A beta plaque dual-mode imaging probe and preparation method and application thereof

Abstract

The invention provides an A beta plaque dual-mode imaging probe as well as a preparation method and application thereof, and belongs to the technical field of fine chemical engineering. According to the present invention, quinoline-malononitrile is adopted as a fluorescent dye parent, dimethylaminobenzene and other units are adopted as an A beta plaque binding group, thiophene and other units are introduced as a pi bridge to prolong the emission wavelength of the probe, and < 18 > F is adopted as a PET imaging nuclide to prepare the A beta plaque dual-mode imaging probe, the probe has the advantages of being long in fluorescence emission wavelength, high in affinity, good in selectivity, high in stability, excellent in biocompatibility and the like, dyeing marking of the A beta plaque can be achieved, and information such as distribution, form and size of the plaque can be accurately reflected.

Description

Technical Field

[0001] The present invention relates to the technical field of fine chemical industry, and in particular to an Aβ plaque dual-mode imaging probe and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease, one of the main pathological features of which is the abnormal deposition of β-amyloid (Aβ) plaques in the brain. Currently, the main technology used to image Aβ plaques is positron emission tomography (PET). PET imaging uses radioactive tracers (such as [ 18 F]-labeled compounds) combined with Aβ plaques can achieve three-dimensional quantitative imaging and have excellent tissue penetration. However, the spatial resolution of PET imaging is low, making it difficult to accurately locate Aβ plaques. Fluorescence imaging technology, with its advantages of high sensitivity and high resolution, has unique advantages in presenting the microstructure of Aβ plaques. However, due to the attenuation and scattering of fluorescence signals in deep tissues, its scope of application is subject to certain limitations. In summary, the current development of Aβ imaging agents based on a single modality is difficult to simultaneously meet the multiple requirements of spatial resolution, sensitivity, quantitative analysis, and tissue penetration.

[0003] Therefore, it is necessary to develop a dual-mode imaging probe for Aβ plaques that integrates the advantages of real-time dynamic monitoring by fluorescence imaging and quantitative detection by PET imaging, so that it can simultaneously provide high-resolution local microscopic information and quantitative imaging data, which will help provide a more comprehensive imaging basis for the pathological research and treatment effect evaluation of Alzheimer's disease. Summary of the invention

[0004] The purpose of the present invention is to provide an Aβ plaque dual-mode imaging probe and a preparation method and application thereof, so as to solve the problem that Aβ imaging agents in the prior art are difficult to simultaneously meet the multiple requirements of spatial resolution, sensitivity, quantitative analysis and tissue penetration.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an Aβ plaque dual-mode imaging probe, wherein the Aβ plaque dual-mode imaging probe has a structure as shown in Formula I:

[0007]

[0008] In formula I, R1 is selected from cyano, carboxyl or R2 is selected from a C1 to C4 hydrocarbon group, wherein n is independently an integer between 0 and 10, and F is selected from18 F or 19 F; R3 is selected from methyl, wherein m is independently 0 or 1, R5 is independently selected from -H, -CH3, -CH2OH, -CH2OTs, -CH2 18 F,-CH2 19 F, -CH2CH3, -CH2CH2OH, -CH2 CH2OTs, -CH2CH2 18 F or -CH2CH2 19 F; R4 is selected from -H, - 18 F or - 19 F.

[0009] Preferably, the R1 is selected from cyano.

[0010] Preferably, R2 is selected from wherein n is independently an integer between 0 and 10, and F is selected from 18 F or 19 F.

[0011] Preferably, R3 is selected from wherein m is independently 0 or 1, R5 is independently selected from -H, -CH3, -CH2OH, -CH2OTs, -CH2 18 F,-CH2 19 F, -CH2CH3, -CH2CH2OH, -CH2 CH2OTs, -CH2CH2 18 F or -CH2CH2 19 F.

[0012] As a preference, when R2 is selected from When n is independently an integer between 0 and 10, R3 is selected from wherein m is independently 0 or 1, R5 is independently selected from -CH2OH, -CH2OTs, -CH2 18 F,-CH2 19 F, -CH2CH2OH, -CH2CH2OTs, -CH2CH2 18 F or -CH2CH2 19 F;

[0013] When R2 is selected from When n is independently an integer between 0 and 10, F is selected from 18 F or 19 F, R3 is selected from wherein m is independently 0 or 1, and R5 is independently selected from -H, -CH3 or -CH2CH3.

[0014] Preferably, the Aβ plaque dual-mode imaging probe has a structure as shown in Formula Ⅰ-1, Ⅰ-2, Ⅱ-1 or Ⅱ-2:

[0015]

[0016]

[0017] The present invention provides a method for preparing the above-mentioned Aβ plaque dual-mode imaging probe, comprising the following steps:

[0018] (1) Under argon protection conditions, using N,N-dimethylformamide as solvent, compound A, compound X and sodium ethoxide react to obtain compound B;

[0019] (2) Under argon protection conditions, using toluene as solvent, compound B and compound Y react to obtain compound C;

[0020] (3) Under argon protection conditions, using dichloromethane as a solvent, compound C and diethylaminosulfur trifluoride reacted to prepare an Aβ plaque dual-mode imaging probe;

[0021] The compound A is

[0022] The compound X comprises 14-bromo-3,6,9,12-tetraoxatetradecan-1-ol, 2-(2-bromoethoxy)ethanol, 13-bromo-2,5,8,11-tetraoxatetradecan or 2-bromoethyl methyl ether;

[0023] The compound Y comprises 5-(4-dimethylaminophenyl)thiophene-2-carboxaldehyde or 5-(4-(2-hydroxyethylmethylamino)phenyl)thiophene-2-carboxaldehyde.

[0024] Preferably, in step (1), the molar ratio of compound A, compound X and sodium ethoxide is 1:1-2:0.8-1.2, the reaction is carried out at room temperature, and the reaction time is 1-3 hours;

[0025] In the step (2), the molar ratio of compound B to compound Y is 1:0.8-1.2, the reaction temperature is 100-120° C., and the reaction time is 2-4 hours.

[0026] Preferably, in step (3), the molar ratio of compound C to diethylaminosulfur trifluoride is 1:0.8-1.2, the reaction temperature is -80--75°C, and the reaction time is 1-3h.

[0027] The present invention also provides a use of the above-mentioned Aβ plaque dual-mode imaging probe in the preparation of an Aβ plaque imaging reagent.

[0028] Beneficial effects of the present invention:

[0029] The present invention uses quinoline-malononitrile as the fluorescent dye matrix, dimethylaminobenzene and other units as Aβ plaque binding groups, introduces thiophene and other units as π bridges to extend the probe emission wavelength, and 18 F was used as a PET imaging nuclide to prepare a dual-mode imaging probe for Aβ plaques. The probe has the advantages of long fluorescence emission wavelength, high affinity, good selectivity, strong stability, and excellent biocompatibility. It can achieve staining and labeling of Aβ plaques and accurately reflect the distribution, morphology, size and other information of the plaques.

[0030] In terms of fluorescence imaging, the probe exhibits weak fluorescence emission in phosphate buffer, but when the phosphate buffer contains Aβ protein, the fluorescence signal is significantly enhanced to 40 times the original. Furthermore, the probe has a "lighting-up" response characteristic and excellent blood-brain barrier penetration properties, enabling it to significantly identify Aβ plaques in 5xFAD mice. In terms of PET imaging, due to the stability of the probe signal, imaging can be effectively performed over a longer time window. In summary, this type of probe can track Aβ plaques for a long time with a high signal-to-noise ratio through fluorescence / PET dual-mode imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The fluorescence spectra of the Aβ plaque dual-mode imaging probe with a structure shown in Formula Ⅰ-1 prepared in Example 1 in mixed solvents of glycerol / water at different ratios;

[0032] Figure 2 The Aβ plaque dual-mode imaging probe with a structure shown in Formula Ⅰ-1 prepared in Example 1 and different concentrations of Aβ 42 Fluorescence spectra of protein aggregates after binding;

[0033] Figure 3 The fluorescence spectra of the Aβ plaque dual-mode imaging probe with a structure shown in Formula I-2 prepared in Example 2 in mixed solvents of glycerol / water at different ratios;

[0034] Figure 4 The Aβ plaque dual-mode imaging probe with a structure shown in Formula I-2 prepared in Example 2 and different concentrations of Aβ 42 Fluorescence spectra of protein aggregates after binding;

[0035] Figure 5 The fluorescence spectra of the Aβ plaque dual-mode imaging probe with a structure shown in Formula II-1 prepared in Example 3 in mixed solvents of glycerol / water at different ratios;

[0036] Figure 6 The Aβ plaque dual-mode imaging probe with the structure shown in formula II-1 prepared in Example 3 and different concentrations of Aβ 42 Fluorescence spectra of protein aggregates after binding;

[0037] Figure 7 The fluorescence spectra of the Aβ plaque dual-mode imaging probe with a structure shown in Formula II-2 prepared in Example 4 in mixed solvents of glycerol / water at different ratios;

[0038] Figure 8 The Aβ plaque dual-mode imaging probe with the structure shown in Formula II-2 prepared in Example 4 and different concentrations of Aβ 42 Fluorescence spectra of protein aggregates after binding;

[0039] Fig. 9 This is a comparison chart of the cytotoxicity of the dual-mode imaging probe for Aβ plaques with a structure as shown in Formula Ⅰ-1 prepared in Example 1 to HepG2 cells at different concentrations;

[0040] Fig.10 Aβ plaque dual-mode imaging probe with a structure as shown in Formula I-1 prepared in Example 1 is used for staining Aβ plaques on brain tissue sections of 5xFAD transgenic mice, wherein adjacent sections are stained with Thioflavin S to confirm the distribution of plaques;

[0041] Fig.11 A fluorescence imaging diagram of Aβ plaques using the dual-mode imaging probe of formula Ⅰ-1 prepared in Example 1;

[0042] Fig.12 The Aβ plaque dual-mode imaging probe with a structure as shown in Formula Ⅰ-1 prepared in Example 1 is used for positron emission tomography imaging of Aβ plaques. DETAILED DESCRIPTION

[0043] The present invention provides an Aβ plaque dual-mode imaging probe, wherein the Aβ plaque dual-mode imaging probe has a structure as shown in Formula I:

[0044]

[0045] In formula I, R1 is selected from cyano, carboxyl or R2 is selected from a C1 to C4 hydrocarbon group, wherein n is independently an integer between 0 and 10, and F is selected from 18 F or 19 F; R3 is selected from methyl, wherein m is independently 0 or 1, R5 is independently selected from -H, -CH3, -CH2OH, -CH2OTs, -CH218 F,-CH2 19 F, -CH2CH3, -CH2CH2OH, -CH2 CH2OTs, -CH2CH2 18 F or -CH2CH2 19 F; R4 is selected from -H, - 18 F or - 19 F. The curved line marks in each group in the present invention are substitution positions.

[0046] In the present invention, the R1 is selected from cyano.

[0047] In the present invention, the R2 is selected from wherein n is independently an integer between 0 and 10, and F is selected from 18 F or 19 F.

[0048] In the present invention, the R3 is selected from wherein m is independently 0 or 1, R5 is independently selected from -H, -CH3, -CH2OH, -CH2OTs, -CH2 18 F,-CH2 19 F, -CH2CH3, -CH2CH2OH, -CH2 CH2OTs, -CH2CH2 18 F or -CH2CH2 19 F.

[0049] In the present invention, when R2 is selected from When n is independently an integer between 0 and 10, R3 is selected from wherein m is independently 0 or 1, R5 is independently selected from -CH2OH, -CH2OTs, -CH2 18 F, -CH2 19 F, -CH2CH2OH, -CH2CH2OTs, -CH2CH2 18 F or -CH2CH2 19 F.

[0050] In the present invention, when R2 is selected from When n is independently an integer between 0 and 10, F is selected from 18 F or 19 F, R3 is selected from wherein m is independently 0 or 1, and R5 is independently selected from -H, -CH3 or -CH2CH3.

[0051] In the present invention, the Aβ plaque dual-mode imaging probe has a structure as shown in Formula Ⅰ-1, Ⅰ-2, Ⅱ-1 or Ⅱ-2:

[0052]

[0053] The present invention provides a method for preparing the above-mentioned Aβ plaque dual-mode imaging probe, comprising the following steps:

[0054] (1) Under argon protection conditions, using N,N-dimethylformamide as solvent, compound A, compound X and sodium ethoxide react to obtain compound B;

[0055] (2) Under argon protection conditions, using toluene as solvent, compound B and compound Y react to obtain compound C;

[0056] (3) Under argon protection conditions, using dichloromethane as a solvent, compound C and diethylaminosulfur trifluoride reacted to prepare an Aβ plaque dual-mode imaging probe;

[0057] The compound A is

[0058] The compound X comprises 14-bromo-3,6,9,12-tetraoxatetradecan-1-ol, 2-(2-bromoethoxy)ethanol, 13-bromo-2,5,8,11-tetraoxatetradecan or 2-bromoethyl methyl ether;

[0059] The compound Y comprises 5-(4-dimethylaminophenyl)thiophene-2-carboxaldehyde or 5-(4-(2-hydroxyethylmethylamino)phenyl)thiophene-2-carboxaldehyde.

[0060] In the present invention, in the step (1), the molar ratio of compound A, compound X and sodium ethoxide is 1:1-2:0.8-1.2, preferably 1:1:1 or 1:2:1; the reaction is carried out at room temperature, and the reaction time is 1-3 hours, preferably 2 hours;

[0061] In the step (2), the molar ratio of compound B to compound Y is 1:0.8-1.2, preferably 1:1, the reaction temperature is 100-120° C., preferably 110° C., and the reaction time is 2-4 h, preferably 3 h.

[0062] In the present invention, in the step (3), the molar ratio of compound C to diethylaminosulfur trifluoride is 1:0.8-1.2, preferably 1:1; preferably, compound C is added to a dichloromethane solvent, and then diethylaminosulfur trifluoride is added dropwise to react, the reaction temperature is -80--75°C, preferably -78°C, and the reaction time is 1-3h, preferably 2h.

[0063] The present invention also provides a use of the above-mentioned Aβ plaque dual-mode imaging probe in the preparation of an Aβ plaque imaging reagent.

[0064] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0065] Example 1

[0066] Compound A (100 mg, 0.40 mmol), 14-bromo-3,6,9,12-tetraoxatetradecane-1-ol (120 mg, 0.40 mmol) and sodium ethoxide (27 mg, 0.40 mmol) were placed in a dry 50 mL round-bottom flask, 10 mL DMF was used as solvent, and the mixture was stirred at room temperature at 25 ° C for 2 h under argon protection. After removing the solvent, the mixture was purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 25:1) to obtain 74 mg of a yellow oily substance (compound B-1), with a yield of 39%.

[0067]

[0068] 1 H NMR (400MHz, CDCl3, ppm) δ: 9.10 (d, J=8.4Hz, 1H, Ph-H), 7.73 (t, J=8.0Hz, 1H, P h-H),7.63(d,J=8.8Hz,1H,Ph-H),7.44(t,J=7.7Hz,1H,Ph-H),6.82(s,1H,Ph-H ),4.54(t,J=4.8Hz,2H,-NCH2CH2O-),3.89(t,J=4.8Hz,2H,-NCH2CH2O-),3.56-3.71(m,20H,PEG-H),2.65(s,3H,-CH3); Massspectrometry(NSI-MS,m / z):[MH] - calcd.for[C 25 H 33 O6N3-H] - 470.2286; found, 470.2290.

[0069]

[0070] Compound B-1 (72 mg, 0.15 mmol) and 5-(4-dimethylaminophenyl)thiophene-2-carboxaldehyde (35 mg, 0.15 mmol) were placed in a dry 50 mL round-bottom flask, 10 mL of toluene was used as the solvent, heated to 110°C under argon protection, and refluxed and stirred at 110°C for 3 h. After removing the solvent, the mixture was purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 25:1) to obtain 42 mg of dark red solid (compound C-1), with a yield of 41%.

[0071] 1 H NMR (400MHz, CDCl3, ppm) δ: 9.11 (d, J = 8.4Hz, 1H, Ph-H), 7.73 (t, J = 7.9Hz, 1H, Ph-H), 7.64 (d, J = 8.8Hz, 1H, Ph-H), 7.50 (d ,J=8.6Hz,2H,Ph-H),7.44(t,J=7.8Hz,1H,Ph-H),7.38(d,J=15.4Hz,1H,alkene-H),7.19(d,J=3.8Hz,1H,thioph-H),7. 14(s,1H,Ph-H),7.12(d,J=15.4Hz,1H,alkene-H),7.12(d,J=3.8Hz,1H,thioph-H),6.71(d,J=8.6Hz,2H,Ph-H),4.55(t Mass spectrometry(ESI-MS,m / z):[M+H] + calcd.for[C 38 H 44 O6N4S+H] + 685.3060; found, 685.3058.

[0072]

[0073] Compound C-1 (27 mg, 0.04 mmol) was placed in a dry 50 mL round-bottom flask, and diethylaminosulfur trifluoride (6 mg, 0.04 mmol) was added dropwise at -78°C under argon protection with 10 mL of dichloromethane as solvent, and the reaction was continued at -78°C for 2 hours. After the reaction was completed, it was quenched with a saturated sodium bicarbonate solution, extracted with dichloromethane, dried, and after removing the solvent, it was purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 25:1) to obtain 13 mg of dark red solid, which is the Aβ plaque dual-mode imaging probe with a structure shown in Formula I-1, with a yield of 48%.

[0074] 1 H NMR (400MHz, CDCl3, ppm) δ: 9.13 (d, J = 8.4Hz, 1H, Ph-H), 7.73 (t, J = 7.9Hz, 1H, Ph-H), 7.60 (d, J = 8.7Hz, 1H, Ph-H), 7.51 (d, J = 8.5Hz, 2H, Ph-H),7.45(t,J=7.7Hz,1H,Ph-H),7.40(d,J=15.5Hz,1H,alkene-H),7.20(d,J=3.6Hz,1H,thioph-H),7.16(s,1H,Ph-H),7.13(d,J=3. 6Hz,1H,thioph-H),7.11(d,J=15.5Hz,1H,alkene-H),6.73(d,J=7.6Hz,2H,Ph-H),4.54(t,J=4.8Hz,2H,-NCH2CH2O-),4.53(dt,J1=3.7 Mass spectrometry(ESI-MS,m / z):[M+H] + calcd.for[C 38 H 43 FN4O5S+H] + 687.3016; found, 687.3014.

[0075] Example 2

[0076] Compound A (100 mg, 0.40 mmol), 2-(2-bromoethoxy)ethanol (68 mg, 0.40 mmol) and sodium ethoxide (27 mg, 0.40 mmol) were placed in a dry 50 mL round-bottom flask, and 10 mL of DMF was used as the solvent. The mixture was stirred at room temperature at 25 ° C for 2 h under argon protection. After removing the solvent, the mixture was purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 25:1) to obtain 62 mg of a yellow oily substance (compound B-2) with a yield of 38%.

[0077]

[0078] 1 H NMR (400MHz, CDCl3, ppm) δ: 9.09 (d, J=8.4Hz, 1H, Ph-H), 7.74 (t, J=7.8Hz, 1H, Ph-H), 7.59 (d,J=8.8Hz,1H,Ph-H),7.44(t,J=7.8Hz,1H,Ph-H),6.81(s,1H,Ph-H),4.52(t,J=5.3Hz,2 H,-NCH2CH2O-),3.89(t,J=5.3Hz,2H,-NCH2CH2O-),3.70(t,J=4.2Hz,2H,-OCH2CH2OH),3. 58(m,4H,-CH2OCH2CH2OCH2-),3.53(t,J=4.2Hz,2H,-OCH2CH2OH),2.64(s,3H,-CH3); Mass spectrometry(ESI-MS,m / z):[MH] - calcd.for[C 19 H 21 O3N3-H] - 404.1581; found, 404.1582.

[0079]

[0080] Compound B-2 (40 mg, 0.12 mmol) and 5-(4-dimethylaminophenyl)thiophene-2-carboxaldehyde (28 mg, 0.12 mmol) were placed in a dry 50 mL round-bottom flask, 10 mL of toluene was used as the solvent, heated to 110°C under argon protection, and refluxed and stirred at 110°C for 3 h. After removing the solvent, the mixture was purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 25:1) to obtain 43 mg of dark red solid (compound C-2), with a yield of 66%.

[0081] 1H NMR (400MHz, CDCl3, ppm) δ: 9.07 (d, J = 8.4Hz, 1H, Ph-H), 7.72 (t, J = 7.6Hz, 1H, Ph-H), 7.59 (d, J = 8.6Hz, 1H, Ph-H), 7.49 ( d,J=8.4Hz,2H,Ph-H),7.42(t,J=7.8Hz,1H,Ph-H),7.36(d,J=15.4Hz,1H,alkene-H),7.17(d,J=3.6Hz,1H,thioph-H),7 .10(s,1H,Ph-H),7.09(d,J=3.6Hz,1H,thioph-H),7.07(d,J=15.4Hz,1H,alkene-H),6.70(d,J=8.2Hz,2H,Ph-H),4.53( Mass spectrometry(ESI-MS,m / z):[M+H] + calcd.for[C 32 H 32 O3N4S+H] + 553.2268; found, 553.2265.

[0082]

[0083] Compound C-2 (30 mg, 0.04 mmol) was placed in a dry 50 mL round-bottom flask, and diethylaminosulfur trifluoride (6 mg, 0.04 mmol) was added dropwise at -78°C under argon protection with 10 mL of dichloromethane as solvent, and the reaction was continued at -78°C for 2 hours. After the reaction was completed, it was quenched with a saturated sodium bicarbonate solution, extracted with dichloromethane, dried, and after removing the solvent, it was purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 25:1) to obtain 6 mg of dark red solid, which is the Aβ plaque dual-mode imaging probe with a structure shown in Formula I-2, with a yield of 25%.

[0084] 1H NMR (400MHz, CDCl3, ppm) δ: 9.12 (dd, J1=0.8Hz, J2=8.4Hz, 1H, Ph-H), 7.73 (t, J=7.4Hz, 1H, Ph-H), 7.59 (d, J=8.7Hz, 1H, Ph-H), 7.50 (d, J=8.7Hz,2H,Ph-H),7.44(t,J=7.6Hz,1H,Ph-H),7.39(d,J=15.4Hz,1H,alkene-H),7.19(d,J=3.8Hz,1H,thioph-H),7.15(s,1H,Ph-H), 7.12(d,J=3.8Hz,1H,thioph-H),7.10(d,J=15.4Hz,1H,alkene-H),6.72(d,J=8.6Hz,2H,Ph-H),4.54(t,J=5.2Hz,2H,-NCH2CH2O-),4.4 Mass spectrometry(ESI-MS,m / z):[M+H] + calcd.for[C 32 H 31 FN4O2S+H] + 555.2225; found, 555.2228.

[0085] Example 3

[0086] Compound A (100 mg, 0.40 mmol), 13-bromo-2,5,8,11-tetraoxatridecane (216 mg, 0.80 mmol) and sodium ethoxide (27 mg, 0.40 mmol) were placed in a dry 50 mL round-bottom flask, and 10 mL of DMF was used as the solvent. The mixture was stirred at room temperature at 25 ° C for 2 h under argon protection. After removing the solvent, the mixture was purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 25:1) to obtain 143 mg of a yellow oily substance (compound B-3) with a yield of 81%.

[0087]

[0088] 1H NMR (400MHz, CDCl3, ppm) δ: 9.04 (dd, J1=1.3Hz, J2=8.5Hz, 1H, Ph-H), 7.74 (m, 1H, Ph-H), 7.62 (d, J=8.6Hz, 1H, Ph-H), 7.42 (m, 1H, Ph-H), 6.77 (s, 1H, Mass spectrometry(NSI-MS,m / z):[M+H] + calcd.for[C 24 H 31 N3O5+H] + 442.2336; found, 442.2329.

[0089]

[0090] Compound B-3 (285 mg, 0.65 mmol) and 5-(4-(2-hydroxyethylmethylamino)phenyl)thiophene-2-carboxaldehyde (169 mg, 0.65 mmol) were placed in a dry 50 mL round-bottom flask, 10 mL of toluene was used as the solvent, heated to 110°C under argon protection, and refluxed and stirred at 110°C for 3 h. After removing the solvent, the mixture was purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 25:1) to obtain 300 mg of dark red solid (compound C-3) with a yield of 67%.

[0091] 1H NMR (400MHz, DMSO-d6, ppm) δ: 9.04 (dd, J1=1.2Hz, J2=8.4Hz, 1H, Ph-H), 8.05 (d, J=9.0Hz, 1H, Ph-H), 7.89 (m, 1H, Ph-H), 7.60 (t, J=7.8Hz, 1H ,Ph-H),7.57(d,J=15.7Hz,1H,alkene-H),7.51(d,J=8.9Hz,2H,Ph-H),7.46(d,J=3.9Hz,1H,thioph-H),7.33(d,J=3.9Hz,1H,thioph-H),7 .31(d,J=15.7Hz,1H,alkene-H),7.00(s,1H,Ph-H),6.73(d,J=9.0Hz,2H,Ph-H),4.73(t,J=5.4Hz,1H,-OH),4.71(t,J=6.0Hz,2H,-NCH2CH2 Mass spectrometry(ESI-MS,m / z):[M+Na] + calcd.for[C 38 H 44 N4O6S+Na] + 707.2874; found, 707.2849.

[0092]

[0093] Compound C-3 (300 mg, 0.42 mmol) was placed in a dry 50 mL round-bottom flask, and diethylaminosulfur trifluoride (63 mg, 0.42 mmol) was added dropwise at -78°C under argon protection with 10 mL of dichloromethane as solvent, and the reaction was continued at -78°C for 2 hours. After the reaction was completed, it was quenched with a saturated sodium bicarbonate solution, extracted with dichloromethane, dried, and after removing the solvent, it was purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 25:1) to obtain 169 mg of dark red solid, which is the Aβ plaque dual-mode imaging probe with a structure shown in Formula II-1, with a yield of 59%.

[0094] 1H NMR (400MHz, CDCl3, ppm) δ: 9.12 (d, J = 8.4Hz, 1H, Ph-H), 7.73 (t, J = 8.0Hz, 1H, Ph-H), 7.60 (d, J = 8.8Hz, 1H, Ph-H), 7.50 (d, J = 8.5Hz, 2H, Ph-H), 7.44 ( t,J=7.7Hz,1H,Ph-H),7.38(d,J=15.4Hz,1H,alkene-H),7.19(d,J=4.0Hz,1H,thioph-H),7.14(d,J=4.0Hz,1H,thioph-H),7.13(s,1H,Ph-H),7.11( d,J=15.4Hz,1H,alkene-H),6.72(d,J=8.6Hz,2H,Ph-H),4.63(dt,J1=5.0Hz,J2=47.1Hz,2H,-NCH2CH2F),4.54(t,J=4.8Hz,2H,-NCH2CH2O-),4.00(t ,J=4.8Hz,2H,-NCH2CH2O-),3.49-3.75(m,18H,PEG-H,-NCH2CH2F),3.34(s,3H,-OCH3),3.08(s,3H,-NCH3); Massspectrometry(ESI-MS,m / z):[M+H] + calcd.for[C 38 H 43 FN4O5S+H] + 687.3016; found, 687.3014.

[0095] Example 4

[0096] Compound A (100 mg, 0.40 mmol), 2-bromoethyl methyl ether (111 mg, 0.80 mmol) and sodium ethoxide (27 mg, 0.40 mmol) were placed in a dry 50 mL round-bottom flask, and 10 mL of DMF was used as the solvent. The mixture was stirred at room temperature at 25 ° C for 2 h under argon protection. After removing the solvent, the mixture was purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 25:1) to obtain 65 mg of a yellow oily substance (compound B-4) with a yield of 53%.

[0097]

[0098] 1H NMR (400MHz, DMSO-d6, ppm) δ: 8.90 (d, J=8.4Hz, 1H, Ph-H), 8.06 (d, J=8.9Hz, 1H, Ph-H), 7.88 (t, J= 8.1Hz,1H,Ph-H),7.59(t,J=7.8Hz,1H,Ph-H),6.80(s,1H,Ph-H),4.66(t,J=5.1Hz,2H,-NCH2CH2O -),3.82(t,J=5.1Hz,2H,-NCH2CH2O-),3.46(t,J=4.2Hz,2H,-OCH2CH2OCH3),3.32(t,J=4.2Hz,2H ,-OCH2CH2OCH3),3.12(s,3H,-OCH3),2.70(s,3H,-CH3); Massspectrometry(NSI-MS,m / z):[M+H] + calcd.for[C 18 H 19 N3O2+H] + 310.1550; found, 310.1543.

[0099]

[0100] Compound B-4 (115 mg, 0.37 mmol) and 5-(4-(2-hydroxyethylmethylamino)phenyl)thiophene-2-carboxaldehyde (96 mg, 0.37 mmol) were placed in a dry 50 mL round-bottom flask, 10 mL of toluene was used as the solvent, heated to 110°C under argon protection, and refluxed and stirred at 110°C for 3 h. After removing the solvent, the mixture was purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 25:1) to obtain 116 mg of dark red solid (compound C-4), with a yield of 57%.

[0101]

[0102] Compound C-4 (116 mg, 0.21 mmol) was placed in a dry 50 mL round-bottom flask, and diethylaminosulfur trifluoride (31 mg, 0.21 mmol) was added dropwise at -78°C under argon protection with 10 mL of dichloromethane as solvent, and the reaction was continued at -78°C for 2 hours. After the reaction was completed, it was quenched with a saturated sodium bicarbonate solution, extracted with dichloromethane, dried, and after removing the solvent, it was purified by silica gel column chromatography (developing solvent: dichloromethane: methanol = 25:1) to obtain 72 mg of dark red solid, which is the Aβ plaque dual-mode imaging probe with a structure shown in Formula II-2, with a yield of 59%.

[0103] 1H NMR (400MHz, CDCl3, ppm) δ: 9.09 (d, J = 8.2Hz, 1H, Ph-H), 7.72 (t, J = 7.6Hz, 1H, Ph-H), 7.58 (d, J = 8.8Hz, 1H, Ph-H), 7.50 (d, J = 8.8Hz, 2H, Ph-H), 7.43 (t, J = 7 .8Hz,1H,Ph-H),7.37(d,J=15.4Hz,1H,alkene-H),7.18(d,J=3.8Hz,1H,thioph-H),7.13(d,J=15.4Hz,1H,alkene-H),7.12(s,1H,Ph-H),7.11(d,J=3.8H z,1H,thioph-H),4.63(dt,J1=5.1Hz,J2=47.1Hz,2H,-NCH2CH2F),4.54(t,J=5.2Hz,2H,-NCH2CH2O-),4.00(t,J=5.2Hz,2H,-NCH2CH2O-),3.70(dt,J1=5. Mass spectrometry(NSI-MS,m / z):[M+H] + calcd.for[C 21 H 31 FN4O2S+H] + 555.2225; found, 555.2236.

[0104] Experimental Example 1

[0105] The fluorescence spectra of the dual-mode imaging probe of Aβ plaques with the structure shown in Formula Ⅰ-1 were tested in different viscosity environments: Ⅰ-1 was dissolved in analytical grade dimethyl sulfoxide to prepare 1.0×10 -3 mol / L stock solution. Then prepare 2mL of glycerol / water mixed solvents with glycerol content of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%. Take 20μL of the stock solution of Ⅰ-1 and add it to the prepared glycerol / water mixed solvents. After mixing evenly, transfer to a quartz cuvette (10mm×10mm) and test its fluorescence spectrum. The results are as follows Figure 1As shown, with 470nm as the excitation wavelength, the maximum emission wavelength of the Aβ plaque dual-mode imaging probe with the structure shown in Formula Ⅰ-1 is about 720nm, which is located in the near-infrared region. And as the glycerol content in the mixed solvent increases, that is, the viscosity increases, the fluorescence intensity increases significantly.

[0106] Experimental Example 2

[0107] Test the Aβ plaque dual-mode imaging probe with the structure shown in Formula Ⅰ-1 and different concentrations of Aβ 42 Fluorescence spectrum of protein aggregate response: The concentration was 1.0×10 -3 mol / L of the stock solution of Ⅰ-1 was diluted to obtain a concentration of 1.0×10 -4 mol / L stock solution. Re-preparation of Aβ 42 The concentration of protein aggregates was 2.0×10 -4 Then, 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, and 100 μL of the above Aβ were added to 1.99 mL, 1.98 mL, 1.97 mL, 1.96 mL, 1.95 mL, 1.94 mL, 1.93 mL, 1.92 mL, 1.91 mL, and 1.90 mL of 10 mM, pH = 7.4 phosphate buffer, respectively. 42 Protein aggregate stock solution, and then add 20 μL of 1.0×10 -4 mol / L of Ⅰ-1 stock solution, incubated at 37℃ with slight shaking for 10min, and then tested its fluorescence spectrum. The test results are as follows Figure 2 As shown, with 470nm as the excitation wavelength, Ⅰ-1 and Aβ 42 There is almost no fluorescence before protein aggregates bind. 42 After protein aggregates bind, there is a significant fluorescence enhancement at 657nm, and along with Aβ 42 As the concentration of protein aggregates increases, the fluorescence intensity of Ⅰ-1 increases.

[0108] Experimental Example 3

[0109] The same method as in Experimental Example 1 was used to test the fluorescence spectra of the Aβ plaque dual-mode imaging probe with the structure shown in Formula I-2 in different viscosity environments. The test results are as follows: Figure 3 As shown in the figure, with 470nm as the excitation wavelength, the maximum emission wavelength of Ⅰ-2 is around 720nm, which is located in the near-infrared region. And with the increase of glycerol content in the mixed solvent, that is, the increase of viscosity, the fluorescence intensity is significantly enhanced.

[0110] Experimental Example 4

[0111] The same method as in Experimental Example 2 was used to test the Aβ plaque dual-mode imaging probe with the structure shown in Formula I-2 in combination with different concentrations of Aβ 42 Fluorescence spectrum of protein aggregate response, test results are as follows Figure 4 As shown, with 470nm as the excitation wavelength, Ⅰ-2 and Aβ 42 There is almost no fluorescence before protein aggregates bind. 42 After protein aggregates bind, there is a significant fluorescence enhancement at 657nm, and along with Aβ 42 As the concentration of protein aggregates increases, the fluorescence intensity of Ⅰ-2 increases.

[0112] Experimental Example 5

[0113] The same method as in Experimental Example 1 was used to test the fluorescence spectra of the Aβ plaque dual-mode imaging probe with the structure shown in Formula II-1 in different viscosity environments. The test results are as follows: Figure 5 As shown in the figure, with an excitation wavelength of 470 nm, the maximum emission wavelength of II-1 is around 720 nm, which is located in the near-infrared region. And with the increase of glycerol content in the mixed solvent, that is, the increase of viscosity, the fluorescence intensity increases significantly.

[0114] Experimental Example 6

[0115] The same method as in Experimental Example 2 was used to test the dual-mode imaging probe of Aβ plaques with the structure shown in Formula II-1. 42 Fluorescence spectrum of protein aggregate response, test results are as follows Figure 6 As shown, with 470nm as the excitation wavelength, Ⅱ-1 and Aβ 42 There is almost no fluorescence before protein aggregates bind. 42 After protein aggregates bind, there is a significant fluorescence enhancement at 657nm, and along with Aβ 42 As the concentration of protein aggregates increases, the fluorescence intensity of Ⅱ-1 increases.

[0116] Experimental Example 7

[0117] The same method as in Experimental Example 1 was used to test the fluorescence spectra of the Aβ plaque dual-mode imaging probe with the structure shown in Formula II-2 in different viscosity environments. The test results are shown in Figure 7 As shown in the figure, with an excitation wavelength of 470 nm, the maximum emission wavelength of II-2 is around 720 nm, which is located in the near-infrared region. And with the increase of glycerol content in the mixed solvent, that is, the increase of viscosity, the fluorescence intensity increases significantly.

[0118] Experimental Example 8

[0119] The same method as in Experimental Example 2 was used to test the Aβ plaque dual-mode imaging probe with the structure shown in Formula II-2 in combination with different concentrations of Aβ42 Fluorescence spectrum of protein aggregate response, test results are as follows Figure 8 As shown, with 470nm as the excitation wavelength, Ⅱ-2 and Aβ 42 There is almost no fluorescence before protein aggregates bind. 42 After protein aggregates bind, there is a significant fluorescence enhancement at 657nm, and along with Aβ 42 As the concentration of protein aggregates increases, the fluorescence intensity of Ⅱ-2 increases.

[0120] Experimental Example 9

[0121] Test the cytotoxicity of the dual-mode imaging probe for Aβ plaques of the structure shown in Formula I-1: HepG2 cells were seeded in a 96-well plate at a density of 1.0×10 4 cells / well, cultured in a constant temperature incubator for 8 hours. Add culture medium containing Ⅰ-1 (concentrations of 0μM, 1μM, 2μM, 4μM, 8μM, 16μM, 32μM, 50μM, 75μM, 100μM, 100μL) to different wells and continue to culture in a constant temperature incubator for 24 hours. Use a needle to aspirate the culture medium in each well, add 100μL of 0.5mg / mL MTT solution, incubate for 4 hours, aspirate the culture medium, add 100μL of dimethyl sulfoxide to each well, and test the absorbance value at 590nm with an enzyme marker. The formula for calculating cell viability is: (OD treated / OD control )×100%. The result is Fig. 9 As shown, when the concentration of Ⅰ-1 reaches 100 μM, the cell survival rate still remains above 80%, indicating that the Aβ plaque dual-mode imaging probe with the structure shown in formula Ⅰ-1 has low cytotoxicity.

[0122] Experimental Example 10

[0123] The Aβ plaque dual-mode imaging probe with the structure shown in Formula Ⅰ-1 was tested for fluorescent staining of brain tissue sections of 5xFAD transgenic mice: the brain tissue sections of 5xFAD transgenic mice were dewaxed and hydrated, and then 100 μL of 100 μM Ⅰ-1 aqueous solution was added to the tissue sections. After incubation at room temperature for 10 minutes, the solution was carefully removed from the edge with dust-free paper and then placed under a microscope for observation. Adjacent sections were stained with the gold standard dye Thioflavin S to determine the distribution of plaques. The results are shown in Figure 2. Fig.10 As shown in the figure, due to the inherent aggregation quenching property of Thioflavin S, the fluorescence signal of some Aβ plaques is not strong enough, and the contrast with the background is insufficient, which easily leads to inevitable "false negative" results. In contrast, Ⅰ-1 has excellent anti-fading properties and can display the Aβ plaque information on the brain tissue sections of 5xFAD transgenic mice with high fidelity, significantly improving the signal quality and detection accuracy.

[0124] Experimental Example 11

[0125] Testing the Aβ plaque dual-mode imaging probe with the structure shown in Formula Ⅰ-1 for Aβ plaque fluorescence imaging: The 10-month-old 5xFAD transgenic mice and normal mice in the control group used in this experiment were purchased from Nanjing Junke Biotechnology Co., Ltd. The mice were housed in sterile cages in a laminar flow hood in a sterile room and fed with food and water treated with high-pressure steam.

[0126] 10-month-old 5xFAD transgenic mice and control normal mice were injected with 2.3 mg / kg of Ⅰ-1 through the tail vein, and then imaged using the PerkinElmer IVIS Spectrum CT small animal in vivo imaging system. During the imaging process, the mice were anesthetized with an oxygen flow of 2.0 L / min containing 2.0% isoflurane, and the excitation wavelength and receiving wavelength were set to 500 nm and 700 nm, respectively. The results are shown in Fig.11 As shown in the figure, before the injection of Ⅰ-1, the fluorescence signals of 5xFAD transgenic mice and normal mice in the control group were weak. 13h after the injection of Ⅰ-1, the fluorescence signal in the brain of 5xFAD transgenic mice was significantly higher than that of normal mice in the control group. This shows that Ⅰ-1 has excellent blood-brain barrier penetration ability and "lighting-up" response characteristics, and can image Aβ plaques through fluorescence mode.

[0127] Experimental Example 12

[0128] Testing the Aβ plaque dual-mode imaging probe with the structure shown in Formula I-1 for Aβ plaque positron emission tomography imaging: 10-month-old 5xFAD transgenic mice and control group normal mice were injected with 10MBq 18 F-labeled Ⅰ-1 was then imaged using the Inviscan IRIS PET-CT small animal in vivo imaging system. During the imaging process, the mice were anesthetized with an oxygen flow of 2.0 L / min containing 2.0% isoflurane. Fig.12 As shown, in the injection 18 After 12 minutes of exposure to F-labeled Ⅰ-1, it was observed that the signal intensity in the hippocampus, cortex and other regions of the brain of 5xFAD transgenic mice was significantly higher than that of normal mice in the control group, indicating that Ⅰ-1 can image Aβ plaques through positron emission tomography.

[0129] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A dual-mode imaging probe for Aβ plaques, characterized in that: The Aβ plaque dual-mode imaging probe has a structure as shown in Formula I: In formula I, R1 is selected from cyano, carboxyl or R2 is selected from a C1 to C4 hydrocarbon group, wherein n is independently an integer between 0 and 10, and F is selected from 18 F or 19 F; R3 is selected from methyl, wherein m is independently 0 or 1, R5 is independently selected from -H, -CH3, -CH2OH, -CH2OTs, -CH2 18 F,-CH2 19 F, -CH2CH3, -CH2CH2OH, -CH2CH2OTs, -CH2CH2 18 F or -CH2CH2 19 F; R4 is selected from -H, - 18 F or - 19 F.

2. The Aβ plaque dual-mode imaging probe according to claim 1, characterized in that: The R1 is selected from cyano.

3. The Aβ plaque dual-mode imaging probe according to claim 1 or 2, characterized in that: The R2 is selected from wherein n is independently an integer between 0 and 10, and F is selected from 18 F or 19 F.

4. The Aβ plaque dual-mode imaging probe according to claim 3, characterized in that: The R3 is selected from wherein m is independently 0 or 1, R5 is independently selected from -H, -CH3, -CH2OH, -CH2OTs, -CH2 18 F,-CH2 19 F, -CH2CH3, -CH2CH2OH, -CH2 CH2OTs, -CH2CH2 18 F or -CH2CH2 19 F.

5. The Aβ plaque dual-mode imaging probe according to claim 4, characterized in that: When R2 is selected from When n is independently an integer between 0 and 10, R3 is selected from wherein m is independently 0 or 1, R5 is independently selected from -CH2OH, -CH2OTs, -CH2 18 F, -CH2 19 F, -CH2CH2OH, -CH2CH2OTs, -CH2CH2 18 F or -CH2CH2 19 F; When R2 is selected from When n is independently an integer between 0 and 10, F is selected from 18 F or 19 F, R3 is selected from wherein m is independently 0 or 1, and R5 is independently selected from -H, -CH3 or -CH2CH3.

6. The Aβ plaque dual-mode imaging probe according to claim 5, characterized in that: The Aβ plaque dual-mode imaging probe has a structure as shown in Formula Ⅰ-1, Ⅰ-2, Ⅱ-1 or Ⅱ-2:

7. The method for preparing the dual-mode imaging probe for Aβ plaques according to claim 6, characterized in that: The steps include: (1) Under argon protection conditions, using N,N-dimethylformamide as solvent, compound A, compound X and sodium ethoxide react to obtain compound B; (2) Under argon protection conditions, using toluene as solvent, compound B and compound Y react to obtain compound C; (3) Under argon protection conditions, using dichloromethane as a solvent, compound C and diethylaminosulfur trifluoride reacted to prepare an Aβ plaque dual-mode imaging probe; The compound A is The compound X comprises 14-bromo-3,6,9,12-tetraoxatetradecan-1-ol, 2-(2-bromoethoxy)ethanol, 13-bromo-2,5,8,11-tetraoxatetradecan or 2-bromoethyl methyl ether; The compound Y comprises 5-(4-dimethylaminophenyl)thiophene-2-carboxaldehyde or 5-(4-(2-hydroxyethylmethylamino)phenyl)thiophene-2-carboxaldehyde.

8. The method for preparing the dual-mode imaging probe for Aβ plaques according to claim 7, characterized in that: In the step (1), the molar ratio of compound A, compound X and sodium ethoxide is 1:1-2:0.8-1.2, the reaction is carried out at room temperature, and the reaction time is 1-3 hours; In the step (2), the molar ratio of compound B to compound Y is 1:0.8-1.2, the reaction temperature is 100-120° C., and the reaction time is 2-4 hours.

9. The method for preparing the dual-mode imaging probe for Aβ plaques according to claim 7 or 8, characterized in that: In the step (3), the molar ratio of compound C to diethylaminosulfur trifluoride is 1:0.8-1.2, the reaction temperature is -80--75°C, and the reaction time is 1-3h.

10. Use of the Aβ plaque dual-mode imaging probe according to any one of claims 1 to 6 in the preparation of an Aβ plaque imaging agent.

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