A beta-amyloid protein probe and its preparation method and application

Abstract

The present invention discloses a β-amyloid protein probe and a preparation method and application thereof. The skeleton of the β-amyloid protein probe can effectively increase the ICT effect of the probe, so that the probe has a larger emission wavelength and a larger fluorescence intensity enhancement factor before and after binding to the Aβ protein. At the same time, it uses a nitrogen-containing six-membered aromatic ring as an electron acceptor, which not only has better electron-withdrawing ability, but also the large steric hindrance of the electron acceptor after the probe enters the hydrophobic cavity of the Aβ protein can strengthen the restriction on molecular torsion, and trigger a more efficient ICT effect with the thiophene-containing π bridge. At the same time, this type of nitrogen-containing six-membered aromatic ring can combine well with the β-sheet structure of the Aβ protein, effectively improving the binding ability of the probe to the Aβ protein and the Aβ protein selectivity. Therefore, the probe can be used for labeling Aβ protein and preparing an imaging agent for labeling Aβ protein.

Description

Technical Field

[0001] The present invention relates to the field of molecular probes, and in particular to a beta-amyloid protein probe and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD), commonly known as senile dementia, is a progressive neurodegenerative disease.

[0003] The pathological characteristics of AD are mainly the abnormal deposition of amyloid β-protein (Aβ). Clinically, accurate diagnosis of AD is difficult and easily affected by subjective factors. At present, the most authoritative means of diagnosing AD are cerebrospinal fluid marker detection and positron emission tomography (PET) scanning. Among them, the detection of cerebrospinal fluid Aβ can stably and reliably detect the earlier pathological changes of AD, but the detection of cerebrospinal fluid Aβ requires invasive lumbar puncture, and patient compliance is poor. PET scanning can achieve non-invasive and accurate early diagnosis, which requires Aβ-related imaging probes to label Aβ protein.

[0004] The β-pleated cavity formed within the Aβ protein is a hydrophobic, non-polar environment, while the exterior of the Aβ protein exhibits a hydrophilic, polar state. Aβ probes with an intramolecular charge transfer (ICT) effect are highly sensitive to environmental polarity. Upon entering the hydrophobic cavity of the Aβ protein from a physiological water environment, the fluorescence properties of the Aβ probe, such as its maximum emission wavelength, undergo significant changes under the action of excitation light, resulting in a fluorescence "on" response. However, the ICT effect of the backbone structure of existing Aβ probes is suboptimal, resulting in a need for further improvement in their optical properties and the fluorescence intensity enhancement factor after binding to the Aβ protein. Summary of the Invention

[0005] One object of the present invention is to provide a β-amyloid protein probe, the skeleton of which can form a coplanar structure that is conducive to electron transport when the environmental polarity changes. At the same time, by utilizing a multi-conjugated structure and suitable electron donors and electron acceptors, the ICT effect of the probe can be significantly enhanced, the optical properties of the probe, such as the maximum emission wavelength, can be improved, and the fluorescence intensity enhancement factor of the probe after binding to the Aβ protein can be increased.

[0006] The present invention is achieved through the following technical solutions:

[0007] A β-amyloid protein probe has the structural formula shown in Formula I:

[0008]

[0009] In formula I, R1 is selected from N,N'-dimethylaryl, methylaminoaryl, N,N'-dimethylnaphthyl, N-azetidinylaryl; R2 is selected from substituted or unsubstituted pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, wherein the substituent is C1-C3 alkyl, hydroxymethyl, hydroxyethyl, fatty acyl, trifluoromethyl, n=0 or 1.

[0010] In this technical solution, the probe's backbone comprises a rotatable thiophene group, an electron donor R1 group attached to one side of the thiophene group, and an electron acceptor R2 group attached to the other side of the thiophene group. This backbone structure utilizes the rotatable thiophene group to enable the probe to exhibit a rotation-coplanar property when in solvents of varying polarity. Specifically, in a hydrophilic physiological environment, molecular rotation results in weak fluorescence from the probe. However, upon binding to the hydrophobic cavity of the Aβ protein, a coplanar structure is formed that facilitates electron transfer, enhancing the probe's ICT effect and effectively increasing the fluorescence intensity enhancement factor after binding to the protein.

[0011] In the present technical solution, the electron donor R1 can be N,N'-dimethylaryl, methylaminoaryl, N,N'-dimethylnaphthyl, N-azetidinylaryl. The electron acceptor R2 is a nitrogen-containing six-membered aromatic ring. Specifically, R2 can be an unsubstituted pyridyl, pyrazinyl, pyrimidinyl or pyridazinyl, or a substituted pyridyl, pyrazinyl, pyrimidinyl or pyridazinyl. In one or more embodiments, the substitution can be monosubstituted or disubstituted. The substituent can be a C1-C3 alkyl, hydroxymethyl, hydroxyethyl, fatty acyl, trifluoromethyl, In some embodiments, the substituent may be a C1-C3 linear alkyl group or a branched alkyl group.

[0012] In this technical solution, by increasing the electron-donating capacity of the electron donors on both sides of the thiophene and the electron-withdrawing capacity of the electron acceptors, and utilizing a multi-conjugated structure to enhance electron transport, the energy gap between the HOMO-LUMO energy levels can be reduced, thereby enabling the probe to have a longer emission wavelength. In some preferred embodiments, the maximum emission wavelength of the probe reaches 720nm. Furthermore, the use of a nitrogen-containing six-membered aromatic ring electron acceptor not only has better electron-withdrawing ability, but also triggers a more efficient ICT effect with the thiophene-containing π bridge after the probe enters the hydrophobic cavity of the Aβ protein. Furthermore, this type of nitrogen-containing six-membered aromatic ring can effectively bind to the β-sheet structure of the Aβ protein, effectively improving the binding ability and Aβ protein selectivity of the probe.

[0013] In this technical solution, experiments have shown that the binding ability of this type of probe to Aβ protein is proportional to its fluorescence intensity. The binding ability of the probe to Aβ protein can be evaluated by the fluorescence intensity after binding to Aβ protein, thereby obtaining a probe with strong fluorescence intensity and high ability to bind to Aβ protein.

[0014] In some preferred embodiments, R2 is selected from substituted pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, wherein the substituent is methyl, ethyl, hydroxyethyl, acyl, trifluoromethyl,

[0015] Furthermore, the R2 is selected from any one of the following groups:

[0016]

[0017] Furthermore, the β-amyloid protein probe is selected from the following compounds:

[0018]

[0019] Furthermore, the β-amyloid protein probe is selected from the following compounds:

[0020]

[0021]

[0022] Furthermore, the fluorine substitution of the β-amyloid probe is 18 F. By replacing the fluorine substitution of the probe with 18 F, Probes that can be used for PET imaging can be obtained.

[0023] Another object of the present invention is to provide a method for preparing any of the aforementioned β-amyloid protein probes, which has a short synthesis path, mild reaction conditions, and readily available raw materials, and is conducive to scale-up production.

[0024] Specifically, a method for preparing a β-amyloid protein probe comprises the following steps:

[0025] Under an inert atmosphere, the aldehyde represented by Formula II, the phosphate derivative represented by Formula III, and a solvent are uniformly mixed, the reaction system is cooled to below -10°C, a base is added dropwise, and the mixture is reacted at below -10°C to obtain a β-amyloid protein probe; or

[0026] Under an inert atmosphere, the aldehyde represented by formula II, the phosphate derivative represented by formula III and the solvent are uniformly mixed, the reaction system is cooled to below -10°C, a base is added dropwise, and the reaction is carried out at below -10°C to obtain an intermediate, the intermediate is dehydroxylated, the intermediate is subjected to a bioorthogonal reaction with a dienophile and then dehydroxylated, or the intermediate is reacted with a dienophile. After the reaction, a β-amyloid protein probe is obtained;

[0027] Formula II: Formula III:

[0028] Wherein, R1 in formula II is selected from N,N'-dimethylaryl, methylaminoaryl, N,N'-dimethylnaphthyl, N-azetidinylaryl, and R3 in formula II is selected from substituted or unsubstituted pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrazinyl, wherein the substituent is C1-C3 alkyl, acyl, trifluoromethyl, protected or unprotected hydroxymethyl or hydroxyethyl, C3-C4 alkynyl; n=0 or 1.

[0029] In this technical solution, some probes, such as probes 3a and 3b, can be directly obtained by reacting an aldehyde represented by Formula II with a phosphate derivative represented by Formula III. In some embodiments, the aldehyde represented by Formula II, the phosphate derivative represented by Formula III, and a solvent are uniformly mixed under an inert atmosphere, and then a base is added dropwise to the reaction system at low temperature, followed by a further reaction at low temperature for 2-3 hours to obtain the amyloid β probe.

[0030] In the present technical solution, for a part of the probes, such as probe 3g, the intermediate can be first obtained by reacting the aldehyde shown in formula II with the phosphate derivative shown in formula III, and some groups of the intermediate, such as the hydroxyethyl group, are protected by a hydroxy protecting group. Therefore, after obtaining the intermediate, the intermediate is dehydroxylated to obtain a β-amyloid protein probe. For a part of the probes, such as probe 3e, the electron acceptor in the obtained intermediate is a tetrazine group, and the tetrazine group is connected to a hydroxyethyl group protected by OTBS. Therefore, after obtaining the intermediate, the intermediate needs to first undergo a bioorthogonal reaction with a dienophile and then be dehydroxylated to obtain a β-amyloid protein probe. For a part of the probes, such as 3f-F and 3g-F, after obtaining an intermediate containing a terminal alkynyl group, the alkynyl group is reacted with the alkynyl group to obtain the β-amyloid protein probe. reaction to obtain a β-amyloid protein probe.

[0031] In some preferred embodiments, R3 is a substituted pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, or tetrazinyl group, wherein the substituent is a C1-C2 alkyl group, an acyl group, a trifluoromethyl group, a hydroxyethyl group, a protected hydroxyethyl group, or a butynyl group.

[0032] In some preferred embodiments, the base is an organic base or an inorganic base. In one or more embodiments, the inorganic base is at least one of sodium carbonate, potassium carbonate, and sodium hydroxide. In one or more embodiments, the organic base can be at least one of sodium tert-butoxide, potassium tert-butoxide, or lithium diisopropylamide.

[0033] In some preferred embodiments, the temperature for adding the base to the reaction system and the reaction temperature after the addition of the base are -35°C to -10°C. Preferably, the reaction temperature is -30°C to -20°C.

[0034] In some preferred embodiments, the solvent is anhydrous tetrahydrofuran.

[0035] Another object of the present invention is to provide a use of any of the aforementioned β-amyloid protein probes, wherein the β-amyloid protein probe is used to bind to Aβ protein to label Aβ protein; or, the β-amyloid protein probe is used to prepare an imaging molecular probe for labeling Aβ protein.

[0036] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0037] 1. The backbone of the amyloid beta protein probe provided by the present invention can effectively increase the ICT effect of the probe, so that the probe has a longer emission wavelength and a greater fluorescence intensity enhancement factor before and after binding to the Aβ protein;

[0038] 2. The present invention uses a nitrogen-containing six-membered aromatic ring as an electron acceptor, which not only has better electron-withdrawing ability, but also the large steric hindrance of the electron acceptor can strengthen the restriction of molecular torsion after the probe enters the hydrophobic cavity of the Aβ protein, triggering a more efficient ICT effect with the thiophene-containing π bridge. In addition, this type of nitrogen-containing six-membered aromatic ring can well bind to the β-sheet structure of the Aβ protein, effectively improving the binding ability of the probe to the Aβ protein and the Aβ protein selectivity;

[0039] 3. The binding ability of the probe of the present invention to the Aβ protein is proportional to its fluorescence intensity. The binding ability of the probe to the Aβ protein can be evaluated by the fluorescence intensity after binding to the Aβ protein, thereby obtaining a probe with strong fluorescence intensity and high binding ability to the Aβ protein;

[0040] 4. The preparation method of the present invention has a short synthesis path, mild reaction conditions, and readily available raw materials, which is conducive to scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0042] Figure 1 A specific embodiment of the present invention 18 F-labeled probe 3e- 18 HPLC spectrum of F, where (A) 3e- 18 F reaction time 15min HPLC radioactive peak, (B) 3e- 18HPLC chromatogram at 254 nm after 15 min of reaction of 3e-F, (C) 3e-F and 3e- 18 F co-injection radioactive peaks, (D) 3e-F and 3e- 18 F co-injection HPLC chromatogram at 254 nm;

[0043] Figure 2 A specific embodiment of the present invention 18 F labeled probe 3h- 18 HPLC spectrum of F, (A) 3h- 18 F reaction time 15min HPLC radioactive peak, (B) 3h- 18 HPLC chromatogram at 254 nm after 15 min of reaction of 3h-F, (C) 3h-F and 3h- 18 F co-injection radioactive peaks, (D) 3h-F and 3h- 18 F co-injection HPLC chromatogram at 254 nm;

[0044] Figure 3 The selectivity of the probe to Aβ protein and HSA protein in a specific embodiment of the present invention is shown;

[0045] Figure 4 The selectivity of 3e-F, 3f-F, and 3h-F for Aβ protein and HSA protein in the specific embodiment of the present invention is shown;

[0046] Figure 5 The in vivo fluorescence imaging results of 3e-F and 3h-F in AD mice are shown in a specific embodiment of the present invention;

[0047] Figure 6 The co-localization results of 3e-F, 3h-F and Aβ plaques in AD mouse brain slices in a specific embodiment of the present invention are shown;

[0048] Figure 7 The probe 3e- in a specific embodiment of the present invention is shown. 18 F and 3h- 18 F is the PET imaging results of AD mice. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples and drawings. The illustrative embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention. The term "connection" used herein, unless otherwise specified, may refer to direct connection or indirect connection via other groups.

[0050] All raw materials of the present invention are not particularly limited in their sources and can be purchased from the market or prepared according to conventional methods well known to those skilled in the art.

[0051] There is no particular limitation on the purity of all raw materials in the present invention. The present invention preferably adopts analytical purity or conventional purity requirements in the field of molecular probes.

[0052] The present invention has no particular limitation on the expression of the substituents, and all expressions familiar to those skilled in the art are adopted. Based on common sense, those skilled in the art can correctly understand the meaning of the substituents according to the expressions.

[0053] All raw materials of the present invention, their brands and abbreviations are conventional brands and abbreviations in the field. Each brand and abbreviation is clear and unambiguous in the field of its relevant use. Those skilled in the art can purchase them from the market or prepare them by conventional methods based on the brand, abbreviation and corresponding use.

[0054] 1. Probe Preparation

[0055] [Example 1]

[0056]

[0057] Under argon protection, aldehyde 1a (15 mg, 0.0582 mmol) and phosphate derivative 2a (18.5 mg, 0.0757 mmol) were added to a round-bottom flask, followed by 3 mL of anhydrous tetrahydrofuran. After the system was cooled to -20 °C, 1 M t BuOK (87 μL, 0.0873 mmol) was added dropwise and reacted at -20°C for 2 h. After the reaction, the product was purified by silica gel chromatography (PE:DCM=2:1) to obtain probe 3a (13.5 mg) in a yield of 67%.

[0058] 1 H NMR(400MHz,Chloroform-d)δ8.44(d,J=1.5Hz,1H),8.39(d,J=1.5Hz,1H),7.75(d,J=15.6Hz,1H),7.40-7.34(m,2H),7.05(d ,J=3.7Hz,1H),7.00(d,J=16.0Hz,1H),6.90(d,J=4.3Hz,1H),6.89-6.81(m,2H),6.73-6.67(m,2H),2.99(s,6H),2.56(s,3H).

[0059] 13C NMR(101MHz,Chloroform-d)δ151.54,150.42,148.33,145.07,144.25,142.60,139.49, 130.05,129.69,127.81,127.14,125.73,125.13,122.60,117.52,112.51,40.53,21.49.

[0060] HRMS(DART-TOF)calculated for C 21 H 21 N3S + [M+H] + m / z 348.1534, found348.1520.

[0061] [Example 2]

[0062]

[0063] 1) Preparation of phosphate derivative 2b

[0064] Under argon, 2-methyl-5-pyrimidinemethanol (400 mg, 3.23 mmol) and acetonitrile (35 mL) were added to a round-bottom flask. SOCl2 (0.7 mL, 9.69 mmol) was slowly added dropwise at 0°C. After completion of the addition, the mixture was allowed to react at room temperature for 12 h. After completion of the reaction, the mixture was concentrated under reduced pressure and used directly in the next step without purification. Under argon, triethyl phosphite (11 mL, 65 mmol) was added, and the mixture was allowed to react at 130°C overnight. After completion of the reaction, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE:EA = 1:1) to obtain the phosphate derivative 2b (543 mg) as a colorless oily liquid in a 69% yield.

[0065] 1 H NMR (400MHz, Chloroform-d) δ8.52(d,J=2.5Hz,2H),4.15-3.91(m,4H),3.01(dd,J=21.4,2.3Hz,2H),2.66(d,J=2.2Hz,3H),1.23(td,J=7.1,2.6Hz,6H).

[0066] 13 C NMR (101MHz, Chloroform-d) δ 166.76 (d, J = 3.6Hz), 157.56, 157.50, 122.93 (d, J = 9.1Hz), 62.55, 62.49, 28.29 (d, J = 140.9Hz), 25.66, 16.47, 16.41.

[0067] HRMS(DART-TOF)calculated for C 10 H 18 N2O3P + [M+H] + m / z 245.2388, found 245.2356.

[0068] 2) Preparation of probe 3b

[0069] Under argon protection, aldehyde 1a (15 mg, 0.0582 mmol) and phosphate derivative 2b (18.5 mg, 0.0757 mmol) were added to a round-bottom flask, followed by 3 mL of anhydrous tetrahydrofuran. After the system was cooled to -20°C, 1 M t BuOK (87 μL, 0.0873 mmol) was added dropwise and reacted at -20°C for 2 h. After the reaction, the product was purified by silica gel chromatography (PE:EA=4:1) to obtain probe 3b (16.5 mg) in a yield of 81%.

[0070] 1 H NMR (400MHz, Chloroform-d) δ8.70 (s, 2H), 7.39-7.34 (m, 2H), 7.12 (d, J = 86.3Hz, 1H), 7.00-6.84 (m, 4H), 6.73-6.65 (m, 3H), 2.99 (s, 6H), 2.74 (s, 3H).

[0071] 13 C NMR(101MHz,Chloroform-d)δ166.26,154.03,150.28,144.61,139.14,129.90, 128.74,127.81,127.67,125.46,124.78,119.77,117.29,112.38,40.40,25.75.

[0072] HRMS(DART-TOF)calculated for C 21 H 21 N3S + [M+H] + m / z 348.1534, found348.1522.

[0073] [Example 3]

[0074]

[0075] 1) Preparation of phosphate derivative 2c

[0076] Under argon, 2-trifluoromethyl-5-chloromethylpyridine (400 mg, 2.04 mmol) and triethyl phosphite (7 ml, 40.8 mmol) were added to a round-bottom flask. The mixture was reacted at 130°C overnight. After completion of the reaction, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE:EA=2:1) to obtain the phosphate derivative 2c (462 mg) as a colorless oily liquid in a yield of 76%. 1 H NMR(400MHz,Chloroform-d)δ8.61(t,J=2.5Hz,1H),7.86(dt,J=8.2,2.5Hz,1H),7.6 4(d,J=8.1Hz,1H),4.15-3.99(m,4H),3.20(d,J=22.0Hz,2H),1.27(t,J=7.1Hz,6H).

[0077] 13 C NMR(101MHz,Chloroform-d)δ150.59(d,J=7.2Hz),146.48,138.26(d,J=5.8Hz),131.42(d ,J=9.4Hz),122.75,120.17-120.00(m),62.41,62.35,30.90(d,J=139.4Hz),16.25,16.19.

[0078] HRMS(DART-TOF)calculated for C 11 H 16 F3NO3P + [M+H] + m / z 298.2220, found 298.2301.

[0079] 2) Preparation of probe 3c

[0080] Under argon protection, aldehyde 1a (20 mg, 0.0777 mmol) and phosphate derivative 2c (28 mg, 0.0932 mmol) were added to a round-bottom flask, followed by 3.8 mL of anhydrous tetrahydrofuran. After the system was cooled to -20°C, 1 M t BuOK (116 μL, 0.116 mmol) was added dropwise and reacted at -20°C for 3 h. After the reaction, the product was purified by silica gel chromatography (PE:EA=5:1) to obtain probe 3c (19.1 mg) in a yield of 61%.

[0081] 1H NMR(400MHz,Chloroform-d)δ8.75(d,J=2.1Hz,1H),7.89(dd,J=8.2,2.2Hz,1H),7.63(d,J=8.2Hz,1H),7.40-7.35(m,2H),7. 32(d,J=16.1Hz,1H),7.04-6.97(m,2H),6.89(dd,J=9.8,6.1Hz,2H),6.81(d,J=16.0Hz,1H),6.74-6.68(m,2H),3.00(s,6H).

[0082] 13 C NMR(101MHz,Chloroform-d)δ150.49,148.17,145.30,139.02,136.01,133.30,130.33,129 .53,127.85,126.56,125.64,124.97,121.88,120.58(d,J=3.0Hz),117.33,112.50,40.51.

[0083] HRMS(DART-TOF)calculated for C 22 H 19 F3N2S + [M+H] + m / z 401.1299,found401.1292.

[0084] [Example 4]

[0085]

[0086] 1) Preparation of phosphate derivative 2d

[0087] Under argon, 4-pyridinemethanol (5 g, 45.8 mmol) and DCM (67 mL) were added to a round-bottom flask. SOCl2 (13 mL, 183 mmol) was slowly added dropwise at 0°C. After completion of the addition, the mixture was allowed to react at room temperature for 4 h. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was diluted with DCM (30 mL) and washed with saturated NaHCO3 solution (40 mL). The DCM layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The mixture was used directly in the next step without purification. A portion of the crude product (1 g) from the previous step was dissolved in 5 mL of toluene and set aside.

[0088] Under argon, a reaction flask was sequentially added with NaH (60%) (316 mg, 7.9 mmol), 10 mL of toluene, and diethyl phosphite (2 mL, 16 mmol). The toluene solution containing the crude product from the previous step was then slowly added dropwise. After the addition was complete, the flask was moved to 80°C and allowed to react for 0.5 h. After completion, the reaction was quenched with water (40 mL), extracted with ethyl acetate (30 mL x 3), and washed with saturated brine (40 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 2:1) to afford the phosphate derivative 2d (1.53 g) as a colorless oily liquid in an 85% yield.

[0089] 1 H NMR (400MHz, Chloroform-d) δ8.48(d,J=5.0Hz,2H),7.26-7.13(m,2H),3.98(p,J=7.3Hz,4H),3.07(d,J=22.4Hz,2H),1.19(t,J=7.0Hz,6H).

[0090] 13 C NMR (101MHz, Chloroform-d) δ149.90, 149.87, 141.43 (d, J = 8.6Hz), 125.16, 125.09, 62.53, 62.46, 33.57 (d, J = 137.4Hz), 16.45, 16.39.

[0091] 2) Preparation of probe 3d

[0092] Under argon protection, aldehyde 1a (40 mg, 0.155 mmol) and phosphate derivative 2d (50 mg, 0.218 mmol) were added to a round-bottom flask, followed by 8 mL of anhydrous tetrahydrofuran. After the system was cooled to -20°C, 1 M t BuOK (232 μL, 0.232 mmol) was added dropwise and reacted at -20°C for 3 h. After the reaction, the product was purified by silica gel chromatography (DCM / MeOH = 20:1) to obtain probe 3d (33.2 mg) in a yield of 64%.

[0093] 1 H NMR(400MHz,Chloroform-d)δ8.55(d,J=5.3Hz,2H),7.41-7.34(m,3H),7.32-7.28(m ,2H),7.03-6.96(m,2H),6.89(dd,J=9.9,6.1Hz,2H),6.77-6.67(m,3H),3.00(s,6H).

[0094] 13 C NMR(101MHz,Chloroform-d)δ150.46,150.19,145.26,144.82,139.14,130.23,1 29.50,127.83,126.61,125.65,125.02,124.50,120.64,117.39,112.49,40.51.

[0095] HRMS(DART-TOF)calculated for C 23 H 21 N2O2S + [M+H] + m / z 333.1425,found333.1420.

[0096] [Example 5]

[0097]

[0098] 1) Preparation of phosphate derivative 2e

[0099] Under argon, methyl 6-hydroxymethylnicotinate (700 mg, 4.18 mmol) and toluene (40 mL) were added to a round-bottom flask. PBr (0.4 mL, 4.18 mmol) was slowly added at 0°C. After complete addition, the mixture was allowed to react at room temperature for 12 h, then at 115°C for 1 h. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was diluted with DCM (30 mL) and washed sequentially with saturated aqueous sodium bicarbonate (30 mL) and saturated aqueous sodium chloride (30 mL). After concentration under reduced pressure, the residue was used directly in the next step without purification. Under argon, triethyl phosphite (14 mL, 80 mmol) was added, and the mixture was allowed to react at 125°C for 10 h. After completion of the reaction, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE:EA = 1:1) to afford the phosphate derivative 2e (378 mg) as a yellow oily liquid in a 31% yield.

[0100] 1 H NMR(400MHz,Chloroform-d)δ9.12(d,J=2.2Hz,1H),8.23(dt,J=8.2,2.0Hz,1H),7.46(dt,J=8.2,2.1Hz,1H), 4.07(tt,J=8.6,6.2Hz,4H), 3.93(d,J=1.7Hz,3H), 3.47(dd,J=22.4,1.7Hz,2H), 1.26(td,J=7.1,1.6Hz,6H).

[0101] 13 C NMR (101MHz, Chloroform-d) δ 164.68, 156.37, 149.66, 136.54, 123.35, 122.95, 61.43, 61.36, 51.38, 35.93 (d, J = 134.5Hz), 15.34, 15.27.

[0102] HRMS(DART-TOF)calculated for C 12 H 19 NO5P + [M+H] + m / z 288.2598,found288.2579.

[0103] 2) Preparation of probe 3e

[0104] Under argon protection, aldehyde 1a (25 mg, 0.097 mmol) and phosphate derivative 2e (33 mg, 0.116 mmol) were added to a round-bottom flask, followed by 5 mL of anhydrous tetrahydrofuran. After the system was cooled to -20°C, 1 M t BuOK (150 μL, 0.150 mmol) was added dropwise and reacted at -20°C for 3 h. After the reaction, the product was purified by silica gel chromatography (CHCl 3 / CCl 4 = 1:1) to obtain probe 3e (15.8 mg) in a yield of 41%.

[0105] 1 H NMR(400MHz,Chloroform-d)δ9.15(d,J=2.1Hz,1H),8.22(dd,J=8.2,2.2Hz,1H),7.87(d,J=15.6Hz,1H),7.40-7.35(m,2H),7.33( d,J=8.2Hz,1H),7.09(d,J=3.7Hz,1H),7.00(d,J=16.0Hz,1H),6.95-6.87(m,3H),6.71(d,J=8.3Hz,2H),3.95(s,3H),3.00(s,6H).

[0106] 13 C NMR(101MHz,Chloroform-d)δ166.34,159.55,151.50,145.92,139.67,138.09,130.85, 130.64,129.27,128.19,126.18,125.70,123.88,121.88,117.84,112.86,52.72,40.87.

[0107] HRMS(DART-TOF)calculated for C 23 H 22 N2O2S + [M+H] + m / z 391.1480,found391.1458.

[0108] [Example 6]

[0109]

[0110] 1) Preparation of aldehyde 1b

[0111] Under nitrogen, azetidine (674 μL, 10 mmol), compound 75 (11.8 ml, 110 mmol), anhydrous potassium carbonate (1.52 g, 11 mmol), and DMSO (10 ml) were added sequentially to the reaction flask. The reaction system was incubated at 100°C under nitrogen for 4.5 h. After completion, water (200 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (50 ml x 3). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 76 (1.8 g) in a 92% yield.

[0112] Under argon protection, compound 76 (577 mg, 3.57 mmol), diethyl 2-(thienylmethyl)phosphonate (1 g, 4.28 mmol), and ultra-dry tetrahydrofuran (40 ml) were added in sequence. After the system was cooled to 0°C, 1M t BuOK (5 mL, 5 mmol) was added dropwise and reacted at 0°C for 3 h. After completion of the reaction, the reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to obtain compound 77 (311.5 mg) in a yield of 36%.

[0113] Under argon protection, compound 77 (260 mg, 1.077 mmol) and anhydrous tetrahydrofuran (11 mL) were added in sequence, the system was cooled to -78 °C, and then 1.6 M n BuLi (1 mL, 1.61 mmol) was added dropwise and reacted at -78°C for 1 h. After 1 h, anhydrous DMF (167 μL, 2.15 mmol) was added dropwise and allowed to react at -78°C for another 2 h. After completion of the reaction, water (50 mL) was added to quench the reaction. The product was extracted with EA (30 mL x 3). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford aldehyde 1b (273 mg, 94%) in a 94% yield.

[0114] 1H NMR(400MHz,Chloroform-d)δ9.81(s,1H),7.62(d,J=4.0Hz,1H),7.39-7.34(m,2H) ,7.12-6.96(m,3H),6.43-6.38(m,2H),3.94(t,J=7.2Hz,4H),2.40(p,J=7.3Hz,2H).

[0115] 13 C NMR (101MHz, Chloroform-d) δ182.51,154.28,152.36,140.42,137.70,133.84,128.31,125.23,124.86,116.50,111.29,52.21,16.89.

[0116] HRMS(DART-TOF)calculated for C 16 H 16 NOS + [M+H] + m / z 270.3700, found 270.3697.

[0117] 2) Preparation of probe 3f

[0118] Under argon protection, aldehyde 1b (20 mg, 0.074 mmol), phosphate derivative 2f (43 mg, 0.111 mmol), and anhydrous tetrahydrofuran (5 mL) were added, and the reaction system was cooled to -20°C. Subsequently, 2M lithium diisopropylamide (LDA) (67 μL, 0.134 mmol) was slowly added dropwise. After the addition was complete, the reaction was allowed to proceed for 3 h. After completion of the reaction, the product was purified by silica gel column chromatography to obtain compound 78 (24.7 mg) in a yield of 65%.

[0119] Compound 78 (24.7 mg, 0.0488 mmol), DCM (0.4 mL), and norbornadiene (50 μL, 0.488 mmol) were added to a reaction flask, sealed, and allowed to react at room temperature for 18 h. After completion, the reaction was concentrated under reduced pressure, and anhydrous tetrahydrofuran (0.4 mL) and 1 M tetrabutylammonium fluoride (TBAF) (488 μL, 0.488 mmol) were added. The reaction was allowed to react at room temperature for 3 h. After completion, water (20 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (25 mL x 3). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain probe 3f (9.5 mg) in a 52% yield.

[0120] 1H NMR(400MHz,Chloroform-d)δ7.77(d,J=15.9Hz,1H),7.47(d,J=8.7Hz,1H),7 .35-7.31(m,2H),7.30(d,J=8.7Hz,1H),7.08(d,J=3.7Hz,1H),7.03-6.96(m, 2H),6.92-6.86(m,2H),6.64-6.58(m,2H),4.15(t,J=5.5Hz,2H),3.67(t,J=6 .2Hz,2H),3.38(t,J=6.6Hz,2H),3.17(t,J=5.5Hz,2H),2.09(p,J=6.5Hz,2H).

[0121] 13 C NMR(101MHz,Chloroform-d)δ160.04,156.63,148.05,145.46,139.18,130.19,130.13,128.11,12 7.94,127.49,126.37,125.87,124.64,123.17,117.71,112.99,61.20,42.67,40.90,37.91,32.02.

[0122] HRMS(DART-TOF)calculated for C 23 H 23 N3OS + [M+H] + m / z 390.5250,found390.1629.

[0123] [Example 7]

[0124]

[0125] 1) Preparation of phosphate derivative 2f-1

[0126] Compound 2f (200 mg, 0.512 mmol), DCM (1.5 mL), and norbornadiene (519 μL, 5.12 mmol) were added to the reaction flask, sealed, and reacted at room temperature for 18 h. After completion of the reaction, the solution was concentrated under reduced pressure and used directly in the next reaction without further treatment.

[0127] 2) Preparation of aldehyde 1c

[0128] Under argon protection, compound 79 (1.3 g, 5.5 mmol), azetidine (40 μL, 6.05 mmol), PD2(DBA)3 (510 mg, 0.55 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (Xantphos) (636 mg, 1.1 mmol) were added to the reaction flask in sequence. t BuONa (1.58 g, 16.5 mmol) and anhydrous 1,4-dioxane solution (14 mL) were added. The reaction system was incubated at 60°C for 2 h. Upon completion, methanol (5 mL) was added to quench the reaction. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 80 (228.6 mg) in a 20% yield.

[0129] Under argon, compound 80 (160 mg, 0.754 mmol), 5-formyl-2-thiopheneboronic acid (153 mg, 0.98 mmol), anhydrous sodium carbonate (159.8 mg, 1.5 mmol), tetrakis(triphenylphosphine)palladium (17.5 mg, 0.015 mmol), anhydrous 1,4-dioxane solution (4.7 mL), and water (3 mL) were added sequentially. After the addition, the reaction system was heated to 100°C and reacted for 12 h. After completion, the reaction was quenched with water (35 mL) and extracted with EA (25 mL x 3). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford aldehyde 1c (31.1 mg) in a 17% yield.

[0130] 1 H NMR(400MHz,Chloroform-d)δ9.82(s,1H),7.68(d,J=3.9Hz,1H),7.57-7.51(m,2H),7 .23(d,J=4.0Hz,1H),6.46-6.39(m,2H),3.95(t,J=7.3Hz,4H),2.41(p,J=7.3Hz,2H).

[0131] 13 C NMR (101MHz, Chloroform-d) δ182.49,156.10,152.59,140.26,138.01,127.39,121.74,121.66,111.19,52.07,16.75.

[0132] HRMS(DART-TOF)calculated for C 27 H 36 N5OSSi + [M+H] +m / z 506.7640,found506.7635.

[0133] 3) Prepare probe 3g

[0134] Under argon protection, aldehyde 1c (20 mg, 0.082 mmol), phosphate derivative 2f-1 (48 mg, 0.123 mmol), and anhydrous tetrahydrofuran (5 mL) were added, and the reaction system was cooled to -20°C. Subsequently, 2M LDA (73 μL, 0.147 mmol) was slowly added dropwise. After the addition was complete, the reaction was continued for 3 h. After completion of the reaction, the product was purified by silica gel column chromatography to obtain compound 81 (19.1 mg) in a yield of 48%.

[0135] Compound 81 (15 mg, 0.0314 mmol), anhydrous tetrahydrofuran (0.4 mL), and 1M TBAF (314 μL, 0.314 mmol) were added sequentially to the reaction flask at room temperature for 3 h. After completion, the reaction was quenched by the addition of water (25 mL). The mixture was extracted with ethyl acetate (15 mL x 3). The organic layers were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain 3 g (9.8 mg) of the probe in an 86% yield.

[0136] 1 H NMR (400MHz, DMSO-d6) δ7.87(d,J=16.0Hz,1H),7.83(d,J=8.8Hz,1H),7.54(d,J=8.7Hz,1H),7.50(d,J=8.2Hz,2H),7.29(d,J=1.5Hz,2H),7.02(d,J= 16.1Hz,1H),6.44(d,J=8.4Hz,2H),4.74(t,J=5.3Hz,1H),3.85(t,J=7.2H z, 4H), 3.78 (q, J = 6.2Hz, 2H), 3.03 (t, J = 6.6Hz, 2H), 2.32 (p, J = 7.2Hz, 2H).

[0137] 13 C NMR(101MHz,DMSO-d6)δ160.01,156.48,152.17,145.86,138.68,131.17,127.73,12 7.50,126.76,124.46,123.65,122.48,122.41,111.84,60.82,52.30,39.35,16.76.

[0138] HRMS(DART-TOF)calculated for C 21 H 21 N3OS+ [M+H] + m / z 364.1484, found364.1471.

[0139] [Example 8]

[0140]

[0141] 1) Preparation of aldehyde 1d

[0142] Under argon, compound 82 (1.85 g, 10 mmol), 30% methylamine solution (6.5 ml, 50 mmol), and copper powder (32 mg, 0.5 mmol) were added sequentially to a sealed tube. The system was heated to 100°C and allowed to react for 19 h. After completion, the reaction was quenched by the addition of water (70 mL). The mixture was extracted with EA (25 mL x 3). The organic layers were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 83 (1.04 g) in a 77% yield.

[0143] Under argon protection, compound 83 (440 mg, 3.25 mmol), diethyl 2-(thienylmethyl)phosphonate (989 mg, 4.22 mmol), and ultra-dry tetrahydrofuran (30 mL) were added in sequence. After the system was cooled to 0°C, 1 M t BuOK (6.5 mL, 6.5 mmol) was added dropwise and the mixture was reacted at -20°C for 3 h. After completion of the reaction, the reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to obtain compound 84 (502.9 mg) in a yield of 67%.

[0144] Under argon, compound 84 (400 mg, 1.85 mmol) and anhydrous tetrahydrofuran (11 mL) were added sequentially. The system was cooled to -78°C, followed by the slow dropwise addition of 1.6 M nBuLi (2.9 mL, 4.62 mmol). The reaction was allowed to proceed at -78°C for 1 h. After 1 h, anhydrous DMF (286 μL, 3.7 mmol) was added dropwise and the reaction was continued at -78°C for 2 h. After completion of the reaction, water (20 mL) was added to quench the reaction, followed by extraction with EA (25 mL × 3), drying over anhydrous NaSO, and concentration under reduced pressure. The residue was purified by silica gel column chromatography to afford aldehyde 1d (342 mg) in a 76% yield.

[0145] 1H NMR(400MHz,Chloroform-d)δ9.82(q,J=3.1,2.7Hz,1H),7.63(p,J=3.0,2.6Hz,1H),7.36(dq,J=8.8,3.0H z,2H),7.12-7.04(m,2H),7.03-6.95(m,1H),6.59(dq,J=8.7,3.0,2.6Hz,2H),2.89(q,J=3.1,2.6Hz,3H).

[0146] 13 C NMR (101MHz, Chloroform-d) δ182.41,154.16,149.98,137.57,133.64,128.52,125.09,124.97,116.31,112.39,30.46.

[0147] HRMS(DART-TOF)calculated for C 14 H 14 NOS + [M+H] + m / z 244.3320, found 244.3329.

[0148] 2) Prepare the probe for 3 hours

[0149] Under argon, aldehyde 1d (80 mg, 0.33 mmol), phosphate derivative 2f (193 mg, 0.495 mmol), and anhydrous tetrahydrofuran (19 mL) were added. The reaction system was cooled to -20°C, and 2M LDA (280 μL, 0.561 mmol) was slowly added dropwise. After the addition was complete, the reaction was continued for 3 h. After completion of the reaction, the product was purified by silica gel column chromatography to obtain compound 85 (81 mg) in a 51% yield.

[0150] Compound 85 (20 mg, 0.0417 mmol), DCM (0.4 mL), and norbornadiene (42 μL, 0.417 mmol) were added to a reaction flask, sealed, and allowed to react at room temperature for 18 h. After completion, the reaction was concentrated under reduced pressure, and anhydrous tetrahydrofuran (0.4 mL) and 1M TBAF (417 μL, 0.417 mmol) were added. The reaction was continued at room temperature for 3 h. After completion, water (25 mL) was added to quench the reaction, followed by extraction with EA (20 mL x 3), drying over anhydrous NaSO, and concentration under reduced pressure. The residue was purified by silica gel chromatography to obtain probe 3h (11.8 mg) in a 78% yield.

[0151] 1H NMR (400MHz, DMSO-d6) δ7.89-7.78(m,2H),7.53(d,J=8.8Hz,1H),7.32(d,J=8.3Hz,2H),7.24(d,J=3.8Hz,1H),7.12-6.96(m ,3H),6.84(d,J=16.0Hz,1H),6.52(d,J=8.2Hz,2H),3.77(q,J=5.6Hz,2H),3.02(t,J=6.6Hz,2H),2.68(s,3H),2.51(s,3H). 13 CNMR(101MHz,DMSO-d6)δ159.58,155.98,150.09,144.71,138.43,130.33,130.19,127.88,127.27,127 .01,125.90,124.04,123.80,123.59,116.22,111.70,60.36,38.89,29.52.HRMS(DART-TOF)calculated for C 21 H 21 N3OS + [M+H] + m / z 364.1484, found364.1476.

[0152] [Example 9]

[0153]

[0154] 1) Preparation of aldehyde 1f

[0155] Under argon, compound 86 (600 mg, 3.22 mmol), 5-formyl-2-thiopheneboronic acid (653 mg, 4.18 mmol), anhydrous sodium carbonate (682 mg, 6.44 mmol), tetrakis(triphenylphosphine)palladium (75 mg, 0.064 mmol), anhydrous 1,4-dioxane solution (20 mL), and water (12.8 mL) were added sequentially. The reaction system was heated to 100°C and allowed to react for 12 h. After completion, water (45 mL) was added to quench the reaction, and the mixture was extracted with EA (25 mL x 3). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford aldehyde 1f (96 mg) in a 15% yield.

[0156] 1H NMR (400MHz, Chloroform-d) δ9.82(s,1H),7.68(d,J=4.0Hz,1H),7.55-7.49(m,2H),7.23(d,J=4.0Hz,1H),6.65-6.59(m,2H),2.89(s,3H). 13 C NMR(101MHz,Chloroform-d)δ182.50,156.02,150.40,140.23,138.01,127.73,121.96,121.61,112.40,30.41.HRMS(DART-TOF)calculated for C 12 H 12 NOS + [M+H] + m / z 218.2940, found 218.2951.

[0157] 2) Probe 3i

[0158] Under argon protection, aldehyde 1f (25 mg, 0.115 mmol), phosphate derivative 2f-1 (67 mg, 0.173 mmol), and anhydrous tetrahydrofuran (10 mL) were added, and the reaction system was cooled to -20°C. Subsequently, 2M LDA (103 μL, 0.207 mmol) was slowly added dropwise. After the addition was complete, the reaction was allowed to proceed for 3 h. After completion of the reaction, the product was purified by silica gel chromatography to obtain compound 87 (32.1 mg) in a yield of 65%.

[0159] Compound 87 (20 mg, 0.0443 mmol), anhydrous tetrahydrofuran (0.4 mL), and 1M TBAF (443 μL, 0.443 mmol) were added sequentially to a reaction flask. The reaction was allowed to proceed at room temperature for 3 h. After completion, the reaction was quenched by the addition of water (25 mL). The mixture was extracted with EA (20 mL × 3), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to afford probe 3i (13.3 mg) in an 89% yield.

[0160] 1H NMR (400MHz, DMSO-d6) δ7.87-7.78(m,2H),7.52(d,J=8.7Hz,1H),7.44-7.39(m,2H),7.23(dd,J=18.8,3.8Hz,2H),6.98(d,J=16.1Hz,1H), 6.58-6.51(m,2H),6.01(q,J=5.0Hz,1H),4.73(t,J=5.2Hz,1H),3.77(td,J=6.6,5.2Hz,2H),3.01(t,J=6.6Hz,2H),2.69(d,J=4.9Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ159.49,156.08,150.11,145.92,137.67,130.75,127.26,127.14,126. 55,123.96,122.88,121.27,120.76,111.84,60.37,38.86,29.53.HRMS(DART-TOF)calculated for C 19 H 19 N3OS + [M+H] + m / z 338.1327, found338.1318.

[0161] [Example 10]

[0162]

[0163] 1) Preparation of aldehyde 1g

[0164] Under argon, compound 88 (5 g, 22.5 mmol), 60% NaH (894 mg, 90 mmol), and DMF (83 mL) were added sequentially. The reaction was allowed to react at room temperature for 1 h. After 1 h, iodomethane (7.6 mL, 122 mmol) was slowly added dropwise. After the addition was complete, the reaction system was returned to room temperature and allowed to react for 3 h. After completion, water (400 mL) was added to quench the reaction, followed by extraction with EA (50 mL x 3), drying over anhydrous Na2SO4, and concentration under reduced pressure. The residue was purified by silica gel column chromatography to afford compound 89 (1.77 g) in a 32% yield.

[0165] Under argon, compound 89 (500 mg, 2 mmol), 5-formyl-2-thiopheneboronic acid (405 mg, 2.6 mmol), anhydrous sodium carbonate (424 mg, 4 mmol), tetrakis(triphenylphosphine)palladium (47 mg, 0.04 mmol), anhydrous 1,4-dioxane solution (13 mL), and water (8 mL) were added sequentially. The reaction system was heated to 100°C and allowed to react for 12 h. After completion, water (90 mL) was added to quench the reaction, and the mixture was extracted with EA (25 mL x 3). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 1g (71 mg) in a 24% yield.

[0166] 1 H NMR(400MHz,Chloroform-d)δ9.88(d,J=3.6Hz,1H),8.01(d,J=3.8Hz,1H),7.75(q,J=3.7Hz,2H),7.66(q, J=2.8,1.7Hz,2H),7.45(d,J=3.9Hz,1H),7.18(dd,J=9.2,2.7Hz,1H),6.89(s,1H),3.10(d,J=3.6Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ182.65,137.77,129.43,127.02,125.48,124.34,123.12,116.87,105.69,40.62.HRMS(DART-TOF)calculated for C 17 H 16 NOS + [M+H] + m / z 282.3810, found 282.3808.

[0167] 2) Preparation of probe 3j

[0168] Under argon protection, aldehyde 1g (20 mg, 0.071 mmol), phosphate derivative 2f (42 mg, 0.107 mmol), and anhydrous tetrahydrofuran (5 mL) were added, and the reaction system was cooled to -20°C. Subsequently, 2M LDA (64 μL, 0.128 mmol) was slowly added dropwise. After the addition was complete, the reaction was allowed to proceed for 3 h. After completion of the reaction, water (40 mL) was added to quench the reaction, and the mixture was extracted with EA (20 mL × 3). The mixture was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 90 (25 mg) in a yield of 69%.

[0169] Compound 90 (20 mg, 0.0386 mmol), DCM (0.4 mL), and norbornadiene (40 μL, 0.386 mmol) were added to a reaction flask, sealed, and allowed to react at room temperature for 20 h. After completion, the reaction was concentrated under reduced pressure, and anhydrous tetrahydrofuran (0.4 mL) and 1M TBAF (386 μL, 0.386 mmol) were added. The reaction was allowed to react at room temperature for 3 h. After completion, water (25 mL) was added to quench the reaction, followed by extraction with EA (15 mL x 3). The organic layers were combined, dried over anhydrous NaSO, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain probe 3j (15.5 mg) in an 85% yield.

[0170] 1 H NMR(400MHz,Chloroform-d)δ8.02(s,1H),7.90(s,1H),7.88(d,J=8.7Hz,1H),7.78(s,1H),7.70(d,J=1.3Hz,2H) ,7.58(d,J=8.8Hz,1H),7.54(d,J=3.8Hz,1H),6.94(d,J=2.5Hz,1H),3.79(t,J=6.6Hz,2H),3.03(d,J=7.7Hz,8H). 13 C NMR(101MHz,DMSO-d6)δ159.70,155.96,148.79,145.04,139.50,134.39,1310.72,128.96,127.45,127.08,126.79,126 .51,126.01,124.23,123.85,123.79,123.71,123.67,116.91,105.51,60.33,40.19,38.89.HRMS(DART-TOF)calculated for C 24 H 23 N3OS + [M+H] + m / z 402.1640, found 402.1629.

[0171] [Example 11]

[0172]

[0173] 1) Preparation of probe 3e-F

[0174] Under argon, probe 3e (24 mg, 0.0615 mmol), LiOH (4 mg, 0.154 mmol), anhydrous tetrahydrofuran (3 mL), water (3 mL), and methanol (50 μL) were added sequentially. The reaction system was heated to 60°C for 1.5 h. After completion, saturated ammonium chloride solution (40 mL) was added to quench the reaction, followed by extraction with EA (20 mL × 3), drying over anhydrous Na2SO4, filtration, and concentration under reduced pressure. The residue was purified by silica gel column chromatography to afford compound 94 (19.6 mg) in an 85% yield.

[0175] Under argon, compound 94 (19.6 mg, 0.052 mmol), 3-butyn-1-amine hydrochloride (16 mg, 0.15 mmol), EDCI (20 mg, 0.1 mmol), DMAP (1 mg, 0.005 mmol), anhydrous DCM (5 mL), and DIPEA (36 μL, 0.2 mmol) were added sequentially. The reaction was allowed to proceed at room temperature for 18 h. After completion, the reaction was quenched by the addition of water (35 mL). The mixture was extracted with EA (25 mL x 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to afford compound 95 (6.3 mg) in a 31% yield.

[0176] Under argon, compound 95 (10 mg, 0.0234 mmol), compound 93 (6.2 mg, 0.0351 mmol), anhydrous copper sulfate (5.8 mg, 0.0234 mmol), sodium ascorbate (9.2 mg, 0.0468 mmol), THPTA (10 mg, 0.0234 mmol), DMF (0.8 mL), and water (0.2 mL) were added sequentially to a reaction flask and allowed to react at room temperature for 3 h. After completion, the reaction was quenched by the addition of water (40 mL), extracted with EA (25 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain probe 3e-F (12.1 mg) in an 88% yield.

[0177] 1H NMR (400MHz, DMSO-d6) δ8.95(d,J=2.3Hz,1H),8.74(t,J=5.6Hz,1H),8.13(dd,J=8.2,2.3Hz,1H),7.90(t,J=7.8Hz, 2H),7.59(d,J=8.2Hz,1H),7.42(d,J=8.5Hz,2H),7.28(d,J=3.8Hz,1H),7.17(d,J=16.1Hz,1H),7.07(d,J=3.8Hz,1 H),6.96(d,J=15.7Hz,1H),6.88(d,J=16.0Hz,1H),6.71(d,J=8.6Hz,2H),4.57-4.51(m,1H),4.48(t,J=5.3Hz,2H), 4.44-4.38(m,1H),3.79(t,J=5.3Hz,2H),3.65-3.60(m,1H),3.56-3.48(m,7H),2.94(s,6H),2.91(t,J=7.5Hz,2H).

[0178] 13 C NMR (101MHz, DMSO-d6) δ164.58,156.82,150.16,148.56,144.59,144.14,138.77,135.55,130.68,129.75,127.72,127.64,127.26,126. 25,125.56,124.18,122.72,121.54,117.03,112.22,83.00(d,J=165.7Hz),69.62,69.53,69.26(d,J=92Hz),49.24,40.15,39.31,25.37.

[0179] 19 F NMR (377 MHz, DMSO-d6) δ 20.07.

[0180] HRMS(DART-TOF)calculated for C 32 H 38 FN6O3S + [M+H] + m / z 605.7534, found605.7541.

[0181] [Example 12]

[0182]

[0183] 1) Preparation of 2g of phosphate derivative

[0184] Under argon, compound 91 (5.5 mL, 34.13 mmol), 4-cyano-1-butyne (270 mg, 3.41 mmol), 3-mercaptopropionic acid (217 μL, 2.05 mmol), and 98% hydrazine hydrate (2.2 mL, 68.2 mmol) were added to a reaction flask in sequence and allowed to react at room temperature for 15 h. After completion, water (300 mL) and sodium nitrite (4.7 g, 68.2 mmol) were added under an ice-water bath. The pH was adjusted to 3-4 with 1 M dilute hydrochloric acid (HCl). After reacting at room temperature for 10 min, the mixture was extracted with EA (40 mL x 3), dried over anhydrous NaSO, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound 92 (456.3 mg) as a red oil in a 52.2% yield.

[0185] Compound 92 (200 mg, 0.7 mmol), DCM (1 mL), and norbornadiene (715 μL, 7 mmol) were added to the reaction flask and allowed to react at room temperature for 12 h. After completion of the reaction, the mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 2 g of the phosphate derivative as a colorless oil.

[0186] 1 H NMR(400MHz,Chloroform-d)δ7.56(dd,J=8.6,1.9Hz,1H),7.38(d,J=8.6Hz,1H),4.16-4.04(m,4H),3.58(d,J=21 .6Hz,2H),3.17(td,J=7.2,1.2Hz,2H),2.71(td,J=7.2,2.6Hz,2H),1.96(t,J=2.7Hz,1H),1.27(t,J=7.1Hz,6H).

[0187] 13 C NMR(101MHz,Chloroform-d)δ160.21(d,J=2.9Hz),154.05(d,J=7.5Hz),127.49(d,J=3.5Hz),126.84(d,J=2.3Hz) ,82.84,69.67,62.56,62.50,34.63,34.10(d,J=135.9Hz),18.34,16.32(d,J=6.2Hz).HRMS(DART-TOF)calculated for C 13 H 20 N2O3P + [M+H] + m / z 283.2878, found 283.2889.

[0188] 2) Preparation of compound 3k

[0189] Under argon protection, aldehyde 1b (40 mg, 0.14 8 mmol), phosphate derivative 2 g (63 mg, 0.222 mmol), and anhydrous tetrahydrofuran (10 mL) were added in sequence, and the reaction system was cooled to -20 ° C, and then 1M t BuOK (300 μL, 0.296 mmol) was added dropwise and reacted for 3 h. After the reaction was completed, the product was purified by silica gel chromatography to obtain compound 3k (48.8 mg) in a yield of 82%.

[0190] 1 H NMR (400MHz, Chloroform-d) δ7.77(d,J=16.0Hz,1H),7.45(d,J=8.7Hz,1H),7.35(d,J=8.7Hz,3H),7.07(d,J=3.7Hz,1H),7.05-6.96(m,2H),6.93-6. 86(m,2H),6.46-6.34(m,2H),3.92(t,J=7.3Hz,4H),3.18(t,J=7.2Hz,2H) ,2.74(td,J=7.2,2.6Hz,2H),2.38(p,J=7.2Hz,2H),1.98(t,J=2.6Hz,1H).

[0191] 13 C NMR(101MHz,Chloroform-d)δ159.31,156.51,151.81,145.25,139.14,130.20,129.82,127.59,127.5 3,126.66,125.78,125.64,123.99,123.34,117.49,111.31,83.07,69.60,52.24,34.77,18.43,16.84.

[0192] HRMS(DART-TOF)calculated for C 25 H 24 N3S + [M+H] + m / z 398.5480, found398.5485.

[0193] 3) Preparation of probe 3f-F

[0194] Under argon, compound 3k (15 mg, 0.0378 mmol), compound 93 (10 mg, 0.0567 mmol), anhydrous copper sulfate (9.4 mg, 0.0378 mmol), sodium ascorbate (15 mg, 0.0756 mmol), THPTA (16.5 mg, 0.0378 mmol), DMF (0.8 mL), and water (0.2 mL) were added sequentially to a reaction flask and allowed to react at room temperature for 3 h. After completion, the reaction was quenched with water (50 mL), extracted with EA (20 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain probe 3f-F (17.5 mg) in an 81% yield.

[0195] 1 H NMR(400MHz,Chloroform-d)δ7.74(dd,J=16.0,2.1Hz,1H),7.47(d,J=2.1Hz,1H),7.40(dd,J=8.8,2.1Hz,1H),7.37-7.32(m,2H ),7.25(d,J=6.0Hz,1H),7.06(t,J=2.9Hz,1H),7.02(dd,J=7.5,2.1Hz,1H),6.98(dd,J=7.7,2.1Hz,1H),6.92-6.85(m,2H),6.41 (dd,J=8.6,2.2Hz,2H),4.60(dq,J=4.0,2.1Hz,1H),4.48(tt,J=5.4,2.0Hz,3H),3.92(td,J=7.2,2.1Hz,4H),3.83(td,J=5.1,2. 1Hz,2H),3.69(dtd,J=29.7,4.1,2.2Hz,2H),3.64-3.55(m,4H),3.38(t,J=7.4Hz,2H),3.28(d,J=7.6Hz,2H),2.44-2.33(m,2H).

[0196] 13 C NMR(101MHz,Chloroform-d)δ160.25,156.26,151.80,146.47,145.25,139.11,130.21,129.80,127.53,127.49,126.80,125.78,125.64,12 4.08,123.35,122.54,117.48,111.32,83.10(d,J=169.0Hz),70.67,7 0.54,70.42(d,J=19.6Hz),69.59,52.25,50.12,35.33,25.04,16.84.

[0197] 19 F NMR(377MHz,Chloroform-d)δ18.69.

[0198] HRMS(DART-TOF)calculated for C 31 H 36 FN6O2S + [M+H] + m / z 575.7274, found575.7280.

[0199] [Example 13]

[0200]

[0201] 1) Preparation of compound 31

[0202] Under argon protection, aldehyde 1d (27 mg, 0.111 mmol), phosphate derivative 2 g (48 mg, 0.166 mmol), and anhydrous tetrahydrofuran (6 mL) were added to the reaction flask in sequence. The reaction system was cooled to -20 °C, and then 1 M t BuOK (222 μL, 0.222 mmol) was added dropwise and reacted for 3 h. After the reaction was completed, the product was purified by silica gel chromatography to obtain compound 31 (20.7 mg) in a yield of 57%.

[0203] 1 H NMR (400MHz, Chloroform-d) δ7.78(d,J=15.9Hz,1H),7.45(d,J=8.7Hz,1H),7.34(dd,J=8.5,4.4Hz,3H),7.07(d,J=3.8Hz,1H),7.03(d,J=15.5Hz,1H),6. 94(d,J=24.7Hz,2H),6.88(d,J=11.0Hz,1H),6.59(d,J=8.3Hz,2H),3.18(t,J =7.2Hz,2H),2.87(s,3H),2.74(td,J=7.3,2.6Hz,2H),1.98(t,J=2.7Hz,1H). 13C NMR(101MHz,Chloroform-d)δ159.44,156.65,149.45,145.38,139.25,130.28,129.95,128.01,12 7.72,126.78,126.05,125.75,124.12,123.47,117.50,112.59,83.21,69.73,34.91,30.71,18.56.

[0204] HRMS(DART-TOF)calculated for C 23 H 22 N3S + [M+H] + m / z 372.5100, found382.5115.

[0205] 2) Preparation of probe 3h-F

[0206] Under argon, compound 31 (18 mg, 0.0485 mmol), compound 93 (13 mg, 0.0727 mmol), anhydrous copper sulfate (12 mg, 0.0485 mmol), sodium ascorbate (19.2 mg, 0.097 mmol), THPTA (21 mg, 0.0485 mmol), DMF (0.8 mL), and water (0.2 mL) were added sequentially to a reaction flask and allowed to react at room temperature for 3 h. After completion, the reaction was quenched by the addition of water (45 mL), extracted with EA (25 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain probe 3h-F (23 mg) in an 89% yield.

[0207] 1H NMR(400MHz,Chloroform-d)δ7.74(d,J=15.9Hz,1H),7.47(s,1H),7.40(dd,J=8.7,1.1Hz,1H),7.33(d, J=8.2Hz,2H),7.26-7.23(m,1H),7.06(d,J=3.7Hz,1H),7.04-6.96(m,2H),6.91-6.85(m,2H),6.59(d,J =8.1Hz,2H),4.62-4.59(m,1H),4.48(td,J=4.5,2.4Hz,3H),3.83(t,J=5.0Hz,2H),3.76-3.71(m,1H),3 .68-3.64(m,1H),3.63-3.56(m,4H),3.38(t,J=7.4Hz,2H),3.27(t,J=7.4Hz,2H),2.87(d,J=1.2Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ160.25,156.27,149.32,146.48,145.22,139.11,130.15,129.76,127.87,127.43,126.74,125.90,125.61 ,124.04,123.41,122.52,117.35,112.45,83.10(d,J=169.2Hz),70.67,70.54,70.42(d,J=19.5Hz),69.60,50.12,35.35,30.58,25.04. 19 F NMR(377MHz,Chloroform-d)δ18.69.HRMS(DART-TOF)calculated forC 29 H 34 FN6O2S + [M+H] + m / z 549.6894, found 549.6889.

[0208] [Example 14]

[0209] In this embodiment, the two-step method is used to prepare 18 F probe, the specific synthesis route is:

[0210]

[0211] 1)[ 18 Radiochemical Synthesis of F]pAz

[0212] Will 18 F- Load onto the activated QMA ion exchange column (QMA column was activated with 10 mL EtOH and 10 mL deionized H2O and dried with 20 mL air) and 222 / K2CO3 solution (33 mg K2CO3 and 390 mg K 222 Dissolve in 21mL MeCN, 6mLH2O) and rinse into 4mL reaction bottle. The content of reaction bottle was measured by activity meter. 18 F]KF / K 222 The dose of KF / K 222 The solution was heated to 110°C and dried under a nitrogen stream. Ultra-dry MeCN (1 mL) was then added and dried under a nitrogen stream at 110°C twice. pAz (6-8 mg) was dissolved in 0.4 mL of ultra-dry MeCN and added to the reaction flask. The mixture was reacted at 90°C for 30 min. After the reaction system cooled to room temperature, 1 mL of H2O / MeCN (1 / 1, v / v) was added to quench the reaction. The reaction solution was then filtered through an organic microporous filter.

[0213] A 20 μL microsyringe was used to draw a mixed solution of 10 μL of the reaction filtrate and 10 μL of the standard reference substance and co-injected it through Radio-HPLC. Finally, the 254 nm channel and 18 F radioactive channel two curves. The radioactive curve is integrated, and the percentage of the corresponding peak area to the total peak area is the synthetic [ 18 F]pAz-responsive RCC. 18 The radiochemical conversion (RCC) of [F]pAz was 97%.

[0214] The reaction filtrate after filtration was prepared by Radio-HPLC, and the [ 18 F]pAz, the collected preparation solution was heated to 96 ° C, 35% of the total volume was blown off under nitrogen flow, and it was directly adsorbed on the Sep-Pak C18 column without dilution, and finally eluted with 0.4 mL of DMF solution [ 18 F]pAz was used for the next reaction.

[0215] 2)[ 18 Radiochemical synthesis of [F] probes

[0216] The probe (2 mg, 0.0045 mmol), sodium ascorbate (1.2 mg, 0.006 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (2.7 mg, 0.0062 mmol) and CuSO4 (0.5 mg, 0.0031 mmol) were added to a mixture containing [ 18F]pAz reaction bottle, add 0.1mL pure water and stir for 30 seconds, then add the 18 A DMF solution of [F]pAz was reacted at 25°C for 15 min. After completion of the reaction, 1.5 mL of H2O / MeCN (1 / 1, v / v) solution was added to quench the reaction, and the reaction filtrate was filtered through an organic microporous filter membrane to obtain the reaction filtrate.

[0217] A 20 μL microsyringe was used to draw a mixed solution of 10 μL of the reaction filtrate and 10 μL of the standard reference substance and co-injected it through Radio-HPLC. Finally, the 254 nm channel and 18 F radioactive channel two curves. The radioactive curve is integrated, and the percentage of the corresponding peak area to the total peak area is the synthetic [ 18 F] The radiochemical conversion rate (RCC) of the probe was greater than 95%.

[0218] By preparing the reaction filtrate, collecting the 18 F probe, use 50mLH2O to dilute the product preparation solution, and adsorb the product dilution solution on Sep-Pak C18 column, and elute with ethanol to obtain 18 F probe, measured by activity meter 18 The F probe dose is calculated 18 The RCY of F probe. Finally, take the purified 18 F probe standard was co-injected by Radio-HPLC to characterize 18 F probe.

[0219] 2. Probe performance test

[0220] [Example 15]

[0221] In this example, the optical properties of probes 3a to 3j, 3e-F, 3f-F, and 3h-F were determined.

[0222] Specifically, the probe was dissolved in DMSO to prepare a 20 mM concentration stock solution, and then diluted with PBS to a 1 μM concentration for testing. The UV maximum absorption wavelength of the probe was detected using a UV spectrophotometer. The probe was diluted with 200 μL of a 95%-5% (v / v) ethanol-water mixed solution to a concentration of 1 μM for testing. The UV maximum absorption wavelength of the probe was used as the excitation wavelength λ ex , and the emission wavelength λ of the probe was detected by fluorescence instrument em .

[0223] The maximum emission wavelength of each probe after binding to Aβ protein was determined by combining the probe (1 μM) with Aβ protein (9 μM) in PBS (200 μL). After 3 minutes, the maximum emission wavelength of the probe-protein binding was measured using a fluorimeter and recorded. An equal volume of DMSO was used to replace the probe as a blank.

[0224] The experimental results, shown in Table 1, show that the commercial probe ThT exhibits a short Stokes shift, while probes 3a through 3j all exhibit longer Stokes shifts, indicating that these probes can achieve higher signal-to-noise ratios and lower spectral overlap during imaging. In particular, probe 3e exhibits a Stokes shift of 266 nm. Furthermore, upon binding to Aβ protein, the maximum emission wavelength exhibits a significant blue shift, demonstrating that probes 3a through 3j exhibit typical ICT-type properties. Their high sensitivity to environmental polarity results in significant changes in fluorescence properties, such as the maximum emission wavelength, when the polarity of the environment changes.

[0225] Table 1: Optical properties of the probes

[0226]

[0227] [Example 16]

[0228] This example tested the specificity of the probe binding to Aβ protein.

[0229] The probe (1 μM) was combined with Aβ protein and HSA protein (9 μM) in PBS solution, respectively. After 3 minutes, the fluorescence intensity at the maximum emission wavelength of the probe-protein binding was detected and recorded using a fluorescence meter. The fluorescence intensity was compared with the probe's own fluorescence intensity at this wavelength to obtain the multiple ratio of the fluorescence intensity enhancement after the probe-protein binding.

[0230] The experimental results are as follows Figure 3 As shown, the fluorescence intensity enhancement ratio after the probes bind to the Aβ protein is significantly greater than the fluorescence intensity enhancement ratio after these probes bind to the HSA protein, especially probes 3b, 3d, 3e, 3f, 3h, and 3j have better Aβ protein selectivity.

[0231] Furthermore, the selectivity of cold fluorine probes 3e-F, 3f-F, and 3h-F, which have similar backbone structures to probes 3e, 3f, and 3h, for Aβ protein was tested. The electron donors of cold fluorine probes 3e-F, 3f-F, and 3h-F are the same as those of probes 3e, 3f, and 3h, respectively, and the electron acceptors have F atoms. Figure 4As shown in the figure, the fluorescence intensity enhancement ratio of the three cold fluorine probes after binding to Aβ protein is significantly greater than that after binding to HSA protein, indicating that the three cold fluorine probes 3e-F, 3f-F and 3h-F have better selectivity for Aβ protein.

[0232] [Example 17]

[0233] This example studies the fluorescence imaging of cold fluorine probes 3e-F and 3h-F.

[0234] Cold fluorine probe 3e-F or 3h-F was administered via the tail vein of WT and AD mice (8 months old) at a concentration of 1 mg / kg (solvent: 20% DMSO, 60% propylene glycol, 20% PBS), and imaging was performed at different time points after administration.

[0235] Brain tissue from AD and WT mice was harvested by perfusion with saline and embedded in OCT gel, with sections cut to 10 μm thick. The OCT gel was washed off the edges of the sections with PBS. After antigen retrieval, the sections were blocked in a blocking buffer containing 10% NGS and 0.1% Triton X-100 for 1 hour at room temperature. The sections were then incubated with primary antibody diluted 1:100 in a primary antibody diluent (10% NGS; PBS) and incubated overnight at 4°C. The sections were washed three times with PBS for 10 minutes each. The sections were then incubated with secondary antibody diluted 1:200 in a secondary antibody diluent (3% NGS; PBS) for 1 hour at room temperature in the dark. After removing the secondary antibody with PBS, the sections were incubated with cold fluoride control molecules and ThT (10 μM) for 1 hour at room temperature in the dark. The cold fluoride probe was then washed with PBS, and DAPI was incubated for 10 minutes at room temperature. Excess dye was then washed with PBS, the sections were mounted, air-dried, and observed under a confocal microscope.

[0236] The experimental results are as follows Figure 5 As shown in Figure 2, cold fluorine probes 3e-F and 3h-F have excellent blood-brain barrier penetration ability and exhibit strong fluorescence effects in the brain of AD mice. Figure 6 As shown, both cold-fluorine probes 3e-F and 3h-F can specifically stain Aβ plaques in brain slices and have excellent colocalization effects with Aβ antibody staining.

[0237] [Example 18]

[0238] Obtained by Example 14 18 F-labeled probe 3e- 18 F and 3h- 18 After F, dynamic PET imaging was performed on AD mice and WT mice to investigate the effect of the thermofluorinated probe 3e- 18 F and 3h- 18 Distribution and metabolism of F in the brains of AD mice and WT mice.

[0239] The probe 3e- 18 F and 3h- 18 F(3e- 18 F and 3h- 18 About 100 μCi of F (dissolved in ethanol) was diluted to 110 μL of physiological saline (10% EtOH) and injected into the tail vein of AD mice and WT mice (n=3).

[0240] The mice were immediately placed in a prone position in a small animal PET scanner, and a PET scan was performed in dynamic scanning mode. All necessary PET data were reconstructed using a 3D-OSEM algorithm based on a Monte Carlo model and then processed by osirix software. Dynamic PET / CT fusion images of the brain were generated. Figure 7 As shown, the thermal fluorine probe 3e- 18 F and 3h- 18 F can be effectively applied to PET imaging of AD mice.

[0241] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A β-amyloid protein probe, characterized in that The β-amyloid probe is selected from the following compounds:

2. A β-amyloid protein probe according to claim 1, characterized in that The β-amyloid probe is selected from the following compounds:

3. A β-amyloid protein probe according to claim 1 or 2, characterized in that, The fluorine substitution of the amyloid β probe is 18 F.

4. The method for preparing a β-amyloid protein probe according to claim 1 or 2, characterized in that: The following steps are involved: Under inert gas protection, the aldehyde represented by Formula II, the phosphate derivative represented by Formula III, and the solvent are uniformly mixed, the reaction system is cooled to below -10°C, a base is added dropwise, and the mixture is reacted at below -10°C to obtain a β-amyloid protein probe; or Under an inert atmosphere, the aldehyde represented by formula II, the phosphate derivative represented by formula III and the solvent are uniformly mixed, the reaction system is cooled to below -10°C, a base is added dropwise, and the reaction is carried out at below -10°C to obtain an intermediate, the intermediate is dehydroxylated, the intermediate is subjected to a bioorthogonal reaction with a dienophile and then dehydroxylated, or the intermediate is reacted with a dienophile. After the reaction, a β-amyloid protein probe is obtained; Formula II: Formula III: Wherein, the values of R1 in Formula II, R3 in Formula III, and n match those of a β-amyloid protein probe according to any one of claims 1 to 2.

5. The method for preparing a β-amyloid protein probe according to claim 4, characterized in that: The base is at least one of sodium carbonate, potassium carbonate, sodium hydroxide, sodium tert-butoxide, potassium tert-butoxide, and lithium diisopropylamide.

6. The method for preparing a β-amyloid protein probe according to claim 4, characterized in that: The temperature at which the base is added dropwise to the reaction system and the reaction temperature after the base is added dropwise are -35°C to -10°C. 7 . Use of the amyloid β protein probe according to any one of claims 1 to 3 in the preparation of an imaging molecular probe for labeling Aβ protein.

Images