or pharmaceutically acceptable salt thereof, wherein:Xi is C or N,B is C or N,Y or Z is independently selected from O, N or C,Wherein Zi represents phenyl, pyridyl, pyridone, N-methyl-2-pyridone, optionally substituted with one or more substituents selected from C1-3 alkyl, hydroxy, C1.3 alkoxy, halogen, a mono, bi or trifluorinated methoxy group; andRi is selected fromor2. The compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof according to claim 1, wherein Zi represents phenyl, pyridyl, pyridone, N-methyl-2-pyridone optionally substituted with one or more substituents selected from methyl, methoxy, hydroxy, flourine, iodine.

3. The compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the compound is represented by Formula la:WhereinXi or X2 each independently selected from C or N,R2 is H or CH3R3 is H or FR4 is H or CH3 or a mono, bi or tri-fluorinated methyl group, provided that if X1=C, then R1 is not4. The compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof according to any of claims 1 or 2, wherein the compound is represented by Formula lb:Wherein R5 or Rg each independently selected from I, OCH3 or a mono, bi or tri-fluorinated methoxy group.

5. The compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof according to any of claims 1 or 2, wherein the compound is represented by Formula IcWhereinY or Z each independently selected from O, N or CR? is H, CH3 or a mono, bi or tri -fluorinated methyl group.

6. A compound represented by Formula (II):or a radioactively labelled compound or pharmaceutically acceptable salt thereof,Wherein:Xi or X2 each independently selected from C or N,Y or Z or B is independently selected from N or C,R9is H or CH3RIO is H or FR11 is H, CH3 or a mono, bi or tri-fluorinated methyl group; provided that if Y = N, Z=O, B=C, and X1=C, then R8 is not7. A compound represented by Formula (III):R13 is H, CH3 or a mono, bi or tri-fluoro methyl group.

8. The compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 7, wherein the compound is selected from the group comprising of:

9. The compound or pharmaceutically acceptable salt thereof according to any of the preceeding claims, wherein the radioactively labelled compounds are radiolabeled with Carbon- 11 (nC), Fluorine-18 (18F) or Tritium (3H) or a radionuclide of Iodine.

10. A composition comprising a compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof according to any of the preceding claims 1 to 9 and at least one pharmaceutically acceptable excipient.

11. The compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 9 or a composition according to claim 10, for use as radioctive tracer compounds in monitoring a disease therapy in an individual.

12. The compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 9 or a composition according to claim 10, for use in the monitoring of an early stage of diseases, preferably Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies.

13. The compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 9 or a composition according to claim 10, for use in in vivo monitoring of an early stage of diseases, preferably Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies.

14. A method for making a radioactively labelled compound according to any of the preceding claims 1 to 8, wherein radioactively unlabelled variant of compounds of claims 1 to 8 are radiolabelled with Carbon-11 (nC), Fluorine-18 (18F), or Tritium (3H), or a radionuclide of Iodine.

15. A method for detection of the aggregation of alpha-synuclein protein, comprising contacting said protein with an amount of radioactively labelled compound according to any of the preceding claims 1 to 9 which is sufficient to be detected by Positron Emission Tomography (PET) by forming at least one PET image and determining the aggregation of alpha-synuclein protein by observing the image.

16. A compound or a radioactively labelled compound or pharmaceutically acceptable salt thereof according to any of claims 1 to 9 or a composition according to claim 10, for use in method oftreating a disease selected from Parkinson’s disease, Multiple System Atrophy, Dementia with Lewy Bodies.

Images