2-styryl benzothiazole derivatives as alpha-synuclein binding compounds

Abstract

The present invention relates to alpha-synuclein binding compounds, diagnostic methods, the use of the alpha-synuclein binding compounds in such diagnostics, and diagnostic compositions comprising the alpha-synuclein binding compounds.

Description

[0001] This invention relates to α-synuclein binding compounds, diagnostic methods, the use of the α-synuclein binding compounds in such diagnostics, and diagnostic compositions comprising the α-synuclein binding compounds. Invention Field

[0002] This invention relates to the field of visualization of the spatial distribution of structures in mammals, and more particularly to the development of small molecules for visualizing brain structures for diagnostic and research purposes, and more specifically, to compounds for the diagnosis and research of synuclein diseases. Background Technology

[0003] Synucleopathies are a complex group of neurodegenerative diseases characterized by the abnormal accumulation of α-synuclein (αSYN) protein aggregates in neurons, nerve fibers, or glial cells. These aggregates form intracellular αSYN fibrils. Typical examples of synucleopathies include Parkinson's disease (PD), multiple system atrophy (MSA), and Lewy body dementia (DLB). The pathogenesis of synucleopathies is not fully understood, and diagnosis is challenging due to the wide variety of symptoms caused by different synucleopathies and other neurodegenerative diseases, such as Alzheimer's disease (AD) characterized by β-amyloid (Aβ) accumulation or Tau protein diseases characterized by highly phosphorylated Tau protein accumulation.

[0004] A crucial prerequisite for the treatment and research of synucleinosis is the ability to diagnose such diseases as early as possible, or in other words, reliable techniques for in vivo detection of αSYN for clinical purposes and reliable techniques for in vitro or ex vivo detection for research purposes.

[0005] Detection of αSYN is challenging because Aβ or Tau fibrils are structurally similar to αSYN fibrils, making them difficult to distinguish. Furthermore, αSYN abundance in the human brain is at least 10 times lower than that of Aβ. Therefore, detection must possess high sensitivity and selectivity for αSYN. Another challenge is that αSYN accumulates intracellularly, making it difficult for drugs, detection agents, or αSYN-binding compounds to approach αSYN as targets in vivo, even if they may bind to or otherwise interact with αSYN. In addition, because αSYN fibrils are located within the brain, drugs or detection agents must cross the blood-brain barrier (BBB).

[0006] There are currently no such drugs or test kits.

[0007] Kaide, S. et al., Synthesis and Evaluation of (18) F-Labeled Chalcone Analogue for Detection of alpha-Synuclein Aggregates in the Brain Using the Mouse Model; ACS Chem Neurosci 2022, 13(20), 2982-2990, disclosed 4-nitrophenyl chalcone derivatives as αSYN-binding compounds. However, brain clearance was not ideal. Furthermore, options for improving the pharmacokinetic characteristics of these 4-nitrophenyl chalcone derivatives through structural optimization such as reducing lipophilicity are very limited.

[0008] Gaur, P. et al., Fluorescent Probe for Selective Imaging of α-Synuclein Fibrils in Living Cells; ACS Chemical Neuroscience 2021, 12(8), 1293–1298, disclosed a 2-styrylbenzothiazole derivative, named RB1, as an αSYN-binding compound. The structure of RB1 is derived from thioflavone T, which is known to bind to Aβ fibrils and be used for detection. Compared to thioflavone T, which does not bind to αSYN, RB1 exhibits limited selectivity for αSYN relative to Aβ. It is speculated that RB1 cannot provide a good signal in the brain due to its structural characteristics, as these characteristics prevent its accumulation in the brain.

[0009] In May 2022, derivatives of the RB1 structure were disclosed at the International Scientific Symposium on Radiopharmaceuticals (iSRS) in Nantes, France. However, these disclosed structures are incomplete, not fully defined, and do not demonstrate any potential applications of these compounds in diagnosing synucleinopathy or even in binding with αSYN. The applicant found that these compounds do not accumulate in the mammalian brain.

[0010] One object of the present invention is to overcome the shortcomings and deficiencies of the prior art. Specifically, one object of the present invention is to provide reliable α-synuclein binding compounds for in vivo, in vitro and ex vivo detection of α-synuclein for diagnostic and research purposes.

[0011] This invention completely solves this problem. Invention Overview

[0013] In one aspect of the invention, the above-mentioned disadvantages are overcome by providing an α-synuclein binding compound having structure 1.

[0014]

[0015] - Where X1-X6 are independently selected from: CR x , N, where each R x x in R is equal to x The subscript of X is combined if at most one X is N in the group consisting of X1 and X2 and in the group consisting of X3 and X4; and

[0016] -Among them, R7, R8 and R x Independently selected from: H, F, Cl, Br, OH, C1-C3 alkyl ether groups, fluorinated C1-C3 alkyl groups, and preferably selected from: H, O-CH3, F; and

[0017] - X7-X9 are independently selected from CH2, O, NH, and S, and are preferably selected from CH2 and O, provided that no heteroatom is directly bonded to the heteroatom.

[0018] In the following text, any R or X, and any R or X containing any subscript (e.g., R1, R...), refers to... x (or X1) should be understood as placeholders, which do not refer to any atom or group of atoms. In the following text, the definition of such placeholders is based on the atom or group of atoms they may represent.

[0019] When referring to αSYN, αSYN binding, or affinity for αSYN, it should be understood to include αSYN fibrils, binding to αSYN fibrils, or affinity for αSYN fibrils, respectively. The term "αSYN fibrils" can refer to αSYN fibrils or other types of non-physiological αSYN accumulations that can form in mammals.

[0020] Surprisingly, the compound having the structure shown in Structure 1 exhibits a higher affinity for αSYN, and more importantly, a higher selectivity relative to Aβ for αSYN, than prior art compounds. It turns out that the affinity and selectivity of the compound having the structure shown in Structure 1 are strongly triggered by the structural modification at the placeholder position in Structure 1.

[0021] Selecting R5 (bonded with X5), R6 (bonded with X6), R7, and R8 independently from the group consisting of H, F, Cl, Br, OH, C1-C3 alkyl ether groups, fluorinated C1-C3 alkyl groups, and fluorinated C1-C3 alkyl ether groups did indeed particularly favorably increase the compound's affinity for αSYN. However, this selection also had a positive impact on other properties of the compound.

[0022] The C1-C3 alkyl ether group is preferably a methyl ether group (O-CH3). The fluorinated C1-C3 alkyl group is preferably 2-fluoroethyl (CH2-CH2-F). The fluorinated C1-C3 alkyl ether group is preferably a 2-fluoroethyl ether group (O-CH2-CH2-F).

[0023] Selecting X7, X8, and X9 independently from the group consisting of CH2, O, NH, and S did indeed improve pharmacokinetics particularly favorably by reducing the lipophilicity of the compound. However, this selection also had a positive impact on other properties of the compound. Avoiding direct adjacency of two heteroatoms ensured that the reactivity of the compound remained at an appropriate level.

[0024] Independently from CR x Select X1 to X6 from the group consisting of R and N (where each R) x x in R is equal to x Combining the subscript of X has the following advantages: by choosing X as N, the lipophilicity of the compound can be further reduced, thereby further improving pharmacokinetics. Choosing X as CR... x This not only significantly improves the compound's affinity for αSYN, but also enhances other properties of the compound. However, this choice also has a positive impact on other properties of the compound. Similarly, avoiding direct proximity of two heteroatoms ensures that the reactivity of the compound remains limited to an appropriate level.

[0025] Compounds of structure 1 have the advantage of readily crossing the blood-brain barrier (BBB). Unbound by this theory, it is presumed that this is at least in part due to the absence of a positively charged quaternary nitrogen atom (N) at the 3-position of the benzothiazole ring. This is more advantageous than known benzothiazole derivatives in the prior art [e.g., Gauretal. (ibid.)], which contain quaternary nitrogen and thus carry a permanently positive charge, which in part prevents them from crossing the blood-brain barrier for brain absorption. Conversely, the ability to cross the blood-brain barrier facilitates the entry of compounds according to the invention into the brain, thereby into tissues known to contain αSYN, and into tissues where αSYN accumulation is known to cause disease.

[0026] Compared to compounds provided by existing technologies, the compound of structure 1 exhibits a high affinity for binding to αSYN fibrils. This advantageously allows for a reduction in the amount of compound used to target αSYN. Hereinafter, "targeting αSYN" should be understood as contacting an αSYN-binding compound with a substance, material, organism, or animal suspected of containing αSYN, or applying an αSYN-binding compound to a substance, material, organism, or animal suspected of containing αSYN, wherein the substance, material, organism, or animal is preferably selected from: mammals, mammal-derived tissues, mammal-derived samples, mammal-derived tissue samples, and artificially generated samples. Preferably, the mammal is a human, more preferably a human suspected of having a synucleinosis.

[0027] Furthermore, the compound of structure 1 exhibits high selectivity for αSYN relative to Aβ. This facilitates the specific targeting of αSYN in brain tissue, where the abundance of Aβ is far higher than that of αSYN, by binding the αSYN-binding compound to αSYN rather than to Aβ. This allows for differentiation between αSYN and Aβ binding to the compound.

[0028] Another advantage is that compounds of structure 1 are well-suited for reducing lipophilicity, thereby further improving pharmacokinetics, as multiple sites in this structure allow for the introduction of hydrophilic structures or substituents without significantly affecting the affinity and selectivity for αSYN.

[0029] In another embodiment, X1 to X6 are CR x .

[0030] Instead of attempting to reduce lipophilicity, X1 to X6 are selected based on CR rather than N. x This can advantageously enhance the affinity or selectivity of compounds for αSYN.

[0031] In another aspect of this implementation, R1 to R4 are H.

[0032] Choosing H for R1 through R4 is advantageous in restricting the position of any substituent (such as F, Cl, Br, OH, C1-C3 alkyl ether groups, and fluorinated C1-C3 alkyl ether groups) that could introduce an -I- effect into the aromatic system to the benzothiazole heterocycle. This has proven particularly effective in enhancing the affinity and selectivity of compounds for αSYN. Furthermore, the position of fluorinated substituents (e.g., fluorinated C1-C3 alkyl, fluorinated C1-C3 alkyl ether groups, or F) is also typically restricted to the benzothiazole heterocycle in this manner, which has been shown to enhance the affinity of αSYN-binding compounds for αSYN and their selectivity for αSYN relative to Aβ.

[0033] In another aspect of this embodiment, R5, R6, and R7 are H.

[0034] Choosing R5, R6, and R7 as H advantageously restricts the position of any substituent that could introduce an -I- effect (e.g., F, Cl, Br, OH, C1-C3 alkyl ether groups, fluorinated C1-C3 alkyl and fluorinated C1-C3 alkyl ether groups) to the R8 position on the benzothiazole heterocycle, which has proven to be the most favorable position, especially considering the compound's affinity and selectivity for αSYN.

[0035] In another aspect of this implementation, R8 is F.

[0036] Of any R-optional substituents that introduce an -I- effect, F has been shown to be the substituent that provides the highest affinity and selectivity for αSYN.

[0037] In another implementation, X7 to X9 are selected, provided that at most one of X7 to X9 is not CH2.

[0038] Advantageously, by selecting only one of X7 to X9 as CH2, O, NH or S, the affinity or selectivity of the compound for αSYN can be increased, rather than reducing lipophilicity.

[0039] In another embodiment, the αSYN-binding compound has structure 2 or structure 3.

[0040]

[0041] αSYN-binding compounds with structure 2 or structure 3 advantageously exhibit the highest affinity and selectivity for αSYN. Furthermore, these αSYN-binding compounds demonstrate favorable pharmacokinetics, whereby, after intravenous injection into the bloodstream of mammals, they distribute to the site of action, primarily the mammalian brain, where they are subsequently metabolized and cleared, thus reducing the burden on the corresponding mammal.

[0042] In one aspect of this embodiment, the compound is fluorescent.

[0043] The fluorescence of αSYN-binding compounds advantageously allows for the detection of the compound by detecting the fluorescence, for example, when targeting αSYN.

[0044] "Detection" should be understood as any form of qualitative or quantitative proof of the presence of an αSYN-binding compound, wherein the presence of the αSYN-binding compound can be visualized or located, for example, in a relative or absolute manner.

[0045] The fluorescence of a compound is advantageous, particularly for detecting, visualizing, or locating αSYN in mammalian tissues, samples derived from mammalian tissues, or artificially generated samples suspected of containing αSYN, under in vitro or ex vivo conditions. This is because the emitted fluorescence is less likely to be covered, shielded, or absorbed by the tissue surrounding the αSYN binding site. Furthermore, fluorescence detection is a commonly used technique in science and medicine. Fluorescence not only aids in the detection itself but also eliminates the need for αSYN-binding compounds to bind to other fluorescent compounds. This binding is a common method, such as with fluorescently labeled compounds or antibodies, but it can adversely interfere with the affinity and / or selectivity of the αSYN-binding compound for αSYN.

[0046] In another aspect of this embodiment, at least one atom of the compound is a radionuclide.

[0047] The presence of radionuclides that are atoms of αSYN-binding compounds advantageously allows for the detection of radiation, for example, when targeting αSYN.

[0048] Radiation detection of compounds is advantageously suited, particularly for the detection, visualization, or localization of αSYN in mammalian tissues, any samples derived from mammalian tissues, or artificially generated samples, whether in vivo or in vitro, where the binding site of the αSYN-binding compound is covered by surrounding tissue or any other material that shields or absorbs fluorescence. Furthermore, the use of radionuclides offers the advantage that, due to the appropriate selection of the radionuclide, the duration (half-life), range, intensity, and type of radiation can be adjusted and adapted to the desired purpose. Radioactivity can overcome the coverage, shielding, or absorption of αSYN-binding compounds by surrounding tissue or other materials at the αSYN binding site. Moreover, radiation detection is a commonly used technique in science and medicine.

[0049] In this embodiment, the αSYN-binding compound containing a radionuclide is preferably used as a so-called "tracer" in positron emission tomography (PET) or single-photon emission computed tomography (SPECT). A tracer is a molecule incorporated into or bound to target cells or target structures in a mammalian body, wherein the molecule can be "tracked" or detected by a method that is advantageously non-invasive, such as PET or SPECT.

[0050] In another aspect of this embodiment, at least one C atom is 11 C radionuclide. 11 C is a radioactive nuclide that emits positrons, thus advantageously allowing the application of αSYN-bound compounds in PET. Furthermore, because... 11C has a short half-life (approximately 20 minutes), making it a short-lived radionuclide, which helps minimize radiation exposure to living organisms. Furthermore, 11 C is widely used in PET imaging using small molecules, so this compound has accumulated empirical value, and appropriate workflows have been established in laboratories and clinics. Furthermore, any αSYN-binding compound disclosed herein contains at least one C atom, therefore this embodiment can be used in combination with any such compound.

[0051] In another aspect of this embodiment, at least one F atom is 18 F is a radioactive nuclide. 18 F is a positron-emitting radioactive nuclide, thus advantageously allowing the application of αSYN-binding compounds in PET. Furthermore, because... 18 F has a short half-life (approximately 110 minutes), making it a short-lived radionuclide, which helps minimize radiation exposure to living organisms. Furthermore, 18 F is widely used in PET imaging using small molecules, therefore, this compound has accumulated empirical value, and appropriate workflows have been established in laboratories and clinics. When transportation or other methods are required to extend the processing time of αSYN-binding compounds, relative to using... 11 C can be preferred. 18 F, because 18 The half-life ratio of F 11 C has a longer half-life.

[0052] Surprisingly, 18 F also leads to an increased affinity of αSYN compounds for αSYN, especially for αSYN compounds with structures 2 and 3.

[0053] On the other hand, the present invention relates to the use of the αSYN binding compound for diagnosing diseases, wherein the diseases are preferably neurodegenerative diseases, more preferably neurodegenerative diseases associated with the presence of non-physiological α-synuclein in mammalian tissues.

[0054] In this article, the term "disease" in medicine should generally be understood as any type of non-physiological condition in various organisms (e.g., mammals), which may be caused by, for example, genetic, tumor, microbial, bacterial, viral, or psychological factors. The term "disease" can also refer to any type of non-physiological condition that may be caused by, for example, physical damage to the organism's body or aging.

[0055] Advantageously, αSYN-binding compounds used for disease diagnosis can diagnose any disease unrelated to the presence of non-physiological α-synuclein in mammalian tissues. This is particularly advantageous for neurodegenerative diseases unrelated to the presence of non-physiological α-synuclein in mammalian tissues.

[0056] αSYN-binding compounds exhibit high affinity for αSYN and high selectivity for αSYN relative to Aβ. Therefore, using αSYN-binding compounds to diagnose neurodegenerative diseases advantageously allows for the differentiation of different types of neurodegenerative diseases, which are often difficult to distinguish due to overlapping pathogenesis and / or symptoms, by demonstrating or refuting the presence of αSYN accumulation in the mammalian brain. This facilitates early diagnosis of neurodegenerative diseases and their types, enabling earlier initiation and appropriate or precise treatment.

[0057] In this embodiment, using αSYN-binding compounds to diagnose neurodegenerative diseases associated with the presence of non-physiological α-synuclein in the tissues of organisms (e.g., mammals) can provide a reliable positive diagnosis of such neurodegenerative diseases by reducing the probability of false negative diagnoses. To date, no other compound has shown reliable αSYN selectivity relative to Aβ, which could lead to misdiagnosis, for example, of neurodegenerative diseases associated with enhanced αSYN accumulation as neurodegenerative diseases associated with enhanced Aβ accumulation.

[0058] The presence of non-physiological αSYN in the tissues of organisms (e.g., mammals) can be an increase or decrease in concentration, spatial distribution, or presence at any site in the tissue, which may lead to acute or chronic disease, or is expected to lead to any type of disease in the future.

[0059] In embodiments where one atom of the αSYN-binding compound is a radionuclide, the high affinity of the αSYN-binding compound for αSYN allows for a reduction in the amount of αSYN-binding compound used when targeting αSYN. This advantageously reduces the risk of exposure to potentially pathogenic or other damaging radiation for mammals (when using the αSYN-binding compound to diagnose diseases by targeting αSYN in an organism, such as a mammal) or users of the αSYN-binding compound (when using the αSYN-binding compound to diagnose diseases by targeting αSYN).

[0060] In another embodiment, the αSYN-binding compound can be used to diagnose synucleinosis in organisms, wherein the synucleinosis is preferably selected from Parkinson's disease, multiple system atrophy, and Lewy body dementia.

[0061] Synucleinopathy is a neurodegenerative disease known to be associated with the presence of non-physiological α-synuclein in the tissues of organisms (e.g., mammals). Therefore, the aforementioned advantages of using this compound to diagnose neurodegenerative diseases associated with the presence of non-physiological α-synuclein in mammalian tissues also apply to synucleinopathy.

[0062] On the other hand, the present invention relates to a diagnostic composition comprising an α-synuclein binding compound according to the invention and a pharmaceutically acceptable carrier.

[0063] Suitable carriers for a particular diagnostic purpose will be clear to those skilled in the art. Further disclosures regarding pharmaceutically acceptable carriers can be found, for example, in Trucillo, P. Drug Carriers: Classification, Administration, Release Profiles, and Industrial Approach. Processes 2021, 9, 470, the full text of which is incorporated herein by reference.

[0064] The diagnostic composition can be applied to any material or substance, preferably to a material or substance that targets αSYN and detects information about αSYN-binding compounds, based on which a disease can be diagnosed, for example by a physician.

[0065] In one embodiment, the diagnostic composition is configured for application to an organism, preferably a mammal.

[0066] Applying diagnostic compositions to organisms facilitates early diagnosis and tracking of disease progression, thereby advantageously allowing for early or preventative treatment or intervention once the disease is diagnosed. Furthermore, treatment can be tailored to the progression of the disease.

[0067] On the other hand, the present invention relates to a method for diagnosing diseases in organisms, wherein the diseases are preferably neurodegenerative diseases, more preferably neurodegenerative diseases associated with the presence of non-physiological α-synuclein in mammalian tissues, the method comprising targeting αSYN and detecting αSYN-binding compounds.

[0068] Targeting may include systemic or local administration of an αSYN-binding compound or a diagnostic compound containing an αSYN-binding compound to an organism (e.g., a mammal), preferably via intravenous injection. The organism is preferably a mammal. The mammal is preferably a human, more preferably a human suspected of having a synucleinosis.

[0069] It should be understood that the above features, as well as the features to be explained below, can be used not only in a given specific combination, but also in other combinations or individually, without departing from the scope of the invention.

[0070] The invention will now be described and explained in further detail with reference to the following non-limiting embodiments and accompanying drawings. Attached Figure Description

[0071] Figure 1 Development of a library of 2-styrylbenzothiazole compounds and the structure of fluorescent probes RB1 and RB2.

[0072] Figure 2 General synthetic routes for N-methylated and non-methylated 2-styrylbenzothiazole: a) DMSO, 18M NaOH aqueous solution, room temperature, 2 h to 24 h; b) MeCN, MeI or MeNs, 80 °C, overnight; c) EtOH, 80 °C, overnight.

[0073] Figure 3 Synthetic routes for analogs with altered π system lengths: a) Pd(PPh3)4, Cs2CO3, piperidine, toluene, 110℃, 5h; b) MeNs, chlorobenzene, 80℃, overnight; c) concentrated H2SO4, acetaldehyde, 0℃, 1h; d) DMSO, 18M NaOH aqueous solution, room temperature, 3h; e) EtOH, 80℃, overnight.

[0074] Figure 4 a) The structure of compounds with promising features and their pathways [ 3 The αSYN binding affinity (average K) determined by H]PiB competition assay i (a) ±SEM, three data points); b) Determination of competitive binding of compounds 14a, 17a, 43, and 44 to Aβ fibrils.

[0075] Figure 5: a) PFSB(9a) and b) 9b 2D 1 H NOESY NMR spectrum. In spectrum (a), H8 and H 2b H 6b The weak interaction between them indicates that the sample is a mixture of (E)-9a and (Z)-9a. This interaction was not detected in spectrum (b), indicating that (Z)-9b is the preferred configuration of the N-methylated compound.

[0076] Figure 6 a) Development of MFSB analogs with lower lipophilicity; b) Through [ 3 H]PiB competition assay (each repeated three times) a= Repeat twice) Determine the binding affinity of PFSB, MFSB, and 15a to αSYN and Aβ fibrils; c) PFSB and MFSB with [ 3 H]MODAG-001 competes with αSYN binding.

[0077] Figure 7 Synthesis of BPi precursors and general synthetic routes for fluorine-18 labeling: a) B2Pin2, KOAc, Pd(dppf)Cl2, DMF, 100℃, 45min; b) Cu(Otf)2, pyridine, n-butanol, [ 18 F]TBAF, DMA, 120℃, 20min.

[0078] Figure 8 :[ 18 Example of analytical QC HPLC analysis of F]PFSB and its non-radioactive reference compounds, using a Luna column 5 μm C18(2). 250 x 4.6 mm, isocratic elution method: 88% MeCN (pH 8) in 25 mM ammonium formate, 1 mL / min. The ratio of the two peaks (retention times ≈ 10.1 and 10.5 min, respectively) depends on the ratio of the E / Z isomers.

[0079] Figure 9 :[ 18 Example of radioactive TLC of F]PFSB, eluent: PE / EtOAc 2:1.

[0080] Figure 10 :[ 18 F] MFSB and its non-radioactive reference compounds: QC HPLC example, using a Luna column, 5 μm C18(2). 250 x 4.6 mm, isocratic elution method: 72% acetonitrile in 25 mM ammonium formate (pH 8), 1 mL / min. The ratio of the two peaks (retention times ≈ 9.1 and 9.7 min) depends on the ratio of the E / Z isomers.

[0081] Figure 11 :[ 18 Example of radioactive TLC of F]MFSB, eluent: PE / EtOAc 1:1.

[0082] Figure 12 :[ 18 F]PFSB and [ 18 F]MFSB in vitro autoradiography on human brain slices, and corresponding IHC staining with αSYN (MSA, PD, and control (Ctrl) tissues) and Aβ (AD tissues). Scale bar represents a 0.5 cm dimension.

[0083] Figure 13 :[ 18 F] In vivo pharmacokinetic characteristics assessment of MFSB: a) Whole-body PET / MR sagittal images at different time points; b) Time-activity curves of brain regions; c) Time-activity curves of the whole brain, kidney, lung and liver. Example

[0084] 1. Materials and Methods

[0085] 1.1 Synthesis of Compounds

[0086] All chemicals were purchased from Sigma Aldrich (St. Louis, Missouri, USA), abcr GmbH (Karlsruhe, Germany), or Carl Roth (Karlsruhe, Germany) and were ready for use without further purification.

[0087] The reaction progress was monitored by thin-layer chromatography (TLC) and / or by analytical HPLC-MS (ESI detector, Agilent, Santa Clara, California, USA) on a 0.20 mm Polygram SIL G / UV254 (silica gel 60) TLC plate (Macherey-Nagel, Düren, Germany) using a selected eluent mixture. The analytical HPLC-MS was equipped with a Luna 5 μm C18 (2) filter. 50 x 2 mm column (Phenomenex, Torrance, California, USA) [gradient: 0–7.60 min (0% to 100% B), 7.60–8.80 (100% B), 8.80–9.30 min (100% to 0% B), 9.30–13.0 min (0% B); solvent A: 0.1% formic acid aqueous solution; solvent B: acetonitrile; 0.4 mL / min], or equipped with a Zorbax Eclipse XBD-C18 50 x 4.6 mm column (Agilent, Santa Clara, California, USA) [gradient: 0–6 min (0% to 100% B); solvent A: H2O:MeCN:formic acid 95:5:0.1 v / v%; solvent B: 0.1% formic acid in MeCN solution; 1 mL / min].

[0088] Purification was performed using an automated rapid chromatography method on an Isolera 4 system (Biotage, Uppsala, Sweden) or a CombiFlash NextGen 300+ (Tele-dyne ISCO, Lincoln, Nebraska, USA).

[0089] 1 H, 13 C and 19 The F NMR spectrum was obtained using an Avance III AV 600 ( 1 H: 600.13MHz; 13 C:150.61MHz) or Avance II AV 400 ( 1 H: 400MHz; 13 C: 101MHz; 19 Data were acquired using a 376 MHz (F) spectrometer (Bruker, Billerica, Massachusetts, USA). All chemical shifts (δ) are expressed in parts per million (ppm) and referenced to the residual solvent peak (CDCl3: δ). H =7.26,δ C =77.16; DMSO-d6:δ H =2.50,δ C =39.52).

[0090] 1.1.1. General Procedure A

[0091] Slowly add 6.50 mL of 18 M NaOH aqueous solution to a DMSO solution of the selected 2-methylbenzothiazole (6.70 mmol) and the selected 4-aminobenzaldehyde or 4-aminocinnamaldehyde (7.37 mmol). Stir the mixture at room temperature for 2 to 24 hours. A yellow precipitate forms. Dilute the mixture with water and allow it to precipitate completely. Filter the precipitate under vacuum to obtain a yellow solid, which is then recrystallized from EtOAc.

[0092] 1.1.2. General Procedure B

[0093] Following the method disclosed in Gaur, P. et al., Fluorescent Probe for Selective Imaging of α-Synuclein Fibrils in Living Cells; ACS Chemical Neuroscience 2021, 12(8), 1293–1298, a solution of the selected 2,3-dimethylbenzothiazolium salt (0.86 mmol) and the selected 4-aminobenzaldehyde or 4-aminocinnamaldehyde (1.83 mmol) in EtOH (7.00 mL) was refluxed overnight. The color changed to red / purple. The precipitate was filtered under vacuum, washed with EtOAc, and a dark solid was given, which was recrystallized with EtOH and / or Et2O.

[0094] 1.1.3. Single Compound

[0095] 4-Methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (1a)

[0096] Synthesized according to general procedure A (46 mg, 60%). f :0.48 (heptane / EtOAc 3:1). 1 H NMR (600MHz, CDCl3) δ7.69–7.63(m,1H),7.47(d,J=8.8Hz,2H),7.39(d,J=16.1Hz,1H),7.30(d,J=16.1Hz,1H),7.2 5–7.20(m,2H),6.91(d,J=8.7Hz,2H),3.28(t,J=5.6Hz,4H),2.75(s,3H),1.70(p,J=5.6Hz,4H),1.66–1.60(m,2H). 13 C NMR (151MHz, CDCl3) δ167.1,153.5,152.6,137.9,134.1,132.7,128.8,126.8,125.6,125.0,119.0,118.9,115.5,49.6,25.7,24.5,18.7.

[0097] 3,4-Dimethyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onthium-4-nitrobenzenesulfonate (1b)

[0098] Synthesized according to standard procedure B (119 mg, 59%). f :0.19 (DCM / MeOH 5%). 1H NMR(400MHz,DMSO-d6)δ8.19(d,J=8.3Hz,2H),8.12(br s,1H),8.01(d,J=15.3Hz,1H),7.89(d,J=8.5Hz,2H),7.83(d,J=8.2Hz,2H),7.66(d,J=15.3Hz ,1H),7.54(s,2H),7.04(d,J=8.7Hz,2H),4.43(s,3H),3.49(s,4H),2.91(s,3H),1.61(s,6H). 13 C NMR(101MHz,DMSO-d6)δ171.5,154.4,153.5,149.6,147.2,140.9,132.8,132.7,127.7 ,127.7,127.3,126.9,123.3,122.3,121.7,113.5,107.0,47.6,40.0,25.0,23.9,20.9.

[0099] 4-Methoxy-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (2a)

[0100] Synthesized according to standard procedure A (10 mg, 5%). f :0.18 (heptane / EtOAc 3:1). 1 H NMR (600MHz, CDCl3) δ7.45(dt,J=8.7,2.9Hz,2H),7.41(dd,J=8.0,0.9Hz,1H),7.38(d,J=16.1Hz,1H),7.31(d,J=16.1Hz,1H),7.28(t,J=8.0H z,1H),6.91(dd,J=8.8,2.7Hz,2H),6.88(dd,J=8.0,0.9Hz,1H),4.05(s,3H),3.27(t,J=5.6Hz,4H),1.70(p,J=5.6Hz,4H),1.65–1.60(m,2H). 13 C NMR (151MHz, CDCl3) δ166.9,153.3,152.6,144.2,137.7,135.8,128.8,126.0,125.6,119.0,115.5,113.6,106.7,56.0,49.6,25.7,24.5.

[0101] 4-Methoxy-3-methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (2b)

[0102] Synthesized according to standard procedure B (46 mg, quant.). R f :0.24 (DCM / MeOH 5%). 1 H NMR (600MHz, DMSO-d6) δ7.97(d,J=15.3Hz,1H),7.87(d,J=8.6Hz,2H),7.83(d,J=8.1Hz,1H),7.66–7.52(m,2H), 7.37(d,J=8.2Hz,1H),7.04(d,J=8.6Hz,2H),4.43(s,3H),4.04(s,3H),3.49(t,J=5.3Hz,4H),1.77–1.51(m,6H). 13 C NMR (151MHz, DMSO-d6) δ170.7,153.5,149.7,149.3,132.7,131.1,128.7,128.6,122.3,115.5,113.5,111.7,106.9,56.9,47.5,39.3,25.1,23.9.

[0103] 4-Fluoro-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (3a)

[0104] Synthesized according to standard procedure A (187 mg, 56%). f :0.43 (heptane / EtOAc 3:1). 1 H NMR(400MHz, CDCl3)δ7.58(dd,J=8.0,1.0Hz,1H),7.51–7.40(m,3H),7.30–7.24(m,2H),7.14(ddd,J=10.5, 8.2,1.0Hz,1H),6.91(dt,J=8.8,8.8,2.7Hz,1H),3.34–3.22(m,4H),1.76–1.66(m,4H),1.66–1.59(m,2H). 13 C NMR (101MHz, CDCl3) δ168.7,155.6(d,J=255.6Hz), 152.7,143.0(d,J=13.4Hz), 139.1,136.9(d,J=3.6Hz), 129.0, 125.6 (d, J = 7.0Hz), 125.1, 118.1, 117.2 (d, J = 4.3Hz), 115.3, 112.0 (d, J = 18.0Hz), 49.4, 25.6, 24.5. 19 F NMR (376MHz, CDCl3) δ-122.8.

[0105] 4-Fluoro-3-methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onthium-4-nitrobenzenesulfonate (3b)

[0106] Synthesized according to standard procedure B (165 mg, 76%). f :0.12 (DCM / MeOH 5%). 1 H NMR (400MHz, DMSO-d6) δ8.19(d,J=8.3Hz,2H),8.14–8.09(m,1H),8.06(d,J=15.3Hz,1H),7.91(d,J=8.6Hz,2H),7.83(d, J=8.4Hz,2H),7.73–7.57(m,3H),7.04(d,J=8.7Hz,2H),4.33(d,J=2.3Hz,3H),3.52(t,J=5.1Hz,4H),1.79–1.49(m,6H). 13 C NMR (101MHz, DMSO-d6) δ172.1,154.4,153.7,151.0,150.5(d,J=252.0Hz),147.2,133.3,130.5(d,J=9.8Hz),129.3,128.3(d, J=8.0Hz),126.9,123.3,122.2,120.1(d,J=4.0Hz),115.9(d,J=20.2Hz),113.4,106.0,47.5,38.3(d,J=11.1Hz),25.1,23.9. 19 F NMR (376MHz, DMSO-d6) δ-125.2.

[0107] 5-Methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (4a)

[0108] Synthesized according to standard procedure A (100 mg, 43%). f :0.44 (heptane / EtOAc 3:1). 1 H NMR (400MHz, CDCl3) δ7.75 (s, 1H), 7.69 (d, J = 8.1Hz, 1H), 7.46 (d, J = 8.6Hz, 2H), 7.42 (d, J = 16.1Hz, 1H), 7.21 (d, J = 16.1Hz, 1H), 7. 16(dd,J=8.1,1.6Hz,1H),6.91(d,J=8.6Hz,2H),3.28(t,J=5.5Hz,4H),2.49(s,3H),1.70(p,J=5.5Hz,4H),1.64(d,J=5.8Hz,4H).13 C NMR (101MHz, CDCl3) δ168.3,154.5,152.6,137.8,136.3,131.2,128.9,126.6,125.5,122.8,121.0,118.6,115.5,49.6,25.7,24.5,21.6.

[0109] 3,5-Dimethyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (4b)

[0110] Synthesized according to standard procedure B (278 mg, 99%). f :0.21 (DCM / MeOH 5%). 1 H NMR(400MHz,DMSO-d6)δ8.17(d,J=8.3Hz,1H),8.01(d,J=15.4Hz,1H),7.96(br s,1H),7.88(d,J=8.8Hz,2H),7.63(d,J=15.4Hz,1H),7.53(dd,J=8.3,1.5Hz,1H),7.0 4(d,J=8.8Hz,2H),4.21(s,3H),3.49(t,J=5.2Hz,4H),2.54(s,3H),1.71–1.50(m,6H). 13 C NMR (101MHz, DMSO-d6) δ171.3,153.5,149.2,142.2,139.3,132.7,128.8,123.9,123.3,122.3,115.9,113.4,107.0,47.5,35.6,25.1,23.9,21.1.

[0111] 5-Methoxy-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (5a)

[0112] Synthesized according to standard procedure A (170 mg, 58%). f :0.29 (heptane / EtOAc 3:1). 1 H NMR (400MHz, CDCl3) δ7.67(d,J=8.7Hz,1H),7.51–7.40(m,4H),7.18(d,J=16.1Hz,1H),6.98(dd,J=8.7,2.5Hz,1H) ,6.91(dt,J=8.8,2.9Hz,2H),3.89(s,3H),3.35–3.21(m,4H),1.77–1.65(m,4H),1.62(ddd,J=7.7,5.9,3.4Hz,2H).13 C NMR (101MHz, CDCl3) δ169.3,159.2,155.4,152.6,137.6,128.9,126.2,125.5,121.7,118.3,115.4,115.0,105.3,55.7,49.5,25.7,24.5.

[0113] 5-Methoxy-3-methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (5b)

[0114] Synthesized according to standard procedure B (222 mg, 97%). f :0.19 (DCM / MeOH 5%). 1 H NMR (400MHz, DMSO-d6) δ8.18(d,J=8.9Hz,1H),7.99(d,J=15.4Hz,1H),7.87(d,J=8.8Hz,2H),7.63(d,J=2.3Hz,1H),7.62(d,J=15 .4Hz,1H),7.32(dd,J=8.9,2.3Hz,1H),7.04(d,J=8.8Hz,2H),4.22(s,3H),3.94(s,3H),3.48(t,J=5.2Hz,4H),1.73–1.51(m,6H). 13 C NMR (101MHz, DMSO-d6) δ171.9,160.5,153.4,148.6,143.4,132.6,124.5,122.3,118.5,116.6,113.5,107.2,99.9,56.3,47.5,35.7,25.0,23.9.

[0115] 5-Fluoro-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (6a)

[0116] Synthesized according to standard procedure A (402 mg, 99%). f :0.45 (heptane / EtOAc 3:1). 1H NMR (400MHz, CDCl3) δ7.73(dd,J=8.8,5.1Hz,1H),7.62(dd,J=9.6,2.5Hz,1H),7.51–7.41(m,3H),7.19(d,J=16.1Hz,1 H),7.09(td,J=8.8,2.5Hz,1H),6.91(dt,J=8.9,3.0Hz,2H),3.35–3.23(m,4H),1.78–1.66(m,4H),1.66–1.58(m,2H). 13 C NMR (101MHz, CDCl3) δ170.6,162.1(d,J=242.8Hz),155.1(d,J=12.1Hz),152.7,138.6,129.7(d,J=1.9Hz),1 29.1, 125.1, 122.1 (d, J = 9.9Hz), 118.0, 115.3, 113.4 (d, J = 25.0Hz), 108.8 (d, J = 23.6Hz), 49.4, 25.7, 24.5. 19 F NMR (376MHz, CDCl3) δ-116.3.

[0117] 5-Fluoro-3-methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (6b)

[0118] Synthesized according to standard procedure B (175 mg, quant.). f :0.19 (DCM / MeOH 5%). 1 H NMR (400MHz, DMSO-d6) δ8.34(dd,J=9.0,5.1Hz,1H),8.12(dd,J=9.7,2.4Hz,1H),8.06(d,J=15.3Hz,1H),7.89(d ,J=8.9Hz,2H),7.64–7.55(m,2H),7.04(d,J=8.9Hz,2H),4.19(s,3H),3.51(t,J=5.2Hz,4H),1.71–1.54(m,6H). 13 C NMR (101MHz, DMSO-d6) δ173.7,162.8(d,J=245.4Hz),154.2,150.5,143.7(d,J=12.6Hz),133.6,126.1(d,J=10.0 Hz), 123.1 (d, J = 2.1Hz), 122.7, 116.1 (d, J = 24.6Hz), 113.9, 107.2, 104.0 (d, J = 28.9Hz), 48.0, 36.3, 25.6, 24.4.19 F NMR(376MHz,DMSO-d6)δ-111.1.

[0119] 6-Methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (7a)

[0120] Synthesized according to standard procedure A (132 mg, 26%). f :0.31 (heptane / EtOAc 3:1). 1 H NMR (400MHz, CDCl3) δ7.89(d,J=8.3Hz,1H),7.69(d,J=1.7Hz,1H),7.53(dt,J=8.8,2.6Hz,1H),7.47(d,J=16.1Hz,1H ),7.35–7.28(m,2H),6.99(d,J=8.4Hz,2H),3.47–3.23(m,4H),2.55(s,3H),1.78(p,J=5.7Hz,4H),1.73–1.62(m,3H). 13 C NMR (151MHz, CDCl3) δ167.1,152.6,152.2,137.6,135.2,134.5,128.8,127.8,125.6,122.2,121.3,118.6,115.5,49.6,25.7,24.5,21.7.

[0121] 3,6-Dimethyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (7b)

[0122] Synthesized according to standard procedure B (273 mg, 97%). f :0.27 (DCM / MeOH 5%). 1 H NMR (600MHz, DMSO-d6) δ8.10(s,1H),8.01(d,J=15.3Hz,2H),8.00(d,J=8.6Hz,1H),7.87(d,J=8.8Hz,2H),7.62(d,J=15.3Hz,1H),7.6 1(dd,J=8.6,1.6Hz,1H),7.04(d,J=8.8Hz,2H),4.22(s,3H),3.49(t,J=5.2Hz,4H),2.51(s,1H),1.70–1.62(m,2H),1.62–1.56(m,4H). 13C NMR (151MHz, DMSO-d6) δ170.5,153.5,149.1,140.1,137.9,132.7,130.1,126.9,123.3,122.3,115.7,113.5,107.0,47.5,35.6,25.1,23.9,20.9.

[0123] 6-Methoxy-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (8a)

[0124] Synthesized according to standard procedure A (95 mg, 19%). f :0.22 (heptane / EtOAc 3:1). 1 H NMR (400MHz, CDCl3) δ7.83(d,J=8.9Hz,1H),7.45(d,J=8.4Hz,2H),7.34(d,J=16.1Hz,1H),7.29(d,J=2.6Hz,1H),7.19(d,J=16.1Hz,1H ), 7.04 (dd, J = 8.9, 2.6 Hz, 1H), 6.91 (d, J = 8.4 Hz, 2H), 3.88 (s, 3H), 3.27 (t, J = 5.4 Hz, 4H), 1.70 (p, J = 5.6 Hz, 4H), 1.62 (q, J = 6.5 Hz, 2H). 13 C NMR (151MHz, CDCl3) δ165.8,157.8,152.5,148.7,137.1,135.7,128.7,125.6,123.2,118.6,115.5,115.4,104.4,56.0,49.6,25.7,24.5.

[0125] 6-Methoxy-3-methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (8b)

[0126] Synthesized according to standard procedure B (89 mg, 32%). f :0.18 (DCM / MeOH 5%). 1 H NMR (400MHz, DMSO-d6) δ8.02(d,J=9.2Hz,1H),7.99–7.91(m,2H),7.86(d,J=8.5Hz,2H),7.60(d,J=15.4Hz,1H) ,7.38(d,J=9.2Hz,1H),7.03(d,J=8.6Hz,2H),4.21(s,3H),3.90(s,3H),3.57–3.44(m,4H),1.82–1.47(m,6H).13 C NMR (101MHz, DMSO-d6) δ169.2,158.9,153.4,148.3,136.1,132.4,128.7,122.4,117.6,117.0,113.5,107.3,106.7,56.2,47.5,35.8,25.0,23.9.

[0127] 6-Fluoro-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (9a–PFSB)

[0128] Synthesized according to standard procedure A (34 mg, 24%). f :0.38 (heptane / EtOAc 3:1). 1 H NMR (400MHz, CDCl3) δ7.87(dd,J=8.9,4.8Hz,1H),7.50(dd,J=8.2,2.6Hz,1H),7.46(d,J=8.4Hz,2H),7.39(d,J=1 6.1Hz, 1H), 7.23–7.12 (m, 2H), 6.91 (d, J = 8.6Hz, 2H), 3.28 (t, J = 5.3Hz, 4H), 1.86–1.66 (m, 4H), 1.67–1.57 (m, 2H). 13 C NMR (101MHz, CDCl3) δ167.9 (d, J = 3.3Hz), 160.5 (d, J = 245.1Hz), 152.7, 150.8 (d, J = 1.7Hz), 138.3, 135.3 (d, J = 11. 0Hz), 128.9, 125.2, 123.4 (d, J = 9.4Hz), 118.1, 115.4, 114.7 (d, J = 24.6Hz), 107.8 (d, J = 26.8Hz), 49.5, 25.7, 24.5. 19 F NMR (376MHz, CDCl3) δ-116.4.

[0129] 6-Fluoro-3-methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (9b)

[0130] Synthesized according to standard procedure B (96 mg, 88%). f :0.16 (DCM / MeOH 5%). 1H NMR (400MHz, DMSO-d6) δ8.25(dd,J=8.3,2.7Hz,1H),8.15(dd,J=9.1,4.3Hz,1H),8.04(d,J=15.3Hz,1H),7.89(d,J=8.8Hz,2H),7 .70(td,J=9.1,2.7Hz,1H),7.62(d,J=15.3Hz,1H),7.04(d,J=8.8Hz,2H),4.22(s,3H),3.50(t,J=5.2Hz,4H),1.70–1.51(m,6H). 13 C NMR (101MHz, DMSO-d6) δ172.1(d,J=1.6Hz),161.0(d,J=246.6Hz),154.1,150.4,139.3,133.4,129.0(d,J=12.0 Hz), 122.7, 118.2 (d, J = 9.5Hz), 117.6 (d, J = 25.6Hz), 113.9, 111.1 (d, J = 28.8Hz), 107.3, 48.0, 36.4, 25.6, 24.4. 19 F NMR (376MHz, DMSO-d6) δ-112.6.

[0131] 7-Methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (10a)

[0132] Synthesized according to standard procedure A (28 mg, 43%). f :0.40 (heptane / EtOAc 3:1). 1 H NMR (400MHz, CDCl3) δ7.79(d,J=8.1Hz,1H),7.54–7.40(m,3H),7.36(t,J=7.7Hz,1H),7.23(d,J=16.1Hz,1H),7.13( d,J=7.3Hz,1H),6.91(d,J=8.4Hz,2H),3.28(t,J=5.3Hz,4H),2.56(s,3H),1.70(p,J=5.4Hz,4H),1.65–1.62(m,2H). 13 C NMR (101MHz, CDCl3) δ167.7,154.0,152.5,138.0,134.7,131.6,128.9,126.4,125.6,125.3,120.1,118.6,115.6,49.7,25.6,24.5,21.6.

[0133] 3,7-Dimethyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (10b)

[0134] Synthesized according to standard procedure B (11 mg, 64%). f :0.19 (DCM / MeOH 5%). 1 H NMR (600MHz, DMSO-d6) δ8.13(d,J=15.3Hz,1H),7.95(d,J=8.4Hz,1H),7.88(d,J=8.8Hz,2H),7.72(dd,J=8.4,7.4Hz,1H),7.66(d,J=15.3H z,1H),7.54(d,J=7.4Hz,1H),7.05(d,J=8.8Hz,2H),4.24(s,3H),3.51(t,J=5.4Hz,4H),2.60(s,3H),1.70–1.62(m,2H),1.62–1.55(m,4H). 13 CNMR(151MHz,DMSO-d6)δ170.7,153.6,150.1,141.8,132.9,132.7,129.3 ,127.9,126.6,122.3,113.7,113.5,106.8,47.5,35.9,25.1,23.9,19.3.

[0135] 7-Methoxy-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (11a)

[0136] The product was synthesized according to general procedure A and purified by rapid chromatography (PE / EtOAc 1% to 20% B) to give a yellow solid product (25 mg, 13%). f :0.41(PE / EtOAc 4:1). 1 H NMR(600MHz, CDCl3)δ7.60(d,J=8.1Hz,1H),7.52–7.45(m,3H),7.39(t,J=8.0Hz,1H),7.24(d,J=15.8Hz,1H), 7.03(s,2H),6.80(d,J=8.0Hz,1H),3.99(s,3H),3.30(t,J=5.5Hz,4H),1.89–1.70(m,4H),1.68–1.62(m,2H). 13C NMR (151MHz, CDCl3) δ168.4,155.6,154.4,154.3,137.6,129.0,127.2,126.8,122.8,119.2,116.0,115.5,105.3,56.1,50.0,25.3,24.0.

[0137] 7-Methoxy-3-methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (11b)

[0138] Synthesized according to standard procedure B (21 mg, 46%). f :0.21 (DCM / MeOH 5%). 1 H NMR (600MHz, DMSO-d6) δ8.12(d,J=15.3Hz,1H),7.87(d,J=8.6Hz,2H),7.75(t,J=8.2Hz,1H),7.69(d,J=8.5Hz,1H),7.63(d, J=15.3Hz,1H),7.31(d,J=8.1Hz,1H),7.05(d,J=8.6Hz,2H),4.21(s,3H),4.06(s,3H),3.46–3.42(m,4H),1.69–1.54(m,6H). 13 C NMR (151MHz, DMSO-d6) δ171.4,153.6,153.6,150.2,143.2,133.0,130.8,122.2,114.7,113.4,108.7,108.4,106.7,56.9,47.5,36.0,25.1,23.9.

[0139] 7-Fluoro-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (12a)

[0140] The product was synthesized according to general procedure A and purified by rapid chromatography (PE / EtOAc 1% to 20% B) to give a yellow solid product (119 mg, 72%). f :0.49(PE / EtOAc 4:1). 1H NMR(600MHz, CDCl3)δ7.75(d,J=8.1Hz,1H),7.50–7.45(m,3H),7.39(td,J=8.1,5.4Hz,1H),7. 21(d,J=16.1Hz,1H),7.05(t,J=8.6Hz,1H),6.98–6.90(m,2H),3.29(t,J=5.5Hz,4H),1.71(br s,4H),1.65–1.60(m,2H). 13 CNMR (151MHz, CDCl3) δ169.0, 157.1 (d, J = 248.7Hz), 157.0 (d, J = 2.7Hz), 152.5, 138.9, 129.1, 127.1 (d, J = 7. 3Hz), 125.3, 121.3 (d, J = 16.6Hz), 118.4 (d, J = 3.5Hz), 117.8, 115.6, 110.5 (d, J = 18.9Hz), 49.7, 25.5, 24.3.

[0141] 7-Fluoro-3-methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (12b)

[0142] Synthesized according to standard procedure B (42 mg, 69%). f :0.19 (DCM / MeOH 5%). 1 H NMR (600MHz, DMSO-d6) δ8.18(d,J=15.1Hz,1H),7.96(d,J=8.4Hz,1H),7.90(d,J=8.6Hz,2H),7.83(q,J=8.0Hz,1H),7 .69–7.57(m,2H),7.06(d,J=8.6Hz,2H),4.22(s,3H),3.54(t,J=5.4Hz,4H),1.65(q,J=5.6Hz,2H),1.63–1.53(m,4H). 13 C NMR(151MHz,DMSO-d6)δ172.2,156.3(d,J=247.7Hz),154.4,151.8,144.9(d,J=15.5Hz),134.1,13 1.4(d,J=7.5Hz),122.6,114.4(d,J=23.1Hz),113.9,113.0,109.5,106.5,48.0,36.7,25.7,24.4.

[0143] 2-(4-(piperidin-1-yl)phenyl)benzo[d]thiazole (13a)

[0144] Under an argon atmosphere, Pd(PPh3)4 (28.0 mg, 0.02 mmol) and Cs2CO3 (140 mg, 0.72 mmol) were added to a 4.00 mL solution of toluene (4.00 mL) containing 140 mg (0.48 mmol). Piperidine (0.12 mL, 1.21 mmol) was added slowly after dilution with 1.00 mL of toluene. The mixture was refluxed for 5 hours. The solution was diluted with water and extracted with EtOAc. The organic phase was dried over MgSO4, evaporated under reduced pressure, and purified by rapid chromatography (PE / EtOAc 1% to 10% B) to give the product (14.0 mg, 10%). f :0.50(PE / EtOAc 5:1). 1 H NMR (600MHz, CDCl3) δ8.01(d,J=8.1Hz,1H),7.98(d,J=8.5Hz,2H),7.85(d,J=7.9Hz,1H),7.47–7.42(m,1H),7.35–7.29(m,1H),7.02(br s, 2H), 3.34 (t, J = 5.5Hz, 4H), 1.77–1.74 (m, 4H), 1.65 (p, J = 5.7Hz, 2H). 13 CNMR (151MHz, CDCl3) δ166.2,154.2,151.7,134.7,129.0,128.3,126.3,124.7,122.7,121.7,121.6,52.7,29.8,25.3.

[0145] 3-Methyl-2-(4-(piperidin-1-yl)phenyl)benzo[d]thiazolyl-3-onium (13b)

[0146] To a solution of 13a (16.0 mg, 0.05 mmol) in chlorobenzene (1.50 mL), methylnosylate (MeNs) (14.0 mg, 0.06 mmol) was added. The mixture was stirred overnight at 80 °C. The precipitate was filtered under vacuum and ground in Et₂O. The yellow solid was further purified by semi-preparative HPLC (0.1% TFA aqueous solution / acetonitrile, 20% to 60% B over 15 min) to separate the product (10.0 mg, 59%) from the aniline N-methylation byproduct. f :0.13 (DCM / MeOH 5%). 1H NMR (600MHz, DMSO-d6) δ8.40(dd,J=8.1,1.2Hz,1H),8.25(d,J=8.4Hz,1H),7.89(ddd,J=8.5,7.2,1.2Hz,1H),7.81(dt,J=9.1,3.2Hz,2H ),7.78(ddd,J=8.2,7.2,1.0Hz,1H),7.19(dt,J=9.1,3.2Hz,2H),4.25(s,3H),3.55–3.53(m,4H),1.72–1.64(m,2H),1.64–1.57(m,4H). 13 C NMR (151MHz, DMSO-d6) δ173.4,153.8,142.7,132.5,129.3,128.1,127.7,123.9,116.9,113.5,111.6,47.4,38.1,24.9,23.8.

[0147] 2-(4-(4-(piperidin-1-yl)phenyl)but-1,3-dien-1-yl)benzo[d]thiazole (14a)

[0148] Synthesized according to standard procedure A (32 mg, 17%). f :0.48(PE / EtOAc 4:1). 1 H NMR (600MHz, CDCl3) δ7.90(d,J=8.1Hz,1H),7.78(d,J=8.0Hz,1H),7.39(t,J=7.7Hz,1H),7.33(d,J=8.3Hz,2H),7.29(t,J=7.5Hz,2H ),7.23(d,J=16.1Hz,1H),6.84(d,J=7.7Hz,2H),6.83–6.66(m,3H),3.20(t,J=5.4Hz,4H),1.64(q,J=5.8Hz,4H),1.59–1.53(m,2H). 13 C NMR (151MHz, CDCl3) δ167.6,154.1,152.2,139.3,138.3,134.4,128.4,126.8,126.3,125.1,124.1,123.4,122.7,121.5,115.6,49.7,25.7,24.4.

[0149] 3-Methyl-2-(4-(4-(piperidin-1-yl)phenyl)but-1,3-dien-1-yl)benzo[d]thiazolyl-3-onium iodide (14b)

[0150] Synthesized according to standard procedure B (109 mg, 87%). f :0.16 (DCM / MeOH 5%). 1 H NMR(600MHz,DMSO-d6)δ8.33(d,J=8.1Hz,1H),8.15(d,J=8.4Hz,1H),7.99(dd,J=14.6, 10.9Hz,1H),7.81(ddd,J=8.5,7.2,1.2Hz,1H),7.71(ddd,J=8.1,7.3,1.0Hz,1H),7.53 (d,J=8.8Hz,2H),7.43(d,J=15.1Hz,1H),7.35(d,J=14.6Hz,1H),7.21(dd,J=15.1,10. 9Hz, 1H), 7.00 (d, J = 8.8Hz, 2H), 4.16 (s, 3H), 3.39 (t, J = 5.1Hz, 4H), 1.63–1.56 (m, 6H). 13 C NMR(151MHz,DMSO-d6)δ170.6,154.5,152.4,150.9,147.4,141.9,130.4,129 .1,127.8,127.3,124.0,122.9,116.3,114.3,112.9,48.0,35.7,25.0,24.0.

[0151] 4-(4-(2-(benzo[d]thiazolyl)vinyl)phenyl)morpholine(15a)

[0152] Synthesized according to standard procedure A (47 mg, 59%). f :0.21 (heptane / EtOAc 3:1). 1 H NMR (600MHz, CDCl3) δ7.96(d,J=8.1Hz,1H),7.84(d,J=8.0Hz,1H),7.51(d,J=8.5Hz,2H),7.46(d,J=16.1Hz,1H),7.45(t,J=7 .4Hz, 1H), 7.35 (t, J = 7.6Hz, 1H), 7.26 (d, J = 16.1Hz, 1H), 6.91 (d, J = 8.5Hz, 2H), 3.87 (t, J = 4.9Hz, 4H), 3.25 (t, J = 4.8Hz, 4H). 13 C NMR (151MHz, CDCl3) δ167.8,154.1,152.1,137.7,134.4,128.9,126.8,126.3,125.1,122.8,121.6,119.2,115.2,66.9,48.5.

[0153] 3-Methyl-2-(4-morpholinylstyryl)benzo[d]thiazol-3-onium iodide (15b)

[0154] Synthesized according to standard procedure B (52 mg, 69%). f :0.13 (DCM / MeOH 5%). 1 H NMR (600MHz, DMSO-d6) δ8.34 (dd, J=8.2, 1.2Hz, 1H), 8.14 (d, J=8.3Hz, 1H), 8. 10(d,J=15.4Hz,1H),7.95(dt,J=9.0,2.9Hz,2H),7.81(ddd,J=8.5,7.2,1.2H z,1H),7.74(d,J=15.4Hz,1H),7.72(ddd,J=8.2,7.2,1.0Hz,1H),7.09(dt,J= 9.0, 3.1Hz, 2H), 4.27 (s, 3H), 3.80–3.70 (m, 4H), 3.42 (dd, J = 5.7, 4.1Hz, 4H). 13 C NMR (151MHz, DMSO-d6) δ171.7,153.8,149.5,142.0,132.4,129.0,127.7,127.1,123.9,123.5,116.2,113.6,108.2,65.8,46.5,35.8.

[0155] 2-(4-Thiomorpholinylstyryl)benzo[d]thiazole(16a)

[0156] Synthesized according to standard procedure A (6 mg, 11%). f :0.39 (heptane / EtOAc 3:1). 1 H NMR(600MHz, CDCl3) δ7.96(d,J=8.1Hz,1H),7.84(d,J=7.9Hz,1H),7.49(d,J=8.5Hz,2H),7.47–7.42(m,2H),7.3 5(td,J=8.0,1.1Hz,1H),7.25(d,J=16.1Hz,1H),6.88(d,J=8.3Hz,2H),3.77–3.67(m,4H),2.74(t,J=5.1Hz,4H). 13C NMR (151MHz, CDCl3) δ167.9,154.0,151.2,137.8,134.3,129.1,126.4,126.1,125.1,122.7,121.6,119.0,116.0,51.2,26.3.

[0157] 3-Methyl-2-(4-thiomorpholinylstyryl)benzo[d]thiazolyl-3-onium iodide (16b)

[0158] Synthesized according to standard procedure B (33 mg, 65%). f :0.13 (DCM / MeOH 5%). 1 H NMR (600MHz, DMSO-d6) δ8.33(dd,J=8.1,1.2Hz,1H),8.13(d,J=8.4Hz,1H),8.08(d,J=15.4Hz,1H),7.97–7.89(m,2H),7.81 (ddd,J=8.5,7.2,1.2Hz,1H),7.74–7.66(m,2H),7.07(d,J=9.0Hz,2H),4.26(s,3H),3.94–3.84(m,4H),2.72–2.63(m,4H). 13 C NMR (151MHz, DMSO-d6) δ171.6,152.3,149.5,142.0,132.8,128.9,127.6,127.0,123.9,122.7,116.1,113.9,107.6,49.5,35.7,25.2.

[0159] 2-(4-(pyrrolidone-1-yl)styryl)benzo[d]thiazole (17a)

[0160] Synthesized according to standard procedure A (15 mg, 15%). f :0.50 (heptane / EtOAc 3:1). 1 H NMR(600MHz, CDCl3) δ7.93(d,J=8.1Hz,1H),7.82(d,J=7.9Hz,1H),7.55–7.38(m,4H),7.31(t,J=7.5 Hz, 1H), 7.18 (d, J = 16.1Hz, 1H), 6.57 (d, J = 8.3Hz, 2H), 3.35 (t, J = 6.1Hz, 4H), 2.03 (heptane, J = 3.4Hz, 4H). 13C NMR (151MHz, CDCl3) δ168.6,154.2,148.9,138.8,134.2,129.2,126.2,124.8,122.8,122.5,121.5,116.7,112.0,47.7,25.6.

[0161] 3-Methyl-2-(4-(pyrrolidone-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (17b)

[0162] Synthesized according to standard procedure B (118 mg, 96%). f :0.16 (DCM / MeOH 5%). 1 H NMR (400MHz, DMSO-d6) δ8.28(d,J=8.0Hz,1H),8.12–7.99(m,2H),7.90(d,J=8.6Hz,2H),7.77(t,J=7.6Hz,1H),7.67( t,J=7.6Hz,1H),7.58(d,J=15.2Hz,1H),6.69(d,J=8.6Hz,2H),4.21(s,3H),3.43–3.35(m,4H),2.00(p,J=3.2Hz,4H). 13 C NMR (101MHz, DMSO-d6) δ171.1,151.0,150.3,141.9,133.1,128.8,127.3,126.7,123.7,121.3,115.8,112.4,105.6,47.6,35.4,24.8.

[0163] 2-(4-(4-fluoropiperidin-1-yl)styryl)benzo[d]thiazole (18a)

[0164] Synthesized according to standard procedure A (84 mg, 57%). f :0.23 (heptane / EtOAc 3:1). 1H NMR (400MHz, DMSO-d6) δ8.04(dd,J=7.9,1.1Hz,1H),7.91(d,J=8.1Hz,1H),7.61(d,J=8. 6Hz,2H),7.54(d,J=16.1Hz,1H),7.48(td,J=7.2,1.0Hz,1H),7.39(td,J=8.2,0.9Hz,1H ),7.36(d,J=16.1Hz,1H),7.00(d,J=8.4Hz,2H),4.87(dtt,J=49.0,7.3,3.6Hz,1H),3.5 8–3.42(m,2H),3.27(ddd,J=12.6,7.7,3.8Hz,2H),2.05–1.87(m,2H),1.83–1.69(m,2H). 13 C NMR(101MHz,DMSO-d6)δ167.7,154.1,151.5,138.3,134.2,129.6,126.8,125.5,125.3, 122.6, 122.5, 118.1, 115.3, 89.0 (d, J = 169.4Hz), 44.5 (d, J = 6.9Hz), 30.9 (d, J = 19.1Hz).

[0165] 2-(4-(4-fluoropiperidin-1-yl)styryl)-3-methylbenzo[d]thiazolyl-3-onium iodide (18b)

[0166] Synthesized according to standard procedure B (143 mg, 87%). f :0.19 (DCM / MeOH 5%). 1 H NMR (400MHz, DMSO-d6) δ8.33(dd,J=8.1,1.2Hz,1H),8.13(d,J=8.4Hz,1H),8.07(d,J=15.5 Hz,1H),7.92(d,J=8.7Hz,2H),7.80(td,J=8.1,7.2,1.3Hz,1H),7.75–7.64(m,2H),7.11(d ,J=8.7Hz,2H),4.93(dtt,J=48.9,7.1,3.5Hz,1H),4.26(s,3H),3.76–3.58(m,2H),3.55–3 .44(m,2H),1.98(dddd,J=24.8,16.1,7.5,3.6Hz,2H),1.78(dtt,J=14.1,7.2,3.8Hz,2H). 13CNMR(101MHz,DMSO-d6)δ172.1,153.5,150.0,142.4,133.2,129.4,128.1,127.5,124.4,12 3.3, 116.6, 114.3, 108.1, 88.7 (d, J = 169.5Hz), 43.6 (d, J = 6.6Hz), 36.2, 31.0 (d, J = 19.4Hz). 19 F NMR (376MHz, DMSO-d6) δ-177.5.

[0167] 4-(2-(benzo[d]thiazolyl-2-yl)vinyl)-N,N-dimethylaniline (19a)

[0168] Synthesized according to standard procedure A (58 mg, 34%). f :0.40 (heptane / EtOAc 3:1). 1 H NMR (600MHz, CDCl3) δ7.94(dt,J=8.0,0.9Hz,1H),7.82(dt,J=7.7,0.9Hz,1H),7.48(dt,J=8.8,2.8Hz,2H),7.45(d,J=16.1Hz,1H),7 .43(ddd,J=8.3,7.2,1.2Hz,1H), 7.32(ddd,J=8.2,7.2,1.2Hz,1H), 7.21(d,J=16.1Hz,1H), 6.72(dt,J=8.9,2.9Hz,2H), 3.03(s,6H). 13 CNMR (151MHz, CDCl3) δ168.4,154.2,151.4,138.5,134.3,129.1,126.2,124.9,123.5,122.6,121.5,117.4,112.2,40.4.

[0169] 2-(4-(dimethylamino)styryl)-3-methylbenzo[d]thiazol-3-onium iodide (19b)

[0170] Synthesized according to standard procedure B (125 mg, 90%). f :0.14 (DCM / MeOH 5%). 1H NMR (400MHz, DMSO-d6) δ8.30(dd,J=8.1,1.2Hz,1H),8.15–7.99(m,2H),7.91(d,J=8.9Hz,2H),7.78(ddd,J=8. 5,7.2,1.2Hz,1H),7.72–7.65(m,1H),7.62(d,J=15.3Hz,1H),6.84(d,J=8.9Hz,2H),4.23(s,3H),3.10(s,6H). 13 C NMR (101MHz, DMSO-d6) δ171.3,153.5,150.1,141.9,132.8,128.8,127.4,126.8,123.8,121.4,115.9,111.9,106.2,39.8,35.5.

[0171] 2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (20)

[0172] Synthesized according to standard procedure A (1.14 g, 53%). f :0.38 (heptane / EtOAc 3:1). 1 H NMR(400MHz, CDCl3)δ7.95(d,J=8.1Hz,1H),7.83(d,J=7.9Hz,1H),7.53–7.39(m,4H),7.33(t,J=7.6Hz,1H),7.23 (d, J=16.2Hz, 1H), 6.91 (d, J=8.5Hz, 2H), 3.28 (t, J=5.6Hz, 4H), 1.70 (p, J=5.6Hz, 4H), 1.63 (q, J=6.5, 5.6Hz, 2H). 13 C NMR (101MHz, CDCl3) δ168.1,154.2,152.6,138.1,134.3,128.9,126.3,125.4,125.0,122.7,121.5,118.4,115.4,49.5,25.7,24.5.

[0173] 3-Methyl-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (RB1)

[0174] Synthesized according to standard procedure B (322 mg, 81%). f :0.17 (DCM / MeOH 5%). 1H NMR (400MHz, DMSO-d6) δ8.31(dd,J=8.2,1.2Hz,1H),8.11(d,J=8.4Hz,1H),8.05(d,J=16.1Hz,1H),7.90(d,J=9.0Hz,2H),7.79 (ddd,J=8.5,7.3,1.3Hz,1H),7.73–7.62(m,2H),7.05(d,J=8.7Hz,2H),4.24(s,3H),3.50(t,J=5.1Hz,4H),1.69–1.51(m,6H). 13 C NMR (101MHz, DMSO-d6) δ171.9,154.1,150.2,142.4,133.4,129.4,128.0,127.4,124.3,122.7,116.5,113.9,107.4,48.0,36.1,25.6,24.4.

[0175] 3-Methyl-2-(4-(4-methylpiperazin-1-yl)styryl)benzo[d]thiazolyl-3-onium iodide (RB2)

[0176] Synthesized according to standard procedure B (159 mg, 97%). f :0.19 (DCM / MeOH 5%). 1 H NMR (400MHz, DMSO-d6) δ8.35(d,J=7.9Hz,1H),8.15(d,J=8.4Hz,1H),8.10(d,J=15.5Hz,1H),7.95(d,J=8.5Hz,2H) ,7.82(t,J=7.9Hz,1H),7.75(d,J=15.5Hz,1H),7.72(t,J=7.7Hz,1H),7.11(d,J=8.5Hz,2H),4.28(s,3H),3.56(br s,4H),2.85(br s,4H),2.53(s,3H). 13 C NMR (101MHz, DMSO-d6) δ171.7,153.0,149.3,141.9,132.4,129.0,127.7,127.1,123.9,123.7,116.2,114.0,108.4,53.3,45.2,44.0,35.9.

[0177] 6-Fluoro-2-(4-(4-(piperidin-1-yl)phenyl)but-1,3-dien-1-yl)benzo[d]thiazole (43)

[0178] Synthesized according to standard procedure A (37 mg, 28%).f :0.47(PE / EtOAc 4:1). 1 H NMR(600MHz, CDCl3) δ7.90(dd,J=8.9,4.8Hz,1H),7.52(dd,J=7.1,1.6Hz,1H),7.40(d,J=8.3Hz,2H),7.33–7.27(m,1H),7.1 9(t,J=8.7Hz,1H),6.92(d,J=8.3Hz,2H),6.88–6.80(m,3H),3.27(t,J=5.4Hz,4H),1.81–1.68(m,4H),1.64(p,J=5.7Hz,2H). 13 C NMR (151MHz, CDCl3)δ

[0179] 167.3, 160.5 (d, J = 245.6Hz), 152.1, 150.8 (d, J = 1.5Hz), 139.3, 138.4, 135.4 (d, J = 11.2Hz), 128.4, 126. 9,124.0,123.5(d,J=9.3Hz),123.1,115.7,114.8(d,J=24.8Hz),107.8(d,J=27.0Hz),49.8,25.6,24.4.

[0180] 6-Fluoro-2-(4-(4-(pyrrolidone-1-yl)phenyl)but-1,3-dien-1-yl)benzo[d]thiazole (44)

[0181] Synthesized according to standard procedure A (24 mg, 36%). f :0.51(PE / EtOAc 4:1). 1 H NMR (600MHz, CDCl3) δ7.92–7.77(m,1H),7.46(d,J=8.3Hz,1H),7.34(d,J=8.1Hz,2H),7.25–7.20( m,1H),7.13(t,J=9.1Hz,1H),6.93–6.68(m,3H),6.52(d,J=8.8Hz,2H),3.31(s,4H),2.00(s,4H). 13C NMR (151MHz, CDCl3) δ 167.7, 162.1 (d, J = 239.1Hz), 150.5, 148.3, 140.1, 139.4, 137.2, 128.8, 127. 8, 123.3 (d, J = 8.4Hz), 122.4, 121.9, 114.8 (d, J = 24.0Hz), 112.1, 107.8 (d, J = 26.8Hz), 47.9, 25.6.

[0182] 4-(4-(2-(6-fluorobenzo[d]thiazolyl)vinyl)phenyl)morpholine(45–MFSB)

[0183] Synthesized according to standard procedure A (21 mg, 69%). f :0.18(PE / EtOAc 4:1). 1 H NMR(600MHz, CDCl3)δ7.90(dd,J=9.0,4.8Hz,1H),7.58–7.47(m,3H),7.43(d,J=16.0Hz,1H),7.24(d,J=16 .0Hz, 1H), 7.19 (td, J = 8.9, 2.6Hz, 1H), 6.96 (d, J = 8.2Hz, 2H), 3.90 (t, J = 4.6Hz, 4H), 3.27 (t, J = 4.2Hz, 4H). 13 C NMR (151MHz, CDCl3) δ 167.6, 160.6 (d, J = 245.5Hz), 151.8, 150.4, 138.0, 135.2, 129.0, 127. 0, 123.5 (d, J = 9.0Hz), 118.9, 115.4, 115.0 (d, J = 25.1Hz), 107.9 (d, J = 27.1Hz), 66.7, 48.7.

[0184] 6-Bromo-2-(4-(piperidin-1-yl)styryl)benzo[d]thiazole (46)

[0185] Synthesized according to standard procedure A (2.80 g, 80%). f :0.51(PE / EtOAc 4:1). 1H NMR (600MHz, CDCl3) δ7.94(s,1H),7.78(d,J=8.6Hz,1H),7.52(d,J=8.6Hz,1H) ,7.46(d,J=8.3Hz,2H),7.43(d,J=16.0Hz,1H),7.18(d,J=16.0Hz,1H),6.92(br s, 2H), 3.29 (t, J = 5.4Hz, 4H), 1.70 (br s, 4H), 1.63 (q, J = 5.7Hz, 2H). 13 C NMR (151MHz, CDCl3) δ168.6,153.0,152.5,138.7,136.0,129.7,129.0,125.3,124.0,123.7,118.4,117.9,115.5,49.6,25.5,24.4.

[0186] 4-(4-(2-(6-bromobenzo[d]thiazolyl)vinyl)phenyl)morpholine(47)

[0187] Synthesized according to standard procedure A (639 mg, 73%). f :0.15(PE / EtOAc 4:1). 1 H NMR (600MHz, CDCl3) δ7.97(d,J=1.9Hz,1H),7.82(d,J=8.6Hz,1H),7.55(dd,J=8.6,1.9Hz,1H),7.53(dt,J=8.7,2.0Hz ,2H),7.48(d,J=16.1Hz,1H),7.25(d,J=16.1Hz,1H),7.00(d,J=8.4Hz,2H),3.92(t,J=4.8Hz,4H),3.30–3.26(m,4H). 13 C NMR (151MHz, CDCl3) δ168.3,151.9,151.0,138.7,135.6,130.1,129.3,126.2,124.2,123.6,118.9,118.7,116.0,66.4,49.2.

[0188] 2-(4-(piperidin-1-yl)styryl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)benzo[d]thiazole (48)

[0189] Pd(dppf)Cl2 (303 mg, 0.40 mmol) was added to a solution of 46 (1.60 g, 4.01 mmol), potassium acetate (786 mg, 8.01 mmol), and pinacol diboronate (1.53 g, 6.01 mmol) in anhydrous DMF (34.0 mL). The mixture was heated at 100 °C for 45 min. It was then poured into water and extracted with EtOAc. The organic phase was dried over MgSO4, evaporated under reduced pressure, and purified by rapid chromatography (PE / EtOAc 5% to 20% B) to give a yellow solid product (1.66 g, 93%). f :0.35(PE / EtOAc6:1). 1 H NMR (600MHz, CDCl3) δ8.31(d,J=1.1Hz,1H),7.93(d,J=8.1Hz,1H),7.87(dd,J=8.1,1.1Hz,1H),7.49–7.44(m,3H),7.23(d ,J=16.1Hz,1H),6.90(d,J=8.4Hz,2H),3.29–3.23(m,4H),1.68(p,J=5.5Hz,4H),1.60(q,J=7.0,6.4Hz,2H),1.37(s,12H). 13 C NMR (151MHz, CDCl3) δ169.6,156.1,152.4,138.5,133.8,132.3,129.0,129.0,128.4,125.4,121.8,118.4,115.4,84.1,49.5,25.5,25.0,24.3.

[0190] 4-(4-(2-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)benzo[d]thiazolyl)vinyl)phenyl)morpholine(49)

[0191] The synthesis was carried out using the same steps as 48 (124 mg, 18%). f :0.17(PE / EtOAc 3:1). 1 H NMR (600MHz, CDCl3) δ8.31(d,J=1.1Hz,1H),7.94(d,J=8.1Hz,1H),7.87(dd,J=8.1,1.1Hz,1H),7.49(d,J=8.7Hz,2H),7.48 (d,J=16.1Hz,1H),7.26(d,J=16.1Hz,1H),6.89(dt,J=8.8,2.9Hz,2H),3.87–3.84(m,4H),3.25–3.21(m,4H),1.36(s,12H).13 C NMR (151MHz, CDCl3) δ169.5,155.6,152.0,138.5,133.6,132.4,130.7,129.0,128.5,126.6,121.8,118.8,115.1,84.2,66.7,48.3,25.0.

[0192] [ 18 F]PFSB hand-synthesized

[0193] [ 18 [F] Fluoride was generated using a PETtrace 890 cyclotron (GE Healthcare, Uppsala, Sweden) and delivered as a target H2O wash. Pretreatment was performed in the following order using a Sep-Pak QMA carb lightcartridge: 10 mL KOTf aqueous solution (90 mg / mL), 10 mL air, 10 mL H2O, 10 mL air. 18 F] fluoride was captured in a QMA column, dried with argon (10 mL through the column), and then eluted with a TBAOTf MeOH solution (10 mg / 1 mL) to obtain [ 18 F]TBAF. The resulting solution was divided into four equal portions in four reaction flasks, and the MeOH was evaporated at 90°C.

[0194] Prepare a DMA stock solution (0.1 mg / μL) of Cu(OTf)₂. To prepare each reaction mixture, dilute 25.5 μL of the stock solution with a selected amount of DMA (510 μL for a and b, 499 μL for c and d, Table 4), then add n-BuOH (60.0 μL) and pyridine (4.90 μL, 60 μmol or 16.0 μL, 200 μmol; Table 4). Add the solution to precursor 48 (9.00 mg, 20.2 μmol or 4.50 mg, 10.1 μmol; Table 4). Sonicate the mixture, add it to the appropriate reaction flask, and heat at 120 °C for 20 min. Quench the reaction with 1 mL of 0.1 M HCl and neutralize with 1 mL of 0.1 M NaOH. Add 500 μL of LiCN to each mixture to avoid product precipitation. The reaction performance was evaluated using radiometric TLC (PE / EtOAc 2:1) and radiometric high-performance liquid chromatography (radioHPLC). This evaluation allows for the optimization of reaction conditions for automated synthesis.

[0195] [ 18 F]PFSB and [ 18 Automated Synthesis of F]MFSB

[0196] The Sep-Pak Plus Light QMA carb column was pretreated in the following order: 10 mL KOTf aqueous solution (90 mg / mL), 10 mL air, 10 mL H2O, 10 mL air. The Sep-Pak Plus Light Alumin N column was pretreated with 5 mL H2O. Sep-Pak Plus tC18 and Sep-Pak Light C18 were pretreated with 10 mL EtOH and 10 mL H2O, respectively. To prepare the reaction mixture, Cu(OTf)2 (2.40 mg, 6.72 μmol) was dissolved in 535.7 μL DMA. n-BuOH (60.0 μL) and pyridine (4.30 μL, 53.8 μmol) were added. This solution was then added to either PFSB precursor 48 (4.00 mg, 8.96 μmol) or MFSB precursor 49 (4.00 mg, 8.92 μmol) and sonicated.

[0197] [ 18 F]fluoride was generated by a PETtrace 890 cyclotron (GE Healthcare, Uppsala, Sweden) and fed into an FxNPro module (GE Heatlhcare, Münster, Germany). It was captured in a QMA column, eluted into the reactor with a MeOH solution of TBAOTf (10 mg / mL), and the solvent was evaporated at 90 °C. The reaction mixture was added, and the reactor was heated at 120 °C for 20 min. The resulting mixture was diluted 10 mL of MeCN / ammonium formate buffer (25 mM, pH 8) 1:1 v / v and captured at the overlap of Alox and tC18 columns. The product was then treated with MeCN (to […]. 18 F]PFSB was 3.4 mL, which is effective against [ 18 [F]MFSB was 2.8 mL) eluted to a solution containing 25 mM ammonium formate (for [ 18 F]PFSB is 1.6 mL, for [ 18 [F]MFSB (2.2 mL) was placed in tube 2. The mixture was injected into the HPLC quantitative loop. Semi-preparative HPLC purification conditions: Luna 5 μm C8(2) 250x 10mm; for [ 18 F]PFSB, in 68% acetonitrile in 25 mM ammonium formate at pH 8 (retention time ≈ 17 min); for [ 18 F]MFSB, in 25mM ammonium formate at pH 8 with 55% acetonitrile (retention time ≈ 12 min); 6 mL / min.

[0198] The product peak was cut off, diluted with water (55 mL), and collected into a C18 column. It was washed with water (5 mL), eluted with EtOH (0.5 mL), prepared with PBS (4.5 mL), and transferred to a product vial. Quality control (QC) was then performed. Figures 8 to 11 The report describes the analytical HPLC conditions and chromatograms.

[0199] Pittsburgh compound-B[ 3 H]PiB and [ 3 Tritiumization of H]MODAG-001

[0200] PiB and MODAG-001 (4-(3-(2-bromopyridin-4-yl)-1H-pyrazol-5-yl)-N,N-dimethylaniline) were tritized by RCTritec AG (Teufen, Switzerland), dissolved in EtOH, and stored at -80°C until use. The radiochemical purity of both radioligands is >99%. 3 H]PiB and [ 3 The molar activity of H]MODAG-001 (A m The values ​​were 21.7 Ci / mmol and 78.9 Ci / mmol, respectively.

[0201] 1.1.4. General Procedure

[0202] 1.1.4.1. General Procedure S1

[0203]

[0204] The suspension of the selected 2-aminobenzothiazole (7.36 mmol) in 50% KOH aqueous solution (28.0 mL) was refluxed for 18 to 42 hours. The mixture was acidified with 37% HCl (20.0 mL). A color change occurred, and a precipitate formed. 2,4-Pentanedione (11.04 mmol) was added, and the mixture was stirred at room temperature for 90 minutes. The mixture was extracted with EtOAc. The organic phase was dried over MgSO4, evaporated under reduced pressure, and purified by rapid chromatography (heptane / EtOAc).

[0205] 1.1.4.2. General Procedure S2

[0206]

[0207] Add 16.5 mmol of iodomethane to a MeCN (25.0 mL) solution of the selected 2-methylbenzothiazole (8.24 mmol). Stir the mixture overnight at 80 °C. Filter the precipitate under vacuum, wash with EtOAc and / or grind in Et2O.

[0208] 1.1.4.3. General Procedure S3

[0209]

[0210] Add methyl p-nitrobenzenesulfonate (1.26 mmol) to a MeCN (2.00 mL) solution of the selected 0.63 mmol 2-methylbenzothiazole. Stir the mixture overnight at 80 °C. Filter the precipitate under vacuum, wash with EtOAc and / or grind in Et2O.

[0211] 1.1.4.4. General Procedure S4

[0212]

[0213] A mixture of 4-fluorobenzaldehyde (46.6 mmol) and the selected secondary amine (39.6 mmol) was diluted with DMF (25.0 mL). K₂CO₃ (58.3 mmol) was added, and the reaction mixture was stirred overnight at 90 °C. The mixture was diluted with water and extracted with EtOAc. The organic phase was dried over MgSO₄, evaporated under reduced pressure, and purified by rapid chromatography (heptane / EtOAc).

[0214] 1.1.5. Synthesis of 2-methylbenzothiazole derivatives, 2,3-dimethylbenzothiazole onium salt derivatives, 4-aminobenzaldehyde derivatives, or 4-aminocinnamaldehyde derivatives for general procedure A or B.

[0215] 2,4-Dimethylbenzo[d]thiazole (21a)

[0216]

[0217] The synthesis was carried out according to the standard procedure S1, with ethylene glycol (0.5 mL / mmol) added to the mixture (393 mg, 33%). f :0.41 (heptane / EtOAc 4:1). 1 H NMR (400MHz, DMSO-d6) δ7.82(pd,J=3.6,0.6Hz,1H),7.28(s,1H),7.27–7.25(m,1H),2.80(s,3H),2.63(s,3H). 13 C NMR (101MHz, DMSO-d6) δ165.6,152.2,134.9,131.4,126.4,124.6,119.2,19.8,18.1.

[0218] 2,3,4-Trimethylbenzo[d]thiazolyl-3-onthium-4-nitrobenzenesulfonate (21b)

[0219]

[0220] Synthesized according to standard procedure S3 (248 mg, 67%). f :0.29(DCM / MeOH 9:1). 1 H NMR (400MHz, DMSO-d6) δ8.30–8.22(m,1H),8.19(d,J=8.4Hz,2H),7.82(d,J=8.3H z, 2H), 7.64 (d, J = 2.1Hz, 1H), 7.62 (s, 1H), 4.36 (s, 3H), 3.13 (s, 3H), 2.93 (s, 3H). 13 C NMR (151MHz, DMSO-d6) δ176.6,154.2,147.3,140.6,132.8,129.5,128.3,127.7,126.9,123.4,122.4,39.9,20.4,17.6.

[0221] 4-Methoxy-2-methylbenzo[d]thiazole (22a)

[0222]

[0223] Synthesized according to standard procedure S1 (223 mg, 20%). f :0.09 (heptane / EtOAc 4:1). 1 H NMR (400MHz, DMSO-d6) δ7.55 (dd, J = 8.0, 1.0 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.01 (dd, J = 8.0, 1.0 Hz, 1H), 3.93 (s, 3H), 2.77 (s, 3H). 13 C NMR (101MHz, DMSO-d6) δ164.7,152.6,142.8,136.7,125.7,113.6,107.4,55.7,19.7.

[0224] 4-Methoxy-2,3-dimethylbenzo[d]thiazol-3-onium iodide (22b)

[0225]

[0226] Synthesized according to the standard procedure S2 (35 mg, 20%). f :0.34(DCM / MeOH 9:1). 1H NMR (600MHz, DMSO-d6) δ7.93(dd,J=8.2,0.9Hz,1H),7.70(t,J=8.2Hz,1H),7.45(dd,J=8.2,0.9Hz,1H),4.35(s,3H),4.05(s,3H),3.09(s,3H). 13 C NMR (151MHz, DMSO-d6) δ176.4,150.4,131.5,131.1,129.5,116.4,112.1,57.5,40.6,17.6.

[0227] 4-Fluoro-2-methylbenzo[d]thiazole (23a)

[0228]

[0229] Synthesized according to standard procedure S1 (431 mg, 36%). f :0.35 (heptane / EtOAc 4:1). 1 H NMR (400MHz, DMSO-d6) δ7.86 (dd, J = 8.1, 1.1 Hz, 1H), 7.41 (td, J = 8.1, 4.9 Hz, 1H), 7.31 (ddd, J = 11.0, 8.1, 1.1 Hz, 1H), 2.82 (s, 3H). 13 C NMR (101MHz, DMSO-d6) δ168.2, 154.4 (d, J = 252.9Hz), 141.2 (d, J = 13.3Hz), 138.1 (d,J=3.8Hz),125.7(d,J=7.1Hz),118.1(d,J=4.1Hz),111.7(d,J=17.9Hz),19.7. 19 F NMR(376MHz,DMSO-d6)δ-123.34.

[0230] 4-Fluoro-2,3-dimethylbenzo[d]thiazolyl-3-onthium-4-nitrobenzenesulfonate (23b)

[0231]

[0232] Synthesized according to standard procedure S3 (188 mg, 78%). f :0.32(DCM / MeOH 9:1). 1H NMR (400MHz, DMSO-d6) δ 8.28–8.20 (m, 1H), 8.18 (d, J = 8.4Hz, 2H), 7.86–7.75 (m, 4H), 4.29 (d, J = 2.7Hz, 3H), 3.16 (s, 3H). 13 C NMR (101MHz, DMSO-d6) δ178.6,154.3,150.8(d,J=253.2Hz),147.2,131.3,130.3(d,J=10.7 Hz), 129.1 (d, J = 7.5Hz), 126.9, 123.3, 120.8 (d, J = 4.6Hz), 116.0 (d, J = 19.1Hz), 39.2, 17.0. 19 F NMR(376MHz,DMSO-d6)δ-125.33.

[0233] 2,3,5-Trimethylbenzo[d]thiazol-3-onium iodide (24b)

[0234]

[0235] Commercially available 24a was synthesized according to the general procedure S2 (460 mg, 61%). f :0.32(DCM / MeOH9:1). 1 H NMR (400MHz, DMSO-d6) δ8.29(d,J=8.4Hz,1H),8.14(d,J=1.5Hz,1H),7.64(dd,J=8.4,1.5Hz,1H),4.17(s,3H),3.15(s,3H),2.57(s,3H). 13 C NMR (101MHz, DMSO-d6)δ

[0236] 176.8,141.8,139.8,129.5,125.8,123.9,116.4,36.0,21.1,17.0.

[0237] 5-Methoxy-2-methylbenzo[d]thiazole (25a)

[0238]

[0239] K₂CO₃ (836 mg, 6.05 mmol) was added to a DMF (8.50 mL) solution of 2-methyl-5-benzothiazolidinol (500 mg, 3.03 mmol). The mixture was stirred at room temperature for 10 minutes. Iodomethane (283 μL, 4.54 mmol) was added dropwise, and the reaction was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with EtOAc. The organic phase was dried over MgSO₄ and evaporated under reduced pressure; it was ready for use without further purification (477 mg, 88%). f :0.16 (heptane / EtOAc 4:1). 1 H NMR (400MHz, DMSO-d6) δ7.87 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 2.5 Hz, 1H), 7.02 (dd, J = 8.8, 2.5 Hz, 1H), 3.83 (s, 3H), 2.76 (s, 3H). 13 C NMR (101MHz, DMSO-d6) δ168.0,158.4,154.3,126.8,122.2,114.1,105.1,55.4,19.8.

[0240] 5-Methoxy-2,3-dimethylbenzo[d]thiazol-3-onium iodide (25b)

[0241]

[0242] Synthesized according to the standard procedure S2 (204 mg, 54%). f :0.40(DCM / MeOH 9:1). 1 H NMR (400MHz, DMSO-d6) δ8.29(d,J=9.0Hz,1H),7.78(d,J=2.4Hz,1H),7.42(dd,J=9.0,2.4Hz,1H),4.17(s,3H),3.97(s,3H),3.13(s,3H). 13 C NMR (101MHz, DMSO-d6) δ177.0,160.6,143.1,125.1,120.4,117.7,100.0,56.4,36.2,17.1.

[0243] 5-Fluoro-2,3-Dimethylbenzo[d]thiazolyl-3-onium iodide (26b)

[0244]

[0245] Commercially available 26a was synthesized according to the general procedure S2 (168 mg, 23%). f :0.32(DCM / MeOH9:1).1 H NMR (400MHz, DMSO-d6) δ 8.48 (dd, J = 9.1, 5.1 Hz, 1H), 8.34 (dd, J = 9.5, 2.5 Hz, 1H), 7.74 (td, J = 9.0, 2.4 Hz, 1H), 4.17 (s, 3H), 3.17 (s, 3H). 13 C NMR (101MHz, DMSO-d6)δ

[0246] 179.5,162.3(d,J=246.7Hz),142.7(d,J=12.8Hz),126.5(d,J=10.1Hz),1 24.7(d,J=2.1Hz), 116.8(d,J=25.0Hz), 104.2(d,J=28.8Hz), 36.4, 17.3. 19 F NMR(376MHz,DMSO-d6)δ-110.17.

[0247] 2,6-Dimethylbenzo[d]thiazole (27a)

[0248]

[0249] Synthesized according to standard procedure S1 (1.50 g, 60%). f :0.37 (heptane / EtOAc 3:1). 1 H NMR (400MHz, DMSO-d6) δ7.80 (p, J = 0.8Hz, 1H), 7.77 (d, J = 8.3Hz, 1H), 7.27 (dd, J = 8.2, 1.6Hz, 1H), 2.76 (s, 3H), 2.42 (s, 3H). 13 C NMR (101MHz, DMSO-d6) δ165.6,151.1,135.3,134.2,127.3,121.5,121.4,20.9,19.6.

[0250] 2,3,6-Trimethylbenzo[d]thiazol-3-onium iodide (27b)

[0251]

[0252] Synthesized according to the standard procedure S2 (378 mg, 50%). f :0.28(DCM / MeOH 9:1). 1H NMR (400MHz, DMSO-d6) δ8.21(t,J=1.2Hz,1H),8.18(d,J=8.7Hz,1H),7.72(dd,J=8.7,1.7Hz,1H),4.17(s,3H),3.14(s,3H),2.53(s,3H). 13 C NMR (101MHz, DMSO-d6) δ176.0,139.8,138.4,130.5,128.7,123.7,116.3,36.1,21.0,17.0.

[0253] 6-Methoxy-2-methylbenzo[d]thiazole (28a)

[0254]

[0255] Synthesized according to standard procedure S1 (1.34 g, 61%). f :0.29 (heptane / EtOAc 3:1). 1 H NMR (400MHz, DMSO-d6) δ7.78 (d, J = 8.9 Hz, 1H), 7.59 (d, J = 2.6 Hz, 1H), 7.05 (dd, J = 8.9, 2.6 Hz, 1H), 3.81 (s, 3H), 2.73 (s, 3H). 13 C NMR (101MHz, DMSO-d6) δ164.0,156.9,147.3,136.5,122.3,114.9,104.7,55.6,19.5.

[0256] 6-Methoxy-2,3-dimethylbenzo[d]thiazol-3-onium iodide (28b)

[0257]

[0258] Synthesized according to the standard procedure S2 (410 mg, 57%). f :0.15(DCM / MeOH 9:1). 1 H NMR (400MHz, DMSO-d6) δ8.18(d,J=9.3Hz,1H),8.00(d,J=2.6Hz,1H),7.47(dd,J=9.3,2.6Hz,1H),4.16(s,3H),3.90(s,3H),3.11(s,3H). 13 C NMR (101MHz, DMSO-d6) δ174.3,159.0,135.8,130.4,118.4,117.6,106.6,56.2,36.2,16.9.

[0259] 6-Fluoro-2-methylbenzo[d]thiazole (29a)

[0260]

[0261] 2-Amino-5-fluorobenzenethiol (300 mg, 2.09 mmol) and 2,4-pentanedione (323 μL, 3.14 mmol) were dissolved in MeCN (1 mL), and p-toluenesulfonic acid (18.0 mg, 0.10 mmol) was added. The mixture was stirred overnight at room temperature. It was diluted with water and extracted with EtOAc. The organic phase was dried over MgSO4, evaporated under reduced pressure, and purified by rapid chromatography (heptane / EtOAc, 0% B to 20% B) to give the product (257 mg, 73%). f :0.29 (heptane / EtOAc 5:1). 1 H NMR (400MHz, DMSO-d6) δ7.94 (dd, J = 8.2, 2.1 Hz, 1H), 7.91 (dd, J = 8.3, 4.3 Hz, 1H), 7.33 (td, J = 9.1, 2.7 Hz, 1H), 2.78 (s, 3H). 13 C NMR(101MHz,DMSO-d6)δ167.1(d,J=3.2Hz),159.4(d,J=241.7Hz),149.7,136.4(d , J=11.7Hz), 123.0 (d, J=9.5Hz), 114.3 (d, J=24.8Hz), 108.3 (d, J=27.1Hz), 19.7. 19 F NMR (376MHz, DMSO-d6) δ-117.24.

[0262] 6-Fluoro-2,3-Dimethylbenzo[d]thiazol-3-onium iodide (29b)

[0263]

[0264] Synthesized according to the standard procedure S2 (73.6 mg, 24%). f :0.17(DCM / MeOH 9:1). 1 H NMR (600MHz, DMSO-d6) δ8.36(dd,J=9.1,4.3Hz,1H),8.32(dd,J=8.3,2.7Hz,1H),7.84(td,J=9.1,2.7Hz,1H),4.19(s,3H),3.15(s,3H). 13C NMR(151MHz,DMSO-d6)δ177.8(d,J=2.5Hz),160.7(d,J=247.2Hz),138.5,130.3(d,J =12.2Hz), 118.8 (d, J = 9.6Hz), 117.9 (d, J = 25.7Hz), 110.9 (d, J = 28.6Hz), 36.5, 17.2. 19 F NMR (376MHz, DMSO-d6) δ-111.66.

[0265] 1-Methyl-3-nitro-2-thiocyanobenzene (30.1)

[0266]

[0267] A suspension of 2-methyl-6-nitroaniline (1.20 g, 7.89 mmol) in water (27.0 mL) was cooled to 0 °C, and 37% HCl (12.0 mL) was added. Sodium nitrite (653 mg, 9.46 mmol) was dissolved in water (5.00 mL) and added dropwise. After stirring at 0 °C for 5 minutes, a solution of potassium thiocyanate (2.30 g, 23.7 mmol) and ferric chloride (III) (640 mg, 3.94 mmol) in water (9.00 mL) was added dropwise. The mixture was stirred at room temperature for 5 hours. It was diluted with water and extracted with EtOAc. The organic phase was dried over MgSO4, evaporated, and used quantitatively without further purification. f :0.54 (heptane / EtOAc 1:1). 1 H NMR (400MHz, DMSO-d6) δ7.96 (d, J = 7.0 Hz, 1H), 7.82 (d, J = 6.9 Hz, 1H), 7.73 (t, J = 7.3 Hz, 1H), 2.66 (s, 3H). 13 C NMR (101MHz, DMSO-d6) δ152.9,144.5,135.4,132.1,123.1,117.0,110.5,21.1.

[0268] 2,7-Dimethylbenzo[d]thiazole (30a)

[0269]

[0270] Under an argon atmosphere, Na₂S (2.05 g, 26.3 mmol) was added fractionally to a 30.1 g (2.55 g, 13.1 mmol) EtOH / H₂O 1:1 v / v (45 mL, 45 mL) solution. The mixture was heated at 65 °C for 3 hours and then extracted with EtOAc. The organic phase was dried over MgSO₄, evaporated under reduced pressure, and purified by rapid chromatography (DCM / MeOH 0% to 2% B). The collected residue was dissolved in MeCN (1 mL) and 2,4-pentanedione (0.06 mL, 0.60 mmol) and p-toluenesulfonic acid (3.00 mg, 0.02 mmol) were added. The mixture was stirred at room temperature for 3 hours. It was diluted with water and extracted with EtOAc. The organic phase was dried over MgSO₄, evaporated, and purified by rapid chromatography (PE / EtOAc 0% to 20% B) to give the product (17.0 mg, 1%). f :0.26(PE / EtOAc 4:1). 1 H NMR (400MHz, CDCl3) δ7.79 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.15 (dt, J = 7.4, 1.0 Hz, 1H), 2.85 (s, 3H), 2.54 (s, 3H). 13 C NMR (151MHz, CDCl3) δ166.7,153.2,136.2,131.7,126.3,125.1,119.9,21.6,20.3.

[0271] 2,3,7-Trimethylbenzo[d]thiazol-3-onium iodide (30b)

[0272]

[0273] Synthesized according to standard procedure S2 (12.0 mg, 40%). f :0.35(DCM / MeOH 9:1). 1 H NMR (600MHz, DMSO-d6) δ8.15(d,J=8.5Hz,1H),7.83(dd,J=8.5,7.5Hz,1H),7.66(dt,J=7.5,1.0Hz,1H),4.21(s,3H),3.20(s,3H),2.66(s,3H). 13 C NMR (151MHz, DMSO-d6) δ176.2,147.0,136.7,133.5,129.7,128.5,114.5,36.6,19.3,17.2.

[0274] 2-Methoxy-6-nitroaniline (31.1)

[0275]

[0276] K₂CO₃ (3.75 g, 27.1 mmol) was added to a MeCN (47.0 mL) solution of 2-amino-3-nitrophenol (4.18 g, 27.1 mmol). Iodomethane (1.86 mL, 29.8 mmol) was added dropwise, and the mixture was stirred at room temperature for 22 hours. The mixture was diluted with water and extracted with EtOAc. The organic phase was dried over MgSO₄, evaporated under reduced pressure, and purified by rapid chromatography (heptane / EtOAc 0% to 20% B) to give an orange solid product (2.63 g, 58%). f :0.39 (heptane / EtOAc 2:1). 1 H NMR (400MHz, CDCl3) δ7.73 (dd, J = 8.9, 1.3 Hz, 1H), 6.88 (dd, J = 7.8, 1.3 Hz, 1H), 6.61 (dd, J = 8.9, 7.8 Hz, 1H), 6.42 (s, 2H), 3.92 (s, 3H). 13 C NMR (101MHz, CDCl3) δ148.3,137.2,131.8,117.5,114.7,113.5,56.4.

[0277] 1-Methoxy-3-nitro-2-thiocyanobenzene (31.2)

[0278]

[0279] The synthesis (quantification) was performed using the same procedure as in 30.1. R f :0.21(PE / EtOAc 3:1). 1 H NMR (600MHz, DMSO-d6) δ7.82–7.70 (m, 2H), 7.64 (dd, J = 8.2, 4.2Hz, 1H), 4.07 (s, 3H). 13 C NMR (151MHz, DMSO-d6) δ158.8,132.9,124.5,117.7,117.3,109.9,106.6,57.5.

[0280] 7-Methoxy-2-methylbenzo[d]thiazole (31a)

[0281]

[0282] The synthesis was carried out using the same procedure as 30a (49.0 mg, 2%). f:0.27(PE / EtOAc 4:1). 1 H NMR (600MHz, DMSO-d6) δ6.79 (t, J = 8.0 Hz, 1H), 6.26 (dd, J = 8.3, 0.9 Hz, 1H), 6.21 (dd, J = 7.8, 0.9 Hz, 1H), 3.70 (s, 3H), 2.11 (s, 3H). 13 C NMR (151MHz, DMSO-d6) δ176.9,153.8,142.9,131.4,117.0,109.1,109.1,57.0,36.9,17.6.

[0283] 7-Methoxy-2,3-dimethylbenzo[d]thiazole-3-onium iodide (31b)

[0284]

[0285] Synthesized according to standard procedure S2 (39.0 mg, 52%). f :38(DCM / MeOH 9:1). 1 H NMR (600MHz, DMSO-d6) δ7.88(s,2H),7.41(s,1H),4.20(s,3H),4.07(s,3H),3.20(s,3H). 13 C NMR (151MHz, DMSO-d6) δ176.9,153.8,142.9,131.4,117.0,109.1,109.1,57.0,36.9,17.6.

[0286] N-(2-bromo-3-fluorophenyl)acetamide (32.1)

[0287]

[0288] Pyridine (6.5 mL, 81.6 mmol) was added to a solution of 2-bromo-3-fluoroaniline (1.55 g, 8.16 mmol) in acetic anhydride (38.5 mL). The mixture was stirred at room temperature for 3 hours. It was then poured into water and extracted with EtOAc. The organic phase was dried over MgSO4, evaporated, and used without further purification (1.75 g, 93%). f :0.18(PE / EtOAc 4:1). 1 H NMR (600MHz, DMSO-d6) δ9.58 (s, 1H), 7.47 (dt, J = 8.1, 1.3 Hz, 1H), 7.39 (td, J = 8.2, 6.2 Hz, 1H), 7.18 (td, J = 8.5, 1.5 Hz, 1H), 2.10 (s, 3H).13 C NMR (151MHz, DMSO-d6) δ 168.7, 158.8 (d, J = 243.7Hz), 138.5, 128.7 (d, J = 9.2Hz), 122.4, 112.7 (d, J = 22.2Hz), 105.0 (d, J = 24.1Hz), 23.3.

[0289] N-(2-bromo-3-fluorophenyl)thioacetamide (32.2)

[0290]

[0291] Lawesson's reagent (1.71 g, 4.22 mmol) was added to a 12 mL solution of THF at 32.1°C (1.40 g, 6.03 mmol) under an argon atmosphere. The mixture was stirred overnight at room temperature, and the precipitate was filtered off. The filtrate was collected and further purified by rapid chromatography (PE / EtOAc 5% to 80% B) to give an orange oily product (1.04 g, 69%). f :0.72(PE / EtOAc 1:1). 1 HNMR (600MHz, DMSO-d6) δ11.54(s,1H),7.47(td,J=8.2,6.1Hz,1H),7.35(td,J=8.5,1.4Hz,1H),7.25(dt,J=8.0,1.4Hz,1H),2.62(s,3H). 13 C NMR(151MHz,DMSO-d6)δ202.4,159.1(d,J=244.9Hz),140.5(d,J=1.9Hz),129.1( d,J=9.3Hz), 125.7(d,J=3.1Hz), 115.3(d,J=22.4Hz), 108.5(d,J=21.4Hz), 33.2.

[0292] 7-Fluoro-2-methylbenzo[d]thiazole (32a)

[0293]

[0294] Under an argon atmosphere, Cs₂CO₃ (1.60 g, 4.90 mmol) and Pd(PPh₃)₄ (189 mg, 0.16 mmol) were added to a 32.2 g (810 mg, 3.26 mmol) solution of 1,4-dioxane (12 mL). The mixture was stirred overnight at 80 °C. It was diluted with water and extracted with EtOAc. The organic phase was dried over MgSO₄, evaporated under reduced pressure, and purified by rapid chromatography (PE / EtOAc 1% to 15% B) to give the product (314 mg, 58%).f :0.53(PE / EtOAc 3:1). 1 H NMR (600MHz, DMSO-d6) δ7.78(d,J=8.1Hz,1H),7.51(td,J=8.1,5.7Hz,1H),7.29(ddd,J=9.4,8.2,1.1Hz,1H),2.83(s,3H). 13 C NMR(151MHz,DMSO-d6)δ168.0,156.2(d,J=246.6Hz),155.9(d,J=2.6Hz),127.4( d,J=7.6Hz), 121.9(d,J=16.7Hz), 118.3(d,J=3.4Hz), 110.4(d,J=18.7Hz), 19.7.

[0295] 7-Fluoro-2,3-Dimethylbenzo[d]thiazol-3-onium iodide (32b)

[0296]

[0297] Synthesized according to standard procedure S2 (47.0 mg, 25%). f :0.19(DCM / MeOH 9:1). 1 H NMR (600MHz, DMSO-d6) δ8.21(d,J=8.5Hz,1H),7.97(q,J=7.5Hz,1H),7.78(t,J=8.9Hz,1H),4.24(s,3H),3.23(s,3H). 13 C NMR (151MHz, DMSO-d6) δ178.4, 155.8 (d, J = 250.7Hz), 143.9 (d, J = 5.6Hz), 131.5 (d, J =8.1Hz), 116.4 (d, J = 23.4Hz), 114.1 (d, J = 17.8Hz), 113.6 (d, J = 3.5Hz), 37.1, 17.6.

[0298] 2-Methylbenzo[d]thiazole (33a)

[0299]

[0300] p-Toluenesulfonic acid (402 mg, 2.34 mmol) was added to a mixture of 2-aminobenzenethiol (5.00 mL, 46.7 mmol) and 2,4-pentanedione (7.20 mL, 70.1 mmol), and stirred overnight. The mixture was diluted with water and extracted with EtOAc. The organic phase was dried over MgSO4, evaporated under reduced pressure, and purified by rapid chromatography (heptane / EtOAc 0% to 20% B) to give an orange oily product (5.74 g, 82%). f :0.23 (heptane / EtOAc 5:1). 1 H NMR(400MHz, DMSO-d6)δ8.02(dt,J=8.0,1.0Hz,1H),7.91(dt,J=8.2,0.9Hz,1H), 7.47(ddd,J=8.2,7.2,1.4Hz,1H), 7.38(ddd,J=8.3,7.3,1.3Hz,1H), 2.79(s,3H). 13 C NMR (101MHz, DMSO-d6) δ166.8, 153.0, 135.2, 125.9, 124.7, 121.9, 19.7.

[0301] 2,3-Dimethylbenzo[d]thiazol-3-onium iodide (33b)

[0302]

[0303] The synthesis was carried out according to the general procedure S2, yielding a pink solid (1.21 g, 50%). f :0.18(DCM / MeOH 9:1). 1 HNMR(400MHz,DMSO-d6)δ8.44(dd,J=8.1,1.2Hz,1H),8.29(d,J=8.4Hz,1H),7.90(ddd ,J=8.5,7.3,1.3Hz,1H),7.81(ddd,J=8.3,7.2,1.1Hz,1H),4.21(s,3H),3.18(s,3H). 13 C NMR (101MHz, DMSO-d6) δ177.2,141.6,129.2,128.7,128.0,124.5,116.7,36.2,17.1.

[0304] 4-(piperidin-1-yl)benzaldehyde (34)

[0305]

[0306] The synthesis was carried out according to the general procedure S4, yielding a white solid (6.45 g, 86%).f :0.18 (heptane / EtOAc 5:1). 1 H NMR (400MHz, DMSO-d6) δ9.68(s,1H),7.67(dt,J=9.8,2.9Hz,2H),7.00(dt,J=9.0,2.7Hz,2H),3.41(dd,J=6.6,3.9Hz,4H),1.64–1.52(m,6H). 13 C NMR (101MHz, DMSO-d6) δ189.9,154.6,131.6,125.4,113.0,47.6,24.8,23.9.

[0307] 4-Morphyrinylbenzaldehyde (35)

[0308]

[0309] Synthesized according to standard procedure S4 (1.77 g, 58%). f :0.19 (heptane / EtOAc 3:1). 1 H NMR (600MHz, DMSO-d6) δ9.74(s,1H),7.73(dt,J=9.0,2.8Hz,2H),7.06(dt,J=8.9,2.6Hz,2H),3.78–3.69(m,4H),3.38–3.32(m,4H). 13 C NMR (151MHz, DMSO-d6) δ190.4,154.9,131.4,126.7,113.2,65.8,46.6.

[0310] 4-Thiomorpholinobenzaldehyde (36)

[0311]

[0312] Synthesized according to standard procedure S4 (73.5 mg, 11%). f :0.22 (heptane / EtOAc 5:1). 1 H NMR (600MHz, DMSO-d6) δ9.70 (s, 1H), 7.71 (dd, J = 9.0, 2.8Hz, 2H), 7.13–6.89 (m, 2H), 3.93–3.69 (m, 4H), 2.75–2.55 (m, 4H). 13 C NMR (151MHz, DMSO-d6) δ190.1,153.3,131.7,125.8,113.4,49.4,24.8.

[0313] 4-(pyrrolidone-1-yl)benzaldehyde (37)

[0314]

[0315] Synthesized according to standard procedure S4 (381 mg, 46%). f :0.37 (heptane / EtOAc 3:1). 1 H NMR (400MHz, DMSO-d6) δ9.64(s,1H),7.67(dt,J=8.8,2.7Hz,2H),6.63(dt,J=8.8,2.6Hz,2H),3.38–3.30(m,4H),1.98–1.93(m,4H). 13 C 1NMR (101MHz, DMSO-d6) δ 189.6, 151.6, 131.7, 124.2, 111.2, 47.3, 24.9.

[0316] 4-(4-Fluoroperidin-1-yl)benzaldehyde (38)

[0317]

[0318] Synthesized according to standard procedure S4 (293 mg, 59%). f :0.21 (heptane / EtOAc 3:1). 1 H NMR (400MHz, DMSO-d6) δ9.71(s,1H),7.70(dt,J=8.9,2.5Hz,2H),7.07(dt,J=8.9,2.5Hz,2H),4.90(dtt,J=48.9,7.1,3.6H z,1H),3.71–3.55(m,2H),3.41(ddd,J=13.5,7.4,3.9Hz,2H),1.94(dddd,J=24.9,12.0,8.1,3.8Hz,2H),1.84–1.62(m,2H). 13 C NMR (101MHz, DMSO-d6) δ190.6, 154.5, 132.1, 126.5, 113.8, 88.8 (d, J = 169.5Hz), 43.7 (d, J = 6.6Hz), 30.8 (d, J = 19.3Hz). 19 F NMR (376MHz, DMSO-d6) δ-177.3.

[0319] 4-(dimethylamino)benzaldehyde (39)

[0320]

[0321] Synthesized according to standard procedure S4 (346 mg, 41%). f :0.33 (heptane / EtOAc 3:1). 1 H NMR (400MHz, DMSO-d6) δ9.67 (s, 1H), 7.68 (dt, J = 9.1, 2.7Hz, 2H), 6.79 (dt, J = 8.9, 2.8Hz, 2H), 3.04 (s, 6H). 13 C NMR (101MHz, DMSO-d6) δ190.3,154.7,132.0,125.0,111.5,40.1.

[0322] 4-(4-methylpiperazin-1-yl)benzaldehyde (40)

[0323]

[0324] Synthesized according to standard procedure S4 (408 mg, 50%). f :0.08 (DCM / MeOH 2%). 1 H NMR(400MHz, DMSO-d6)δ9.71(s,1H),7.70(dt,J=9.0,2.8Hz,2H),7.04(dt,J=9.0, 2.5Hz, 2H), 3.37 (dd, J=6.4, 3.8Hz, 4H), 2.42 (dd, J=6.6, 3.6Hz, 4H), 2.21 (s, 3H). 13 C NMR (101MHz, DMSO-d6) δ190.2,154.7,131.4,126.2,113.3,54.2,46.3,45.7.

[0325] 2-(4-Bromophenyl)benzo[d]thiazole (41)

[0326]

[0327] A solution of 2-aminobenzylthiol (750 mg, 5.99 mmol) and 4-bromobenzaldehyde (1.11 g, 5.99 mmol) in DMSO (6.00 mL) was heated at 140 °C for 75 min. The mixture was diluted with water and extracted with EtOAc. The organic phase was dried over MgSO4, evaporated under reduced pressure, and recrystallized from Et2O to give a white solid product (569 mg, 33%). f :0.70(PE / EtOAc 5:1). 1HNMR(600MHz,DMSO-d6)δ8.16(dt,J=8.2,0.8Hz,1H),8.07(dt,J=8.0,0.8Hz,1H),8.03(dt,J=8.5,2.5H z,2H),7.77(dt,J=8.5,2.5Hz,2H),7.56(ddd,J=8.2,7.2,1.3Hz,1H),7.48(ddd,J=8.2,7.2,1.2Hz,1H). 13 C NMR (151MHz, DMSO-d6) δ166.1,153.5,134.5,132.4,132.0,129.0,126.8,125.8,124.9,123.0,122.4.

[0328] 3-(4-(piperidin-1-yl)phenyl)propenal (42)

[0329]

[0330] A solution of 34 (800 mg, 4.23 mmol) in 96% H₂SO₄ (3 mL) was cooled to 0 °C, and acetaldehyde (0.71 mL, 12.7 mmol) was added dropwise. The mixture was stirred at 0 °C for 1 hour. It was then poured into water, neutralized with 18 M NaOH aqueous solution, and extracted with EtOAc. The organic phase was dried over MgSO₄, evaporated under reduced pressure, and purified by rapid chromatography (PE / EtOAc 5% to 10% B) to give the product (390 mg, 43%). f :0.51(PE / EtOAc 3:1). 1 H NMR(600MHz,DMSO-d6)δ9.54(d,J=7.9Hz,1H),7.59–7.52(m,3H),6.95(dt,J=8.9, 3.1Hz, 2H), 6.60 (dd, J=15.6, 7.9Hz, 1H), 3.34 (d, J=5.4Hz, 4H), 1.60–1.56 (m, 6H). 13 C NMR (151MHz, DMSO-d6) δ193.7,153.9,152.9,130.6,124.0,122.7,114.1,47.9,24.9,24.0.

[0331] 3-(4-(pyrrolidone-1-yl)phenyl)propenal (50)

[0332]

[0333] The synthesis was carried out using the same procedure as 42 (513 mg, 64%). f:0.32(PE / EtOAc 4:1). 1 H NMR (600MHz, DMSO-d6) δ9.51 (d, J = 8.0Hz, 1H), 7.58–7.51 (m, 3H), 6.59–6.53 (m, 3H), 3.32–3.28 (m, 4H), 2.00–1.92 (m, 4H). 13 C NMR (151MHz, DMSO-d6) δ193.3,154.5,149.7,130.8,122.7,120.8,111.7,47.3,24.9.

[0334] 1.1.6. Biological Evaluation

[0335] 1.1.6.1. Calculation of BBB score and CNS MPO

[0336] All required properties (cLogP, cLogD, TPSA, molecular weight, pKa) were calculated using Chemicalize (ChemAxon, Budapest, Hungary) and entered into Excel spreadsheets provided by Gupta, M. et al., The Blood–Brain Barrier (BBB) ​​Score; Journal of Medicinal Chemistry 2019, 62(21), 9824-9836 and Wager, TT et al., Moving beyond Rules: The Development of a Central Nervous System Multiparameter Optimization (CNS MPO) Approach To Enable Alignment of Druglike Properties; ACS Chemical Neuroscience 2010, 1(6), 435-449. All values ​​are reported in Table 5.

[0337] 1.1.6.2. Preparation of αSYN and Aβ1-42 fibrils

[0338] Aβ 1-42The method for generating fibrils is adapted from Bagchi, D.P. et al., Binding of the radioligand SIL23 to alpha-synuclein fibrils in Parkinson disease braintissue establishes feasibility and screening approaches for developing a Parkinson disease imaging agent; PLoS One 2013, 8(2), e55031, and described in Kuebler, L. et al., [ 11 [C]MODAG-001—towards a PET tracer targeting α-synuclein aggregates; European Journal of Nuclear Medicine and Molecular Imaging 2020, 48, 1759-1772. Synthetic lyophilized human Aβ with a purity >90% was used. 1-42 Peptide (5 mg) (EMC Microcollections, Tuebingen, Germany) was dissolved in DMSO (221.5 μL), followed by the addition of deionized water (4.1 mL) and 1 M Tris-HCl (111 μL, pH 7.6) to achieve a final monomer concentration of 250 μM. Aggregation was induced by incubation at 37 °C and 800 rpm for 72 hours using an Eppendorf thermostatic mixer. The resulting fibrils were sonicated for 3 minutes in a water bath (Elmasonic S 60H, ElmaSchmidbauer GmbH, Singen, Germany). The final product was aliquoted, frozen on dry ice, and stored at -80 °C until use.

[0339] 1.1.6.3. Determination of fibril binding

[0340] Human recombinant αSYN (for […]) diluted in phosphate buffered solution (PBS; Gibco DPBS, calcium-free, magnesium-free, Thermo Fisher Scientific, Waltham, MA, USA) 3 H]PiB is 180nM, for [ 3 H]MODAG-001 (50 nM) and synthetic human Aβ 1-42 fibrils (for [ 3 H]PiB is 2μM, for [ 3H]MODAG-001 (1 μM) was determined by saturation binding assay. 3 H]PiB and [ 3 H]MODAG-001's K d Value. The fibrils were incubated in a 96-well micro-detection low-binding plate (Ratiolab GmbH, Dreieich, Germany). The 96-well micro-detection low-binding plate exhibits gradually increasing concentrations in 30 mM Tris-HCl, 0.1% bovine serum albumin, and 0.05% Tween 20. 3 H]PiB (up to 56nM) and [ 3 [H]MODAG-001 (up to 36 nM), total volume 200 μL / well. The tracer was incubated with the corresponding non-radioactive compound (1.5 μM PiB or 0.5 μM MODAG-001). Stock solutions of PiB and MODAG-001 were prepared by dissolving the compounds in DMSO to 1 mM, ensuring that the DMSO concentration in the final assay was ≤0.15%.

[0341] By targeting [ 3 H]PiB and [ 3 Competitive binding assays of H]MODAG-001 were performed to determine the binding affinity (K) of the newly developed 2-styrylbenzothiazole. i The test compound was dissolved in DMSO to a stock concentration of 1 mM, ensuring that the DMSO concentration in the final determination was ≤1%. Adding 6 nM [ 3 H]PiB and 1nM[ 3 H]MODAG-001 competing test compound concentrations (0.6 nM–10 μM). αSYN and Aβ 1-42 The concentration of fibrils is as described above.

[0342] The culture plates were incubated on a shaker (MaxQ 6000, Thermo Fisher Scientific Inc., Marietta, Ohio, USA) at 50 rpm at 37°C for 2 hours and covered with removable sealing tape (PerkinElmer, Waltham, Massachusetts, USA). Vacuum filtration and readings were performed according to the method disclosed in Kuebler, L. et al., [11C] MODAG-001—towards a PET tracer targeting α-synuclein aggregates; European Journal of Nuclear Medicine and Molecular Imaging 2020, 48, 1759-1772. Radioactivity and [ 3 H]PiB、[ 3 The relationship between the concentrations of H]MODAG-001 or non-radioactive test compounds is plotted. Nonlinear regression analysis in GraphPad Prism (GraphPad Software, Inc., version 8.4.0, La Jolla, USA) was used to fit the data points.

[0343] 1.1.6.4. Autoradiography and Immunohistochemistry

[0344] Postmortem human brain slices (10 μm thick) were obtained from the Munich Neurobiobank (NBM, Munich, Germany) and analyzed from the subject cases. Tissue samples were collected with informed consent, following guidelines (No. 345-13) of the Ethics Committee of the Ludwig-Maximilians-University in Munich, Germany. The use of brain tissue samples in this study was approved by the Ethics Committee of the Faculty of Medicine at the University of Tübingen (Ethics Approval No.: 813 / 2018BO2). Table 1 summarizes the subject information from which samples were obtained and used in the experiments.

[0345] Table 1. Overview of human brain tissue used in autoradiography and immunohistochemistry experiments. The degree of pathology in each subject's case was analyzed by the Munich Neurobiobank and indicated by the symbols "+" and "-". The number of "+" symbols indicates the degree of pathology, increasing sequentially from low (+), medium (++), to high (+++); "-" indicates no pathology. Abbreviations: Aβ, β-amyloid; αSYN, α-synuclein; AD, Alzheimer's disease; CB, cerebellum; Ctrl, control; F, female; FC, frontal cortex; LBD, Lewy body dementia; M, male; MSA, multiple system atrophy; PD, Parkinson's disease; PMI, postmortem interval; pTau, phosphorylated Tau.

[0346]

[0347] To perform autoradiography, brain slices were thawed for one hour and then pre-incubated in BSA buffer at room temperature for 25 minutes. To determine total binding (TB), brain slices were mixed with tracer (10 nM […]. 18 F]PFSB, 10nM[ 18 Incubation with MFSB. Serial sections were incubated with the corresponding non-radioactive compounds (10 μM PFSB, 10 μM MFSB) to determine non-specific binding (NSB). The non-radioactive compounds were dissolved in DMSO to 10 mM, resulting in a final DMSO concentration ≤0.1%. Incubation was performed at room temperature for one hour, followed by washing with cold BSA buffer (three times, ten minutes each), and then immersion in cold deionized water three times. After drying under UV light, brain sections were exposed to a storage phosphor screen (Molecular Dynamics, Caesarea, Israel) for 18 hours, and then scanned using a phosphorescence imaging system (STORM 840, Molecular Dynamics, Sunnyvale, California, USA).

[0348] Quantitative data analysis was performed by plotting four regions of interest (ROIs) in the relevant regions of the slices and drawing an ROI for background subtraction next to them (ImageJ 1.8.0_172, National Institutes of Health, Bethesda, Maryland, USA) (Schneider, CA et al., NIH Image to ImageJ: 25 years of image analysis; Nat Methods 2012, 9(7), 671-5). Specific binding (SB) was obtained by subtracting NSB from TB. The SB disease / SB control ratio was calculated by dividing the SB in diseased tissue by the corresponding SB in control tissue.

[0349] Following the autoradiography procedure, brain sections were stored at -20°C. IHC was performed on the same tissue sections used for autoradiography of TB. After thawing, sections were fixed for 20 minutes at room temperature with 4.5% paraformaldehyde (PFA, SAV Liquid Production GmbH, Flintsbach am Inn, Germany). Antigen retrieval was performed after washing in PBS (2 x 5 min). For αSYN pSeR129 staining, brain sections were boiled in sodium citrate buffer (10 mM, pH 6, Sigma Aldrich Chemie GmbH, Darmstadt, Germany) and incubated at room temperature in the boiled buffer for 30 minutes; brain sections to be stained for Aβ were incubated in 97% formic acid at room temperature for 10 minutes. After washing, quenching was performed for 20 minutes (1 mL quenching solution = 890 μL Tris-buffered saline (TBS), 100 μL methanol, and 10 μL 30% hydrogen peroxide). Subsequently, brain slices were washed and equilibrated in TBS (2 x 5 min) and TBS supplemented with 0.1% Triton-X and 1% BSA (hereinafter referred to as TBS-X) (1 x 5 min). Afterwards, they were blocked at room temperature for 60 min in TBS supplemented with 0.3% Triton-X and 10% normal goat serum. The slices were then incubated overnight at 4°C with either mouse anti-phosphorylated αSYN pSeR129 monoclonal antibody (1:5000 in TBS-X, clone pSyn#64; 015-25191, FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) or mouse anti-β-amyloid 17-24 antibody (1:6000 in TBS-X, clone 4G8, 800708, BioLegend, Amsterdam, Netherlands).

[0350] The next day, brain sections were washed with TBS-X (3 x 10 min) and incubated with secondary antibody (EnVision+ / HRP Dual LinkRabbit / Mouse, K406189-2, Agilent Technologies, Waldbronn, Germany) at room temperature for 30 min. After washing with TBS-X (2 x 10 min) and TBS (1 x 10 min), the samples were incubated with 3,3'-diaminobenzidine (1:50, Agilent Technologies, Waldbronn, Germany) for 10 min. After washing with distilled water (2 x 5 min), the brain sections were incubated in hematoxylin (Merck kGaA, Darmstadt, Germany) for 45 sec, and then rinsed with running tap water for 10 min. To dehydrate and clarify the tissue, samples were washed with 70% EtOH (1 min), 95% EtOH (2 x 1 min), 100% EtOH (2 x 1 min), and xylene (2 x 2 min). Slides were mounted with Eukitt fast-curing mounting medium (FlukaAnalytical, Munich, Germany). The stained tissue was scanned at 40x magnification using a NanoZoomer 2.0HT (Hamamatsu Photonics KK, Hamamatsu, Japan).

[0351] 1.1.6.5. In vivo PET / MR imaging

[0352] Animal experiments were conducted in accordance with the European Directive on the Protection and Use of Laboratory Animals (EU Council Directive 2010 / 63 / UE) and the German Animal Welfare Act, and were approved by the local official body (Tübingen District Government, R3 / 19G). Three healthy wild-type female C57BL / 6J mice (20.3±0.9g; 9 weeks old) were purchased from Charles River Laboratories (Sulzbach, Germany) and housed in animal enclosures with a 12:12 hour diurnal cycle, a temperature of 22°C, and a humidity of 40-60%, with free access to standard diet and tap water.

[0353] Mice (n=3) were anesthetized with 1.5% isoflurane evaporated in 100% oxygen at a flow rate of 0.8 L / min. The body temperature of the mice was maintained at 37°C using a feedback temperature control device. PET imaging was performed on an Inveon dedicated microPET system (Inveon D-PET, Siemens, Knoxville, Tennessee, USA). Five seconds after the start of PET acquisition, mice were intravenously injected with 9.9 ± 0.6 MBq of […]. 18 F]MFSB (A during injection) m=46.2±2.5 GBq / μmol). One hour of dynamic acquisition was divided into 39 time frames (12×5s, 6×10s, 6×30s, 5×60s, and 10×300s). Transmission measurements were performed for 13 minutes using a cobalt-57 point source for attenuation correction. Subsequently, anatomical magnetic resonance (MR) scans were performed using a 7Tesla MR scanner (ClinScan, Bruker BioSpin MRI GmbH, Ettlingen, Germany) with a whole-body rat volume coil and Paravision software (v6.0.1, Bruker, Ettlingen, Germany) using T2-weighted Turbo-RARE MRI sequences.

[0354] PET images were reconstructed using the OSEM3D / SP-MAP reconstruction algorithm and registered with whole-body MRI scans. Based on MR anatomy in PMOD (PMOD Technologies, Faellanden, Switzerland, version 4.2), volumes of interest (VOIs) of relevant organs were hand-drawn, and VOIs of different regions of the mouse brain were extracted using atlases provided by PMOD to calculate the corresponding time-activity curves (TACs). The normalized uptake value (SUV) was calculated as the ratio of detected activity to injected activity and body weight (SUV = (TAC(kBq / cc) / injected activity(kBq)) * body weight(g)).

[0355] 2. Results

[0356] 2.1. Development and in vitro evaluation of libraries based on 2-styrylbenzothiazole

[0357] A library of 2-styrylbenzothiazole compounds was designed and developed to prepare six classes of compounds ( Figure 1 The effects of structural modifications on their affinity for αSYN protofibrils were investigated, ultimately yielding lead compounds. All compounds exhibited fluorescence. Methyl, methoxy, and fluorine substitutions were performed at different positions on the benzothiazole ring (compounds 1a-12b). The length of the conjugated system was varied by directly attaching the phenyl moiety to the benzothiazole or by adding two additional carbon atoms (compounds 13a-14b). The effect of aniline nitrogen substitution on binding affinity was also investigated (compounds 15a-19b). Furthermore, for each structural modification, the effect of removing N-methylation was studied by synthesizing non-N-methylated analogs.

[0358] Depending on the availability of starting materials, different substituted benzothiazoles (compounds 21a-33a) were prepared via a dedicated synthetic route. All benzothiazole compounds were N-methylated via reaction with methyl iodide or methyl p-nitrobenzenesulfonate, according to their reactivity (compounds 21b-33b). Figure 2 Various 4-aminobenzaldehydes (compounds 34-40) were synthesized via base-activated substitution of 4-fluorobenzaldehyde with different amines. The benzothiazole and benzaldehyde moieties were combined via condensation reactions to generate RB1, RB2, and 35 other analogues. Figure 2 Pd(PPh3)4 catalyzes the amination of 2-(4-bromophenyl)benzothiazole (41), followed by N-methylation, to give ThT analogs 13a and 13b. 4-Piperidinecinnamaldehyde (42) is synthesized from the reaction of the corresponding benzaldehyde with acetaldehyde under strongly acidic conditions, followed by coupling with 2-methylbenzothiazole to generate 1,3-butadiene derivatives (compounds 14a and 14b). Figure 3 ).

[0359] pass[ 3 H]PiB competitive assays screened the binding affinity of the entire compound library for αSYN fibrils (Tables 2 and 3). The results showed that the affinity fluctuated significantly with structural modifications. The affinity of some analogs was significantly increased, with the 6-methoxy derivative 8b showing a higher K... i The value is as high as 14.7 ± 5.1 nM.

[0360] Table 2. By [ 3 The binding affinity (mean K) of nonmethylated compounds determined by the H]PiB competition assay i ±SEM (two data points) and calculated BBB score and CNS MPO. (For 9a, 14a, 15a, 17a, and 18a, based on three data points; for 12a, based on a single data point.)

[0361]

[0362]

[0363] Table 3. By [ 3 The binding affinity (average K) of methylated compounds determined by H]PiB competitive assay. i ±SEM, two data points).

[0364]

[0365]

[0366] As shown in Tables 2 and 3, different structural modifications exhibit different effects on neutral and cationic compounds. These different effects may be related to the configuration of their double bonds. Nonionic derivatives were shown to be a mixture of E- and Z-isomers, while N-methylated analogs preferentially exhibited the Z-configuration. 1 The 1H NOESY NMR spectrum is shown in Figure 5.

[0367] To cross-match binding properties with compounds predicted to reach the brain, BBB scores and CNS MPO (Central Nervous System Multiparameter Optimization) were calculated. Calculated BBB scores for all nonionic compounds ranged from 4.62 to 4.90 (range: 0 to 6), and CNS MPO scores ranged from 3.0 to 3.5 (range: 0 to 5). Since these two parameters did not differ significantly among analogues, they were not considered in the selection of lead compounds.

[0368] Based on their affinity for αSYN, the most promising compounds were selected for further evaluation, and […]. 3 The selectivity of the analogs for Aβ fibrils was evaluated in a competitive binding assay using H]PiB. No significant competition for the tracer was detected in any of the tested analogs, indicating that the 2-styrylbenzothiazole compounds in this library are characterized by high selectivity for αSYN relative to Aβ. Figure 4 b, 6b).

[0369] 2.2. Optimization by combining promising features

[0370] Based on the observations, certain structural features were found to facilitate binding with αSYN fibrils. To further optimize this process, compounds 43 and 44 were developed by combining these components: due to K... i Because of its low value and ease of fluoro-18 labeling, 6-fluorobenzothiazole was chosen, and a longer π system (n=2) was employed, with N-pyrrolidine substitution ( Figure 4 a). Although the binding rate with Aβ remains low ( Figure 4 b), but for [ 3 Competitive binding assays of H]PiB showed a decrease in affinity for αSYN fibrils (K). i-43 213.9±121.5 nM, K i-44 The value is 85.0 ± 41.0 nM, therefore the optimization is considered unsuccessful.

[0371] In addition to attempting to enhance binding affinity, the combination of favorable moieties also focused on improving pharmacokinetics. Since most compounds in the library had cLogP ≥ 5.5 (Table 5), their lipophilicity was a concern. Therefore, a more hydrophilic derivative was designed. N-morpholine compound 15a exhibited a similar affinity to its N-piperidine analog 20, along with a significantly lower cLogP (4.57, Table 5) and the highest CNS MPO among the evaluated compounds (3.5, Table 5). Therefore, since compound 9a (PFSB) was selected from the first group of analogs for radiolabeling and further evaluation, the N-morpholine moiety was chosen to be combined with 6-fluorobenzothiazole to generate MFSB (45, Figure 6a). [ 3 The competitive binding assay of H]PiB demonstrated that its affinity was not only comparable to PFSB, but also increased by 2.5 times (K). i (10.3 ± 4.7 nM). The selectivity for αSYN relative to Aβ remained unchanged. Figure 6 b).

[0372] Table 5. Chemical properties required to calculate the BBB score and CNS MPO for all nonionic compounds.

[0373]

[0374] Due to scaffold similarity, all fibril binding assays were performed using [ 3 H]PiB was used as a competitor to the radiolabeled compound to assess the impact of overall library structural modifications on binding to αSYN. To evaluate their affinity in a non-relative manner, PFSB and MFSB were also compared with [ 3 H]MODAG-001 underwent a competitive evaluation. 3 H]MODAG-001 has a low K content of 0.6±0.1 nM. d The K value is the current standard for preclinical imaging of αSYN. Both compounds showed moderate to good competition, with MFSB exhibiting K... i 4.6 times lower than PFSB (K i -PFSB: 142.1±16.9 nM, K i -MFSB: 30.8±10.5 nM, Figure 6 c).

[0375] 2.3.[ 18 F]PFSB and [ 18 F]MFSB radiolabeling

[0376] From the first group of compounds, PFSB was selected for radiolabeling and further evaluation. Brominated analogs (46) were synthesized according to standard procedures. Figure 2 ), and in the Pd(dppf)Cl2-catalyzed boronization reaction, a pinacol borate precursor (48) is generated for use with fluorine-18 ( 18 F) mark ( Figure 7 A copper-mediated radiofluorination reaction (CMRF) was established by optimizing the amounts of precursor and pyridine (Table 4). Since the precursor loading did not appear to affect the reaction efficiency, a lower loading was chosen due to poor solubility. Increasing the pyridine concentration negatively impacted the radiochemical conversion (RCC). Based on these results, entry (b) was selected as the starting point for automation. [The process involves generating...] 18 F]PFSB, radiochemical yield (RCY) was 5.8% ± 1.3, molar activity (Am The value was 36.5 ± 8.5 GBq / μmol.

[0377] The same procedure is applied to morpholine analogs. 18 F]MFSB precursor synthesis (49) and radiolabeling ( Figure 7 The tracer was obtained with an RCY of 11.6% ± 2.9%. m The concentration was 41.2 ± 12.0 GBq / μmol. The analytical results for the two tracers are as follows: Figures 8 to 11 As shown.

[0378] Table 4. 18 CMRF optimization of F]PFSB: variations in precursor and pyridine dosages and corresponding RCC. The amounts of Cu(OTf)2, n-BuOH, and DMA remained constant in all experiments.

[0379]

[0380] 2.4. External autoradiography and in vivo PET / MR imaging

[0381] To verify the results of the fibril binding assay, human brain slices were subjected to […]. 18 F]PFSB in vitro autoradiography. Experiments confirmed previously observed binding characteristics: the tracer exhibited an affinity for αSYN pathology, and selectivity for αSYN relative to Aβ. Figure 12 Brain slices from MSA patients showed exceptionally high specific binding (SB), with a ratio of 3.94 ± 0.30 compared to controls studying the same brain regions (Table 6). Conversely, Alzheimer's disease (AD) samples did not show increased binding in areas where Aβ plaques were detected by immunohistochemistry (IHC) (AD / frontal cortex SB ratio: 1.06). Figure 12 ). 18 Autoradiography of F]MFSB confirmed the selective binding of the 2-styrylbenzothiazole compound in the library to αSYN, as the AD / SB ratio in the gray matter region remained well maintained. Figure 12 ).

[0382] Table 6. 18 F]PFSB and [ 18 F]MFSB in vitro autoradiography quantitative analysis. When using [ 18 In the F]MFSB experiment, high NSB hindered the accurate quantification of the disease / control SB ratio in MSA slices.

[0383]

[0384] 2.5. Blood-brain barrier (BBB) ​​crossing and in vivo detection

[0385] To evaluate [ 18 The pharmacokinetic characteristics of F]MFSB, will [ 18 F]MFSB was injected into three healthy wild-type mice, and its distribution over 60 minutes was assessed by dynamic PET / MR. The experiment demonstrated [ 18 F]MFSB successfully crossed the BBB and was observed to begin clearing ( Figure 13 High uptake (SUV≈5) was detected in the lungs and liver within the first 2 minutes, followed by rapid to moderate clearance. Significant uptake was also observed in the kidneys, indicating that renal excretion began within the first few minutes. Figure 13 This indicates that [ 18 F]MFSB can cross the BBB and can be detected in vivo as an α-synuclein-binding compound. Therefore, α-synuclein-binding compounds, as 2-styrylbenzothiazole derivatives, are highly promising diagnostic compounds, at least in the context of neurodegenerative diseases.

[0386] 3. Conclusion

[0387] The inventors have provided high-affinity and high-selectivity α-synuclein binding compounds suitable for the diagnosis and research of synucleinosis.

Claims

1. An α-synuclein-binding compound having structure 1, - Where X1-X6 are independently selected from: CR x , N, where each R x x in R is equal to x The subscript of X is combined if at most one X is N in the group consisting of X1 and X2 and in the group consisting of X3 and X4; and -Among them, R7, R8 and R x Independently selected from: H, F, Cl, Br, OH, C1-C3 alkyl ether groups, fluorinated C1-C3 alkyl groups, and preferably selected from: H, O-CH3, F; and - X7-X9 are independently selected from CH2, O, NH, and S, and are preferably selected from CH2 and O, provided that no heteroatom is directly bonded to the heteroatom.

2. The α-synuclein binding compound according to claim 1, wherein X1 to X6 are CR x .

3. The α-synuclein binding compound according to claim 2, wherein R1 to R4 are H.

4. The α-synuclein binding compound according to any one of claims 2 to 3, wherein R5, R6 and R7 are H.

5. The α-synuclein binding compound according to any one of claims 2 to 4, wherein R8 is F.

6. The α-synuclein binding compound according to any of the preceding claims, wherein at most one of X7 to X9 is not CH2.

7. The α-synuclein binding compound according to any one of the preceding claims, having structure 2 or structure 3.

8. The α-synuclein binding compound according to any of the preceding claims, wherein the compound is fluorescent.

9. The α-synuclein binding compound according to any of the preceding claims, wherein at least one atom of the compound is a radionuclide.

10. The α-synuclein binding compound according to claim 9, wherein at least one C atom is 11 C radionuclide.

11. The α-synuclein binding compound according to any one of claims 9 or 10, wherein at least one F atom is 18 F is a radioactive nuclide.

12. The α-synuclein binding compound according to any one of the preceding claims, for use in diagnosing diseases of an organism, wherein the disease is preferably a neurodegenerative disease, more preferably a neurodegenerative disease associated with the presence of non-physiological α-synuclein in mammalian tissues.

13. The α-synuclein binding compound according to any of the preceding claims, used for the purpose of claim 12, wherein the diagnosis is a diagnosis of synucleinosis, wherein the synucleinosis is preferably selected from: Parkinson's disease, multiple system atrophy, and Lewy body dementia.

14. A diagnostic composition comprising the α-synuclein binding compound of any of the preceding claims and a pharmaceutically acceptable carrier.

15. The diagnostic composition of claim 14, wherein the diagnostic composition is configured for administration to an organism, wherein the organism is preferably a mammal.

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