Photostable tau probe

Abstract

The present invention belongs to the technical field of biomedicine. Disclosed are the preparation of a photostable Tau probe and the use thereof. As a Tau probe with high selectivity and high sensitivity, the probe compound of the present invention is expected to become an important detection method for clinical diagnosis of diseases associated with the Tau protein.

Description

A photostable tau probe TECHNICAL FIELD

[0001] The present application relates to a preparation and application of a photostable tau probe BMP. BACKGROUND

[0002] Tauopathies are a group of neurodegenerative diseases characterized by abnormal accumulation of Tau protein in the brain, including Alzheimer's disease and more than 10 primary tauopathies. Alzheimer's disease (AD) is one of the most common forms of dementia worldwide, characterized by the gradual accumulation of amyloid-beta (Aβ) plaques and hyperphosphorylated Tau protein, which forms neurofibrillary tangles in the brain. Tau PET tracers are essential for imaging Tau pathology in Alzheimer's disease (AD) and other neurodegenerative diseases, enabling visualization and quantitative analysis of Tau aggregates. This contributes to early detection and staging of the disease, monitoring of progression, and evaluation of treatment efficacy.

[0003] Currently, [18F]-FlorTaucipir (AV-1451) is the only Tau-PET tracer approved by the US FDA in 2020. However, first-generation tracers such as AV-1451 and PBB3 have low sensitivity and insufficient specificity, leading to binding with non-target structures. In addition, their metabolites also interfere with imaging. The second-generation Tau tracer PM-PBB3 (APN-1607) has better sensitivity, binding capacity, brain penetration, and pharmacokinetics. However, PM-PBB3 is structurally unstable and prone to double bond isomerization, and PM-PBB3 also binds to amyloid fibers (such as transmembrane protein 106B) off-target. These challenges highlight the need to develop more sensitive, specific, and easy-to-apply Tau imaging agents.

[0004] There is still an urgent need in the art for a Tau probe with good stability and high sensitivity. SUMMARY

[0005] To address the limitations of existing Tau tracers, we synthesized a BMP fluorescent molecule by strategically introducing substituents at different positions of the butadiene double bond of PM-PBB3. Compared to PM-PBB3, the BMP molecule exhibits better photostability and significantly improved sensitivity and selectivity for Tau pathology in APP / PS1 / Tau triple transgenic mouse brain sections, especially in the superficial cortical layers 2 / 3. Characterization of BMP molecule binding identifies its main target epitope and confirms its in vivo blood-brain barrier penetration ability. For this purpose, the present application discloses a preparation and application of a photostable tau probe BMP.

[0006] The first aspect of the present application provides a compound, a stereoisomer thereof, or a salt thereof as described below:

[0007] [Corrected according to Rule 26 15.07.2025]

[0008] In some embodiments, a hydrogen, carbon, or fluorine atom at any position in the compound is independently replaced with an isotope thereof. Alternatively, one hydrogen atom at any position in the compound is replaced with deuterium, or C at any position is replaced with 13 C, or F at any position is replaced with 18 F.

[0009] In some embodiments, a fluorine atom in the compound is replaced with 18 F.

[0010] In some embodiments, the salt is one of hydrochloride, sulfate, trifluoroacetate, acetate, citrate, preferably hydrochloride. More preferably, the hydrochloride salt has the structure:

[0011]

[0012] In some embodiments, the salt is a pharmaceutically acceptable salt.

[0013] In some embodiments, the pharmaceutically acceptable salt refers to a derivative of the compound of the present application obtained by converting an existing acid or base group into a salt form (e.g., by reacting a free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic salts of basic residues (such as amines); alkali or organic salts of acidic residues (such as carboxylic acids); and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, hydrochloride, iodide, 2-hydroxyethanesulfonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pantothenate, pectinate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, trifluoroacetate, valerate, and the like.

[0014] The second aspect of the present application provides a composition comprising the compound, the stereoisomer thereof, or the salt thereof according to the first aspect of the present application.

[0015] The third aspect of the present application provides a diagnostic reagent comprising the compound, stereoisomer or salt thereof according to the first aspect of the present application.

[0016] The fourth aspect of the present application provides use of the compound, stereoisomer or salt thereof according to the first aspect of the present application or the composition according to the second aspect of the present application or the diagnostic reagent according to the third aspect of the present application in the preparation of a diagnostic reagent for a Tau protein-related disease.

[0017] In some embodiments, the Tau protein-related disease is selected from one or more of the following diseases: Alzheimer's disease (AD), frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), primary age-related Tauopathy (PART) and argyrophilic grain disease (AGD).

[0018] The fifth aspect of the present application provides a radiolabel of a BMP molecule, which is obtained by replacing the F atom in BMP-2 or BMP-6 with 18 F, for Tau protein PET imaging. Advantages

[0019] The disclosed light-stable Tau probe BMP has the characteristics of novel structure, strong stability, high specificity and excellent selectivity; when the F atom in BMP-2 or BMP-6 is replaced with 18 F, the molecule can also serve as a highly potential PET diagnostic reagent. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Comparison of staining of BMP-2 and BMP-6 on brain sections of APP / PS1 / Tau triple transgenic mice.

[0021] Figure 2 Monitoring of the light stability of PM-PBB3 and BMP-6 by HPLC.

[0022] Figure 3 Fluorescent response of PM-PBB3 and BMP-6 to Tau aggregates and aβ.

[0023] Figure 4 Comparison of staining of PM-PBB3 and BMP-6 on brain sections of APP / PS1 / Tau triple transgenic mice (** P<0.01, scale bar: 500 µm in whole brain image, 100 µm in high magnification image).

[0024] Figure 5 Representative in vivo labeling of BMP-6 in a triple transgenic mouse model (4R Tauopathy) and wild-type control group (scale bar: 500 pm top, 100 pm bottom).

[0025] Figure 6 NMR spectra of BMP compounds: BMP-6 (Figures 6a-6b); BMP-2 (Figures 6c-6d). DETAILED DESCRIPTION

[0026] The specific embodiments of the present application are described in detail below with reference to the following examples, which are implemented on the basis of the technical solutions described in the present application, and give detailed embodiments and specific operation processes, but the protection scope of the present application is not limited to the following examples.

[0027] The following examples further illustrate the present application. In the technical solutions described in the present application, if the types of components, names of materials, connection structures, preparation methods, materials, structures or component ratios are not explicitly described, they are considered as common technical features disclosed in the prior art. All raw materials, if not specified by synthesis method, are purchased from manufacturers such as Explore Platform, Aladdin, Sigma-Aldrich, etc., and are all analytical pure.

[0028] Example 1 Synthesis of BMP-2 hydrochloride

[0029]

[0030] Step 1) Synthesis of C-2

[0031] To a solution of compound A (0.76 g, 2.4 mmol) in anhydrous THF (20 mL) under nitrogen atmosphere at 0 °C, NaH (60% dispersion in mineral oil, 160 mg, 4.0 mmol) was added, and stirred in an ice bath for 30 minutes. Then a solution of compound B (0.52 g, 2.0 mmol) in THF (5 mL) was added at 0 °C through a syringe. The reaction mixture was stirred at 25 °C for 12 hours, and then quenched with ice water (30 mL). The mixture was extracted with ethyl acetate (50 mL x 3), and the combined organic layers were washed with brine and dried over anhydrous Na2S04. After filtration, the solution was concentrated under vacuum. The residue was purified by silica gel column chromatography to obtain compound C-2 as a yellow solid (300 mg, yield: 36%).

[0032] 1H NMR (400 MHz, Chloroform-d) δ 8.49 (d, J = 2.3 Hz, 1H), 8.44 (d, J = 16.1 Hz, 1H), 7.91 (s, 1H), 7.86 (dd, J = 8.7, 2.4 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.32 (d, J = 2.5 Hz, 1H), 7.09 (dd, J = 9.0, 2.6 Hz, 1H), 6.86 (d, J = 16.2 Hz, 1H), 6.57 (s, 1H), 3.89 (s, 3H), 3.43 (s, 3H), 2.22 (d, J = 1.0 Hz, 3H), 1.54 (s, 9H).

[0033] 13 C{ 1 H} NMR (101 MHz, Chloroform-d) δ 157.76, 148.77, 146.88, 140.79, 136.50, 134.36, 129.96, 128.61, 128.09, 123.76, 121.74, 118.93, 115.86, 103.82, 81.54, 77.48, 77.16, 76.84, 55.93, 34.34, 28.44, 21.27.

[0034] HRMS (ESI, m / z): calculated for C 24 H 28 N3O3S ([M+H] + ) 438.185139; found 438.18459.

[0035] Step 2) Synthesis of D-2

[0036] To a suspension of compound C-2 (400 mg, 0.94 mmol) in dry CH2Cl2(10 mL) was added BBr3(1.0 M in CH2Cl2, 2 mL, 2 mmol) slowly dropwise at -78 °C under nitrogen atmosphere. Then, the reaction was stirred at 25 °C for 12 h. The reaction was quenched with ice water (30 mL) and neutralized with saturated aqueous NaHCO3solution. All organic solvents were evaporated under reduced pressure and the product was precipitated from the mixture. After filtration, the solid was washed with water and dried under vacuum. The crude product was purified by silica gel column chromatography with DCM / EA / MeOH (from 400:100:5 to 200:100:3) as eluent to give D-2 as an orange solid (224 mg, yield: 77%).

[0037] 1 H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.22 (d, J = 16.1 Hz, 1H), 8.16 (d, J = 2.4 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.69 (dd, J = 8.9, 2.4 Hz, 1H), 7.35 (d, J = 2.5 Hz, 1H), 7.03 – 6.81 (m, 3H), 6.59 – 6.45 (m, 2H), 2.81 (d, J = 4.8 Hz, 3H), 2.22 – 2.08 (m, 3H).

[0038] 13 C{ 1 H} NMR (101 MHz, DMSO-d6) δ 160.94, 159.31, 155.38, 148.77, 147.29, 141.05, 135.71, 133.51, 132.03, 123.16, 122.41, 120.66, 118.87, 115.97, 106.34, 27.91, 20.74.

[0039] HRMS (ESI, m / z): calculated for C 18 H 18 N3OS ([M+H] + ) 324.117059; found 324.11651.

[0040] Step 3) Synthesis of BMP-2

[0041] A mixture of compound D-2 (200 mg, 0.65 mmol), 1,2-epoxy-3-fluoropropane (500 mg, 6.5 mmol) and K2CO3(180 mg, 1.3 mmol) in DMF (5 ml) was taken in a sealed tube and stirred at 55 °C for 12 h. After concentration under vacuum, the crude was purified by silica gel column chromatography using MeOH / DCM (from 5% to 10%) as eluent to get compound BMP-2 as an orange solid (128 mg, yield: 51%).

[0042] 1 H NMR (400 MHz, DMSO-d6) δ 8.27 (d, J = 16.1 Hz, 1H), 8.17 (d, J = 2.3 Hz, 1H), 7.93 (d, J = 8.9 Hz, 1H), 7.70 (dd, J = 8.8, 2.4 Hz, 2H), 7.66 (d, J = 2.6 Hz, 1H), 7.13 (dd, J = 8.9, 2.6 Hz, 1H), 7.01 – 6.88 (m, 2H), 6.60 – 6.51 (m, 2H), 5.52 (d, J = 5.1 Hz, 1H), 4.58 (qd, J = 9.6, 4.4 Hz, 1H), 4.53 – 4.39 (m, 1H), 2.82 (d, J = 4.8 Hz, 3H), 2.16 (d, J = 1.3 Hz, 3H).

[0043] 13 C{ 1 H} NMR (101 MHz, DMSO-d6) δ 162.33, 159.38, 156.26, 148.93, 148.39, 141.71, 135.64, 133.53, 132.36, 123.15, 122.38, 120.64, 118.67, 116.12, 108.60, 105.29, 85.30, 83.63, 68.84, 67.53, 27.93, 20.77.

[0044] 19 F{ 1 H} NMR (472 MHz, DMSO-d6) δ -205.58.

[0045] HRMS (ESI, m / z): calculated for C21 H 23 FN3O2S ([M+H] + ) 400.149502; found 400.14895.

[0046] Step 4) Synthesis of BMP-2 hydrochloride

[0047] Compound BMP-2 (500 mg, 1.18 mmol) was dissolved in anhydrous dichloromethane (10 mL) and stirred until completely dissolved. Hydrochloric acid dioxane solution (4 M, 1.5 mL, 6 mmol) was slowly added dropwise to the BMP-2 solution at room temperature, and stirring was continued during the dropwise addition. After the dropwise addition was completed, the reaction mixture was continuously stirred for 30 minutes. It was observed that the product gradually precipitated out of the solution to form a solid precipitate. The reaction mixture was filtered through a Buchner funnel to collect the precipitated solid. The solid was washed with a small amount of cold anhydrous ether (5 mL x 2) to remove residual solvents and impurities. The filtered solid was placed in a vacuum drying oven and dried at 40 °C for 12 hours to obtain BMP-2 hydrochloride as a yellow solid (425 mg, 87%).

[0048] 1 H NMR (500 MHz, DMSO-d6) δ 8.27 (d, J = 1.5 Hz, 1H), 7.96 – 7.91 (m, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.53 – 7.46 (m, 1H), 7.42 (d, J = 1.6 Hz, 1H), 7.19 (dt, J = 16.0, 1.2 Hz, 2H), 7.04 (dd, J = 7.5, 1.5 Hz, 1H), 6.66 (d, J = 7.5 Hz, 1H), 4.89 (q, J = 2.7 Hz, 2H), 4.68 (dd, J = 16.9, 7.6 Hz, 2H), 4.57 (d, J = 6.9 Hz, 1H), 4.26 (s, 1H), 4.21 – 4.07 (m, 1H), 4.04 (s, 1H), 4.03 (d, J = 1.4 Hz, 1H), 2.62 (t, J = 2.6 Hz, 3H), 2.28 (d, J = 1.0 Hz, 3H).

[0049] 13 C{ 1H} NMR (125 MHz, DMSO-d6) δ 166.32, 155.39, 151.30, 147.88, 144.93, 138.18, 136.18, 133.21, 132.98, 130.79, 124.69, 124.28, 115.48, 106.74, 106.28, 85.76, 83.54, 70.95, 69.37, 28.88, 19.39.

[0050] 19 F{ 1 H} NMR (472 MHz, DMSO-d6) δ -205.58.

[0051] HRMS (ESI, m / z): calculated for C21H23FN3O2S ([M] + ) 400.14895; found 400.14894.

[0052] Example Two: Synthesis of BMP-6 hydrochloride salt

[0053]

[0054] Step 1) Synthesis of C-6

[0055] To a solution of compound A (0.76 g, 2.4 mmol) in anhydrous THF (20 mL) was added NaH (60% dispersion in mineral oil, 160 mg, 4.0 mmol) at 0 °C under nitrogen atmosphere and stirred in an ice bath for 30 min. Then a solution of compound B (0.52 g, 2.0 mmol) in THF (5 mL) was added by syringe at 0 °C. The reaction mixture was stirred at 25 °C for 12 h, then quenched with ice water (30 mL). The mixture was extracted with ethyl acetate (50 mL x 3), the combined organic layers were washed with brine and dried over anhydrous Na2S04. After filtration, the solution was concentrated under vacuum. The residue was purified by silica gel column chromatography to give compound C-6 as a yellow solid (500 mg, yield: 59%).

[0056] 1H NMR (400 MHz, CDC13) δ 8.42 (d, J = 2.2 Hz, 1H), 7.91 (d, J = 8.9 Hz, 1H), 7.78 (dd, J = 8.8, 2.4 Hz, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.32 (d, J = 2.5 Hz, 1H), 7.08 (dd, J = 9.0, 2.5 Hz, 1H), 6.95 (d, J = 16.0 Hz, 1H), 6.84 (s, 1H), 6.80 (d, J = 16.0 Hz, 1H ), 3.89 (s, 3H), 3.42 (s, 3H), 2.51 (s, 3H), 1.54 (s, 9H).

[0057] 13 C{ 1 H} NMR (101 MHz, CDC13) δ 162.65, 157.79, 154.70, 154.4, 148.39, 146.61, 141.91, 136.74, 133.68, 133.55, 128.28, 127.58, 124.88, 123.70, 118.92, 115.98, 103.73, 81.54, 55.93, 34.33, 28.44, 14.80.

[0058] HRMS (ESI, m / z): calculated for C 24 H 28 N3O3S ([M+H] + ) 438.185139; found 438.18459.

[0059] Step 2) Synthesis of D-6

[0060] To a suspension of compound C-6 (400 mg, 0.94 mmol) in anhydrous CH2Cl2(10 mL) was added BBr3(1.0 M CH2Cl2solution, 2 mL, 2 mmol) slowly dropwise at -78 °C under nitrogen atmosphere. Then, the reaction was stirred at 25 °C for 12 h. The reaction was quenched with ice water (30 mL) and neutralized with saturated aqueous NaHC03solution. All organic solvents were evaporated under reduced pressure and the product was precipitated from the mixture. After filtration, the solid was washed with water and dried under vacuum. The crude product was purified by silica gel column chromatography with DCM / EA / MeOH (from 400:100:5 to 200:100:3) as eluent to give D-6 as an orange solid (224 mg, yield: 77%).

[0061] 1 H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H), 8.14 (s, 1H), 7.78 (s, 1H), 7.71 (s, 1H), 7.35 (s, 1H), 6.97 (s, 2H), 6.82 (d, J = 29.7 Hz, 3H), 6.49 (s, 1H), 2.80 (s, 3H), 2.44 (s, 3H).

[0062] 13 C{ 1 H} NMR (101 MHz, DMSO-d6) δ 161.46, 159.11, 155.40, 148.39, 147.14, 142.62, 135.88, 133.43, 129.51, 128.09, 123.05, 121.54, 120.75, 116.11, 106.31, 27.95, 14.49.

[0063] HRMS (ESI, m / z): calculated for C 18 H 18 N3OS ([M+H] + ) 324.117059; found 324.11651.

[0064] Step 3) Synthesis of BMP-6

[0065] A mixture of compound D-6 (200 mg, 0.65 mmol), 1,2-epoxy-3-fluoropropane (500 mg, 6.5 mmol) and K2CO3(180 mg, 1.3 mmol) in DMF (5 mL) was taken in a sealed tube and stirred at 55ºC for 12 h. After concentration under vacuum, the crude was purified by silica gel column chromatography with MeOH / DCM (from 5% to 10%) as eluent to get compound BMP-6 as an orange solid (128 mg, yield: 51%).

[0066] 1 H NMR (400 MHz, DMSO-d6) δ 8.15 (d, J = 2.3 Hz, 1H), 7.85 (d, J = 9.0 Hz, 1H), 7.72 (dd, J = 8.9, 2.4 Hz, 1H), 7.66 (d, J = 2.6 Hz, 1H), 7.12 (dd, J = 8.9, 2.6 Hz, 1H), 6.95 (d, J = 16.0 Hz, 1H), 6.87 (q, J = 5.2, 4.8 Hz, 1H), 6.85 (d, J = 16.3 Hz),,6.81 (s, 1H), 6.49 (d, J = 8.8 Hz, 1H), 5.54 (d, J = 5.0 Hz, 1H), 4.57 (qd, J = 9.6, 4.4 Hz, 1H), 4.52 – 4.37 (m, 1H), 4.15 – 4.07 (m, 1H), 4.07 – 3.99 (m, 2H), 2.80 (d, J = 4.8 Hz, 3H), 2.46 (s, 3H).

[0067] 13 C{ 1 H} NMR (101 MHz, DMSO-d6) δ 162.83, 159.18, 156.25, 148.54, 148.19, 143.26, 135.83, 133.45, 129.87, 127.99, 123.00, 121.39, 120.72, 116.23, 108.31, 105.25, 84.49 (d, J = 168.4 Hz), 68.80 (d, J = 7.7 Hz), 67.64 (d, J = 19.0 Hz), 27.98, 14.57.

[0068] 19 F{ 1 H} NMR (472 MHz, DMSO-d6) δ -205.58.

[0069] HRMS (ESI, m / z): calculated for C 21 H 23 FN3O2S ([M+H] + ) 400.149502; found 400.14895.

[0070] Step 4) Synthesis of BMP-6 hydrochloride salt

[0071] Compound BMP-6 (500 mg, 1.18 mmol) was dissolved in anhydrous dichloromethane (10 mL) and stirred until completely dissolved. Hydrochloric acid dioxane solution (4 M, 1.5 mL, 6 mmol) was slowly added dropwise into the BMP-6 solution at room temperature, and stirring was continued during the dropwise addition. After the dropwise addition was completed, the reaction mixture was stirred for another 30 minutes. The product was observed to gradually precipitate out of the solution as a solid. The reaction mixture was filtered through a Buchner funnel to collect the precipitated solid. The solid was washed with a small amount of cold anhydrous ether (5 mL x 2) to remove residual solvents and impurities. The filtered solid was placed in a vacuum drying oven and dried at 40 °C for 12 hours to obtain BMP-6 hydrochloride salt as a light yellow solid (425 mg, 87%).

[0072] 1H NMR (500 MHz, CDCl3) δ 8.46 (d, J = 1.5 Hz, 1H), 8.22 (dq, J = 8.1, 2.6 Hz, 1H), 7.87 – 7.81 (m, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.61 (dq, J = 8.0, 2.6 Hz, 1H), 7.42 (d, J = 1.5 Hz, 1H), 7.29 – 7.22 (m, 2H), 7.22 – 7.15 (m, 2H), 7.04 (dd, J = 7.5, 1.5 Hz, 1H), 4.68 (dd, J = 16.9, 7.6 Hz, 2H), 4.57 (d, J = 6.9 Hz, 1H), 4.21 – 4.07 (m, 1H), 4.07 – 4.00 (m, 2H), 2.98 (t, J = 2.7 Hz, 3H), 2.10 (t, J = 1.0 Hz, 3H).

[0073] 13 C{ 1 H} NMR (125 MHz, CDCl3) δ 166.48, 155.57, 153.89, 147.97, 144.88, 138.13, 136.14, 132.84, 129.64, 126.63, 124.26, 115.44, 114.01, 106.65, 85.68, 83.54, 70.81, 69.37, 29.33, 17.77.

[0074] 19 F{ 1 H} NMR (472 MHz, DMSO-d6) δ -205.58.

[0075] HRMS (ESI, m / z): calculated for C21H23FN3O2S ([M] + ) 400.14895; found 400.14894.

[0076] Example Three: Preliminary Test of Fluorescence Imaging Ability of BMP-2 Hydrochloride and BMP-6 Hydrochloride in Mouse Brain Slices

[0077] Synthesis of probes BMP-2 and BMP-6 as a pair of cis-trans isomers, first test this pair of compounds in the fluorescence imaging ability of APP / PS1 / Tau triple transgenic mice brain sections.

[0078] Harvesting of APP / PS1 / Tau triple transgenic mouse brain sections: APP / PS1 / Tau triple transgenic mice (8-11 months old) were subjected to heart perfusion under isoflurane anesthesia, first perfused with PBS, then perfused with PBS containing 8% formaldehyde (pH 7.4). The removed brain tissue was post-fixed in the same solution for 2 days. Then, the brain tissue block was quickly frozen and sectioned with a Leica microtome, and brain sections of 14 μm thickness were used for immunostaining or probe staining. First, the free-floating brain sections were blocked in PBS solution containing 5% goat serum, 1% bovine serum albumin and 0.4% Triton X-100 for 1 hour. PBS solution of BMP-2 hydrochloride (20 μM) and BMP-6 hydrochloride (20 μM) was used to stain the above brain slices, and the Olympus fluorescence microscope (BX53) equipped with DP74 digital camera was used for imaging, and the fluorescence images were analyzed using ImageJ. The imaging results show that BMP-6 hydrochloride has better imaging contrast than BMP-2 hydrochloride (Figure 1), and is more suitable for further study.

[0079] Example Four: Light stability experiment of BMP-6

[0080] One major challenge in the clinical application of Tau PET tracers is their sensitivity to photoisomerization, especially in PM-PBB3. Our test shows that the purity of PM-PBB3 decreases to below 50% after only 2 hours of light exposure (Figures 2a, 2c). In contrast, BMP-6 in free base form still maintains >98% purity even after 6 hours (Figures 2b, 2d), showing better light stability.

[0081]

[0082] The photostability test was performed according to the following protocol: 1 mg of PM-PBB3 or BMP-6 was dissolved in 1 mL of methanol and placed at room temperature under indoor light (300 lux). The BMP-6 and PM-PBB3 samples were analyzed immediately after preparation using an Agilent 1260 HPLC (DAD detector). After exposure to ordinary indoor light (about 300 lux) at different time points, the purity of BMP-6 and PM-PBB3 was detected again using the same HPLC method. The HPLC analysis used a Diamonsil C18 (3 μm, 150 x 4.6 mm) column, the mobile phase was water / acetonitrile (50:50, v / v), the flow rate was 1.0 mL / min, and the ultraviolet detection wavelength was 365 nm.

[0083] Example Five: Experiment of the binding selectivity of the hydrochloride salt of BMP-6 to tau protein aggregates and aβ in solution

[0084] Test method: Cell culture reagents were purchased from Beyotime or Invitrogen. Tau protein, aβ monomers, and amylin were purchased from GL Biochem. The solvents used were all of analytical grade.

[0085] Preparation of tau, aβ, and amylin aggregates: Tau aggregates: Dissolve the tau protein in phosphate buffer solution (PBS) and add sodium heparin as an inducer. Stir vigorously at room temperature for 72 hours. aβ monomers: Dissolve in PBS and stir at 1200 rpm at room temperature for 72 hours to obtain pre-aggregated aβ. Amylin monomers: Dissolve in PBS (pH 7.4) and stir vigorously at room temperature for 72 hours to form amylin aggregates. Add PM-PBB3 (20 μM) and the hydrochloride salt of BMP-6 (20 μM), respectively, and test the fluorescence intensity. Compared with PM-PBB3, BMP-6 showed superior affinity and selectivity to tau aggregates (Figure 3).

[0086] Example Six: Experiment of the detection of tau protein in mouse brain sections by the hydrochloride salt of BMP-6

[0087] APP / PS1 / Tau triple transgenic mice (8-11 months old) were heart perfused under isoflurane anesthesia, first with PBS and then with PBS containing 8% formaldehyde (pH 7.4). The removed brain tissues were post-fixed in the same solution for 2 days. Then, brain tissue blocks were snap-frozen and sectioned using a Leica microtome, and 14-μm-thick brain sections were used for immunostaining or probe staining. First, free-floating brain sections were blocked in PBS containing 5% goat serum, 1% bovine serum albumin, and 0.4% Triton X-100 for 1 h. Rabbit-derived antibodies pAβ, Αβ40, and Αβ42 were purchased from Cell Signaling Technology (Danvers, MA, USA). Phospho-Tau antibodies were purchased from Thermo Fisher Scientific. Cy3-labeled goat anti-mouse and goat anti-rabbit secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0088] Primary antibody incubation was performed overnight at 4°C, and secondary antibody incubation was performed for 1.5 h, with extensive washing between each step using PBS. Hydrochloride salt solution of BMP-6 or PM-PBB3 staining (20 μΜ) was performed after immunostaining image acquisition using an Olympus fluorescence microscope (BX53) equipped with a DP74 digital camera. Fluorescence images were analyzed using ImageJ. Linear analysis was used to compare staining intensity and to assess colocalization between BMP-6 staining and pTau antibody immunostaining. The rectangular tool was used to measure the mean fluorescence intensity of Tau pathology and Αβ plaque staining within defined areas. Image acquisition settings were kept consistent between different samples to ensure semi-quantitative comparability of results. Semi-quantitative analysis was performed on images of probe BMP-6 and PM-PBB3 staining in the cerebral cortex.

[0089] By comparing the staining patterns of BMP-6 and PM-PBB3 in adjacent brain sections, it was found that both showed similar staining patterns for Tau pathology and Ab plaques (Fig. 4a, 4b). However, the signal intensity of Ab plaques was significantly higher in PM-PBB3 staining, mainly located in deep cortical layers 4 / 5 (Fig. 4a vs 4b, blue arrows). High magnification images showed that phosphorylated Tau or tangles were mainly located in cortical layers 2, 3 and 5, showing similar laminar distribution characteristics as Alzheimer's disease, frontotemporal dementia and progressive supranuclear palsy. In the superficial cortical layers 2 / 3, BMP-6 showed stronger intraneuronal Tau pathology signals than PM-PBB3 (Fig. 4b vs 4a, red arrows). This difference was less obvious in the deep cortical layer 5, where Tau pathology showed a fibrillary appearance (Fig. 4a, 4b, yellow arrows), indicating early formation and late development stages of Tau pathology. In contrast, BMP-6 stained fewer and weaker Ab plaques (Fig. 4b vs 4a). Semi-quantitative line and frame analysis of fluorescence intensity confirmed that BMP-6 showed stronger pTau staining, but fewer and weaker Ab plaques (Fig. 4c, 4d). This made BMP-6 4.9 times more selective for pTau than PM-PBB3 (Fig. 4e).

[0090] Example Seven: In vivo detection of Tau protein in mouse models by hydrochloride salt of BMP-6

[0091] In vivo probe labeling experiments were performed using APP / PS1 / Tau triple transgenic mice (8-11 months old) and their wild-type controls (n = 3 per group). The hydrochloride salt of BMP-6 was dissolved in saline and injected intraperitoneally (5 mg / kg), and the mice were heart perfused 2 hours after injection. Subsequently, the brain tissue was rapidly processed, including 1 hour of post-fixation, and brain sections were immediately imaged.

[0092] Ex vivo imaging showed that BMP-6 was particularly remarkable in labeling neuronal Tau pathology and Ab plaques in the cortex and hippocampus of transgenic mice, especially in the entorhinal cortex, perirhinal cortex, piriform cortex and hippocampus (Fig. 5a, 5b). The differential labeling of BMP-6 between transgenic mice and WT mice strongly suggested that the probe could penetrate the BBB and target Alzheimer's disease-related pathology present in the transgenic model. The probe had the best pharmacokinetic properties, allowing it to stay in Tau-rich brain regions for a long time, thus contributing to clearer imaging. The low background signal and good signal-to-noise ratio in the transgenic mouse model showed the ability of BMP-6 to be applied in in vivo imaging.

[0093] The foregoing description of the embodiments has been presented for the purpose of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed. Modifications and variations are possible in light of the above teachings or can be acquired from practice of the application. As well, the description is presented in the context of the preferred embodiments as a number of alternatives. It is not intended to limit the application to the precise form described.

Claims

1. A compound, a stereoisomer thereof, or a salt thereof, as described below: 。。 2. The compound according to claim 1, a stereoisomer thereof, or a salt thereof, wherein any hydrogen, carbon or fluorine atom in the compound is independently replaced with an isotope thereof.

3. The compound, stereoisomer thereof, or salt thereof according to claim 2, wherein the fluorine atom is replaced with 18 F.

4. The compound, stereoisomer, or salt thereof of any one of claims 1-3, wherein: the salt is one of hydrochloride, sulfate, trifluoroacetate, acetate, citrate, and preferably one of the following structures: 。。 5. A composition comprising the compound, the stereoisomer thereof, or the salt thereof according to any one of claims 1 to 4.

6. A diagnostic reagent comprising the compound, the stereoisomer thereof, or the salt thereof according to any one of claims 1 to 4.

7. Use of the compound, the stereoisomer thereof, or the salt thereof according to any one of claims 1 to 4, or the composition according to claim 5, or the diagnostic reagent according to claim 6, in the manufacture of a diagnostic reagent for a Tau protein-related disease.

8. The use according to claim 7, wherein the Tau protein-related disease is selected from one or more of Alzheimer's disease (AD), frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), primary age-related Tauopathy (PART), and argyrophilic grain disease (AGD).

Images