Beta amyloid protein probe as well as preparation method and application thereof

Abstract

The invention discloses a beta amyloid protein probe and a preparation method and application thereof.A skeleton of the beta amyloid protein probe can effectively improve the ICT effect of the probe, so that the probe has larger emission wavelength and has larger fluorescence intensity enhancement multiple before and after being combined with A beta protein, and meanwhile, the fluorescence intensity enhancement multiple is larger after being combined with A beta protein. A nitrogen-containing six-membered aromatic ring is used as an electron acceptor, so that the probe has better electron-withdrawing capability, large steric hindrance of the electron acceptor can enhance limitation on molecular torsion after the probe enters a hydrophobic cavity of A beta protein, a more efficient ICT effect is triggered by the probe and a thiophene-containing pi bridge, and meanwhile, the probe can be used for detecting the A beta protein. The nitrogen-containing six-membered aromatic ring can be well combined with a beta-shaped sheet structure of the A beta protein, and the combining capacity of the probe and the A beta protein and the selectivity of the A beta protein are effectively improved, so that the probe can be applied to marking of the A beta protein and preparation of imaging agents for marking the A beta 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. As the population ages, the number of people with AD will continue to increase, and the necessity of early diagnosis and timely treatment of AD is becoming increasingly prominent.

[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-folded cavity formed inside the Aβ protein is a hydrophobic non-polar environment, while the outside of the Aβ protein is hydrophilic and polar. The Aβ probe with intramolecular charge transfer (ICT) effect is highly sensitive to environmental polarity. When entering the hydrophobic cavity of the Aβ protein from the physiological water environment, under the action of excitation light, the fluorescence properties such as the maximum emission wavelength of the Aβ probe will change significantly, and the phenomenon of fluorescence "on" response will occur. However, the ICT effect of the skeleton structure of the existing Aβ probe is poor, resulting in its optical properties and the fluorescence intensity enhancement factor after binding to the Aβ protein needing to be further improved. 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 transfer when the environmental polarity changes, and at the same time, by utilizing a multi-conjugated structure, as well as 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, acyl, trifluoromethyl, n=0 or 1.

[0010] In this technical solution, the skeleton of the probe includes a rotatable thiophene group, an electron donor R1 group connected to one side of the thiophene group, and an electron acceptor R2 group connected to the other side of the thiophene. This skeleton structure utilizes the rotatable thiophene group to exhibit the characteristics of rotation-coplanarity when the probe is in solvents of different polarities, that is, in a hydrophilic physiological environment, the rotation of the molecule makes the fluorescence of the probe weaker, and after combining with the hydrophobic cavity of the Aβ protein, it can form a coplanar structure that is conducive to electron transmission, thereby enhancing the ICT effect of the probe and effectively increasing the enhancement multiple of the fluorescence intensity after the probe binds 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 mono- or di-substituted. The substituent can be a C1-C3 alkyl, hydroxymethyl, hydroxyethyl, acyl, trifluoromethyl, In some embodiments, the substituent may be a C1-C3 straight-chain alkyl group or a branched-chain alkyl group.

[0012] In this technical solution, by increasing the electron-donating ability of the electron donors on both sides of the thiophene and the electron-withdrawing ability of the electron acceptors, and by utilizing the multi-conjugated structure to enhance the electron transmission ability, the energy level gap between the HOMO-LUMO energy levels can be reduced, so that the probe has a larger emission wavelength. In some preferred embodiments, the maximum emission wavelength of the probe reaches 720nm. At the same time, the electron acceptor using the nitrogen-containing six-membered aromatic ring not only has a better electron-withdrawing ability, but also triggers a more efficient ICT effect with the π bridge containing thiophene after entering the hydrophobic cavity of the Aβ protein; moreover, this type of nitrogen-containing six-membered aromatic ring can be well combined with the β-sheet structure of the Aβ protein, effectively improving the binding ability of the probe with the Aβ protein and the selectivity of the Aβ protein.

[0013] In the present technical solution, it was found through experiments 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] Further, the R2 is selected from any one of the following groups:

[0016]

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

[0018]

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

[0020]

[0021]

[0022] Furthermore, the fluorine substitution of the amyloid β 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] In an inert atmosphere, after 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 a β-amyloid protein probe; or

[0026] In 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 to obtain a bioorthogonal reaction 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, 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 the technical scheme, for some probes, such as probes 3a and 3b, they can be directly obtained by reacting the aldehyde shown in formula II with the phosphate derivative shown in formula III. In some embodiments, the aldehyde shown in formula II, the phosphate derivative shown in formula III, and the solvent are uniformly mixed under an inert atmosphere, and then a base is added dropwise to the reaction system at low temperature, and then reacted at low temperature for 2 to 3 hours to obtain the β-amyloid protein probe.

[0030] In the technical scheme, for a part of the probes, such as probe 3g, the aldehyde shown in formula II can be used to react with the phosphate derivative shown in formula III to first obtain an intermediate, and some groups of the intermediate, such as the hydroxyethyl group, are protected by a hydroxyl protecting group. Therefore, after obtaining the intermediate, the intermediate is dehydroxylated to obtain the 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 the 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 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 alkali to the reaction system and the reaction temperature after adding the alkali 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 an application 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 skeleton 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 larger emission wavelength and a larger fluorescence intensity enhancement factor before and after binding with 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 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. In addition, this type of nitrogen-containing six-membered aromatic ring can be well combined with the β-sheet structure of the Aβ protein, effectively improving the binding ability of the probe with 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 easily available raw materials, which is conducive to large-scale 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 For the specific embodiment of the present invention 18 F labeled probe 3e- 18 HPLC spectrum of F, where (A) 3e- 18 F HPLC radioactive peak at 15min reaction time, (B) 3e- 18HPLC chromatogram at 254 nm after 15 min of reaction of 3e-F and 3e- 18 F co-injection radioactive peaks, (D) 3e-F and 3e- 18 F 254 nm chromatogram of co-injection HPLC;

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

[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, 3h-F and 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 in a specific embodiment of the present invention are shown;

[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- 18 F and 3h- 18 F is the PET imaging results of AD mice. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the present invention more clear, 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 in this article can be directly connected or indirectly connected via other groups unless otherwise specified.

[0050] All raw materials of the present invention have no particular limitation on 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 meanings according to the expressions.

[0053] All raw materials of the present invention, their brands and abbreviations are conventional brands and abbreviations in the field, and each brand and abbreviation is clear and definite in the field of its related use. Those skilled in the art can purchase them from the market or prepare them by conventional methods according to the brands, abbreviations and corresponding uses.

[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) with 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, found 348.1520.

[0061] [Example 2]

[0062]

[0063] 1) Preparation of phosphate derivative 2b

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

[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 mixture was purified by silica gel chromatography (PE:EA=4:1) to obtain probe 3b (16.5 mg) with 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, found 348.1522.

[0073] [Example 3]

[0074]

[0075] 1) Preparation of phosphate derivative 2c

[0076] Under argon protection, 2-trifluoromethyl-5-chloromethylpyridine (400 mg, 2.04 mmol) and triethyl phosphite (7 ml, 40.8 mmol) were added to a round-bottom flask, and the mixture was reacted at 130°C overnight. After the reaction was completed, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography (PE:EA=2:1) ​​to obtain a colorless oily liquid phosphate derivative 2c (462 mg) with 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) with 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, found 401.1292.

[0084] [Example 4]

[0085]

[0086] 1) Preparation of phosphate derivative 2d

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

[0088] Under argon protection, add NaH (60%) (316 mg, 7.9 mmol), 10 mL toluene, diethyl phosphite (2 mL, 16 mmol) to a reaction bottle in sequence, and then slowly drop the toluene solution containing the crude product of the previous step. After the addition is complete, move the reaction bottle to 80 ° C for 0.5 h. After the reaction is completed, add water (40 mL) to quench the reaction, extract with ethyl acetate (30 mL × 3), wash with saturated brine (40 mL), combine the organic phases, dry the organic phases with anhydrous sodium sulfate, filter and concentrate under reduced pressure, and purify the residue by silica gel column chromatography (PE: EA = 2: 1) to obtain a colorless oily liquid phosphate derivative 2d (1.53 g) with a yield of 85%.

[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 mixture was purified by silica gel chromatography (DCM / MeOH=20:1) to obtain probe 3d (33.2 mg) with 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, found 333.1420.

[0096] [Example 5]

[0097]

[0098] 1) Preparation of phosphate derivative 2e

[0099] Under argon protection, 6-hydroxymethylnicotinate (700 mg, 4.18 mmol) and toluene (40 mL) were added to a round-bottom flask, and PBr3 (0.4 mL, 4.18 mmol) was slowly added at 0 ° C. After the addition was complete, the mixture was moved to room temperature for reaction for 12 h, and then moved to 115 ° C for reaction for 1 h. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was diluted with DCM (30 mL) and then washed with saturated sodium bicarbonate aqueous solution (30 mL) and saturated sodium chloride aqueous solution (30 mL) in turn. After concentration under reduced pressure, the residue was directly put into the next step without purification. Triethyl phosphite (14 mL, 80 mmol) was added under argon protection, and the mixture was reacted at 125 ° C for 10 h. After the reaction was completed, the mixture was concentrated under reduced pressure. After purification by silica gel column chromatography (PE: EA = 1: 1), a yellow oily liquid phosphate derivative 2e (378 mg) was obtained with a yield of 31%.

[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, found 288.2579.

[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 mixture was purified by silica gel chromatography (CHCl3 / CCl4=1:1) to obtain probe 3e (15.8 mg) with 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, found 391.1458.

[0108] [Example 6]

[0109]

[0110] 1) Preparation of aldehyde 1b

[0111] Under nitrogen protection, 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 to the reaction bottle in sequence. The reaction system was reacted at 100 ° C for 4.5 h under nitrogen protection. After the reaction was completed, water (200 mL) was added to quench the reaction, and ethyl acetate was extracted (50 ml × 3). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 76 (1.8 g) with a yield of 92%.

[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, 1 M t BuOK (5 mL, 5 mmol) was added dropwise and reacted at 0°C for 3 h. After the reaction was completed, 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) with 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 (1mL, 1.61mmol) was added dropwise and reacted at -78℃ for 1h. After 1h, anhydrous DMF (167μL, 2.15mmol) was added dropwise and reacted at -78℃ for 2h. After the addition was completed, water (50mL) was added to quench the reaction, and EA was extracted (30mL×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 aldehyde 1b (273mg, 94%) with a yield of 94%.

[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 completed, the reaction was allowed to proceed for 3 h. After the reaction was completed, the mixture was purified by silica gel column chromatography to obtain compound 78 (24.7 mg) with 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 the reaction bottle, sealed, and reacted at room temperature for 18 h. After the reaction was completed, the mixture was concentrated under reduced pressure, and anhydrous tetrahydrofuran (0.4 mL) and 1M tetrabutylammonium fluoride (TBAF) (488 μL, 0.488 mmol) were added thereto. The reaction was carried out at room temperature for 3 h. After the reaction was completed, water (20 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (25 mL×3). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel plate chromatography to obtain probe 3f (9.5 mg) with a yield of 52%.

[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, found 390.1629.

[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 bottle, which was sealed and reacted at room temperature for 18 h. After the reaction was completed, the solution was concentrated under reduced pressure and used directly in the next step without 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 bottle in sequence. t BuONa (1.58 g, 16.5 mmol), anhydrous 1,4-dioxane solution (14 mL). The reaction system was reacted at 60°C for 2 h. After the reaction was completed, 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) with a yield of 20%.

[0129] Under argon protection, 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 in sequence. After the addition, the reaction system was heated to 100 ° C and reacted for 12 h. After the reaction was completed, water (35 mL) was added to quench the reaction, EA was extracted (25 mL × 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 aldehyde 1c (31.1 mg) with a yield of 17%.

[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 N5OSS + [M+H] +m / z 506.7640, found 506.7635.

[0133] 3) Preparation of 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 completed, the reaction was continued for 3 h. After the reaction was completed, it was purified by silica gel column chromatography to obtain compound 81 (19.1 mg) with 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 to the reaction bottle in sequence. The reaction was allowed to react at room temperature for 3 h. After the reaction was completed, water (25 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (15 mL×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 with a yield of 86%.

[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, found 364.1471.

[0139] [Example 8]

[0140]

[0141] 1) Preparation of aldehyde 1d

[0142] Under argon protection, compound 82 (1.85 g, 10 mmol), 30% methylamine solution (6.5 ml, 50 mmol), copper powder (32 mg, 0.5 mmol) were added to a sealed tube in sequence, and the system was heated to 100°C and reacted for 19 h. After the reaction was completed, water (70 mL) was added to quench the reaction, and EA was extracted (25 mL × 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) with a yield of 77%.

[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 reacted at -20°C for 3 h. After the reaction was completed, 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) with a yield of 67%.

[0144] Under argon protection, compound 84 (400 mg, 1.85 mmol) and anhydrous tetrahydrofuran (11 mL) were added in sequence, the system was cooled to -78°C, and then 1.6M nBuLi (2.9 mL, 4.62 mmol) was slowly added dropwise. After the addition was complete, the mixture was reacted at -78°C for 1 h. After 1 h, anhydrous DMF (286 μL, 3.7 mmol) was added dropwise. After the addition was complete, the mixture was reacted at -78°C for 2 h. After the reaction was completed, water (20 mL) was added to quench the reaction, the mixture was extracted with EA (25 mL×3), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain aldehyde 1d (342 mg) with a yield of 76%.

[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 protection, aldehyde 1d (80 mg, 0.33 mmol), phosphate derivative 2f (193 mg, 0.495 mmol), anhydrous tetrahydrofuran (19 mL) were added, the reaction system was cooled to -20 ° C, and then 2M LDA (280 μL, 0.561 mmol) was slowly added dropwise. After the addition was completed, the reaction was continued for 3 hours. After the reaction was completed, it was purified by silica gel column chromatography to obtain compound 85 (81 mg) with a yield of 51%.

[0150] Compound 85 (20 mg, 0.0417 mmol), DCM (0.4 mL), and norbornadiene (42 μL, 0.417 mmol) were added to the reaction bottle, and the reaction was carried out at room temperature for 18 h after sealing. After the reaction was completed, the mixture was concentrated under reduced pressure, and anhydrous tetrahydrofuran (0.4 mL) and 1M TBAF (417 μL, 0.417 mmol) were added thereto. The reaction was carried out at room temperature for 3 h. After the reaction was completed, water (25 mL) was added to quench the reaction, and 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 plate chromatography to obtain probe 3h (11.8 mg) with a yield of 78%.

[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, found 364.1476.

[0152] [Example 9]

[0153]

[0154] 1) Preparation of aldehyde 1f

[0155] Under argon protection, 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 in sequence. The reaction system was heated to 100 ° C and reacted for 12 h. After the reaction was completed, water (45 mL) was added to quench the reaction, EA was extracted (25 mL×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 aldehyde 1f (96 mg) with a yield of 15%.

[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. Then 2M LDA (103 μL, 0.207 mmol) was slowly added dropwise. After the addition was completed, the reaction was allowed to react for 3 hours. After the reaction was completed, the mixture was purified by silica gel plate chromatography to obtain compound 87 (32.1 mg) with 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 to the reaction bottle in sequence. The reaction was allowed to react at room temperature for 3 h. After the reaction was completed, water (25 mL) was added to quench the reaction, and 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 obtain probe 3i (13.3 mg) with a yield of 89%.

[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, found 338.1318.

[0161] [Example 10]

[0162]

[0163] 1) Preparation of aldehyde 1g

[0164] Under argon protection, compound 88 (5 g, 22.5 mmol), 60% NaH (894 mg, 90 mmol), and DMF (83 mL) were added in sequence. The mixture was reacted at room temperature for 1 h. After 1 h of reaction, iodomethane (7.6 mL, 122 mmol) was slowly added dropwise. After the addition was complete, the reaction system was moved to room temperature for 3 h. After the reaction was completed, water (400 mL) was added to quench the reaction, and the mixture was extracted with EA (50 mL × 3), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 89 (1.77 g) with a yield of 32%.

[0165] Under argon protection, 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 in sequence. The reaction system was heated to 100 ° C and reacted for 12 h. After the reaction was completed, water (90 mL) was added to quench the reaction, EA was extracted (25 mL × 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) with a yield of 24%.

[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 the reaction was complete, water (40 mL) was added to quench the reaction, and the product 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 give 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 the reaction bottle, sealed, and reacted at room temperature for 20 h. After the reaction was completed, the mixture was concentrated under reduced pressure, and anhydrous tetrahydrofuran (0.4 mL) and 1M TBAF (386 μL, 0.386 mmol) were added thereto. The reaction was carried out at room temperature for 3 h. After the reaction was completed, water (25 mL) was added to quench the reaction, and EA was extracted (15 mL×3). The organic layers were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel plate chromatography to obtain probe 3j (15.5 mg) with a yield of 85%.

[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 protection, 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 in sequence. The reaction system was heated to 60 ° C for 1.5 h. After the reaction was completed, saturated ammonium chloride solution (40 mL) was added to quench the reaction, EA was extracted (20 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain compound 94 (19.6 mg) with a yield of 85%.

[0175] Under argon protection, 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 in sequence. The reaction was allowed to proceed at room temperature for 18 h. After completion of the reaction, water (35 mL) was added to quench the reaction, and the mixture was 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 compound 95 (6.3 mg) with a yield of 31%.

[0176] Under argon protection, 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 to the reaction bottle in sequence and reacted at room temperature for 3 h. After the reaction was completed, water (40 mL) was added to quench the reaction, EA was extracted (25 mL×3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure, and the residue was purified by silica gel plate chromatography to obtain probe 3e-F (12.1 mg) with a yield of 88%.

[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, found 605.7541.

[0181] [Example 12]

[0182]

[0183] 1) Preparation of phosphate derivative 2g

[0184] Under argon protection, 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 the reaction bottle in sequence, and the mixture was reacted at room temperature for 15 h. After the reaction was completed, water (300 mL) and sodium nitrite (4.7 g, 68.2 mmol) were added under an ice-water bath, and the pH value was adjusted to 3-4 with 1 M dilute hydrochloric acid (HCl). After reacting at room temperature for 10 minutes, the mixture was extracted with EA (40 mL×3), dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain red oily compound 92 (456.3 mg) with a yield of 52.2%.

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

[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), anhydrous tetrahydrofuran (10 mL) were added in sequence, 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 compound was purified by silica gel chromatography to obtain compound 3k (48.8 mg) with 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, found 398.5485.

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

[0194] Under argon protection, 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 to the reaction bottle in sequence and reacted at room temperature for 3 h. After the reaction was completed, water (50 mL) was added to quench the reaction, EA was extracted (20 mL×3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure, and the residue was purified by silica gel plate chromatography to obtain probe 3f-F (17.5 mg) with a yield of 81%.

[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, found 575.7280.

[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 bottle in sequence, and 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, it was purified by silica gel plate chromatography to obtain compound 31 (20.7 mg) with 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, found 382.5115.

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

[0206] Under argon protection, compound 3l (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 to the reaction bottle in sequence and reacted at room temperature for 3 h. After the reaction was completed, water (45 mL) was added to quench the reaction, EA was extracted (25 mL×3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure, and the residue was purified by silica gel plate chromatography to obtain probe 3h-F (23 mg) with a yield of 89%.

[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, based on the two-step method 18 F probe, the specific synthesis route is:

[0210]

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

[0212] Will 18 F- Load onto an activated QMA ion exchange column (the 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, 6mL H2O) and rinse into a 4mL reaction bottle. The activity meter was used to measure the content of [ 18 F]KF / K 222 The dose of KF / K 222 The solution was heated to 110°C, and dried by bubbling under nitrogen flow. Then, ultra-dry MeCN (1 mL) was added, and the mixture was dried by bubbling under nitrogen flow at 110°C, and repeated twice. pAz (6-8 mg) was dissolved in 0.4 mL ultra-dry MeCN solution and added to the reaction bottle, and 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, and the reaction solution was filtered using an organic microporous filter membrane.

[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 RCC of the pAz reaction. 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, used for the next reaction.

[0215] 2)[ 18 Radiochemical synthesis of the [F] probe

[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 0.1 mL of pure water and stir for 30 seconds, then add the 18 The DMF solution of [F]pAz was reacted at 25°C for 15 min. After the reaction was completed, 1.5 mL of H2O / MeCN (1 / 1, v / v) solution was added to quench the reaction, and the reaction filtrate was obtained by filtering with an organic microporous filter membrane.

[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 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, adsorb the product dilution 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 by 18 The RCY of the F probe. 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 mother solution, and then the mother solution was diluted to a 1 μM concentration with PBS for testing. The ultraviolet maximum absorption wavelength of the probe was detected using an ultraviolet spectrophotometer. The probe was diluted to a 1 μM concentration with 200 μL of a 95%-5% (v / v) ethanol-water mixed solution for testing. The ultraviolet 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 detected by combining the probe (1 μM) with Aβ protein (9 μM) in PBS solution. The total volume of the detection solution was 200 μL. After 3 minutes, the maximum emission wavelength of the probe after binding to the protein was detected and recorded using a fluorescence instrument. The probe part was replaced by an equal volume of DMSO as a blank subtraction.

[0224] The experimental results are shown in Table 1. It can be seen that the commercial probe ThT has a shorter Stokes shift; probes 3a to 3j all have longer Stokes shifts, indicating that the probes can have a higher signal-to-noise ratio and lower spectral overlap when imaging, especially the Stokes shift of probe 3e reaches 266nm. At the same time, after the probes bind to the Aβ protein, the maximum emission wavelength shows a significant blue shift, indicating that probes 3a to 3j have the properties of typical ICT probes. Their high sensitivity to environmental polarity enables the fluorescence properties of the probes, such as the maximum emission wavelength, to change significantly when the environmental polarity changes.

[0225] Table 1: Optical properties of the probes

[0226]

[0227] [Example 16]

[0228] This example detects 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 probe binds 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, to 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 carry F atoms. Figure 4As shown, 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 into WT and AD mice (8 months old) via tail vein 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 tissues of AD and WT mice were obtained by perfusion with saline, and sections were embedded with OCT gel, with a thickness of 10 μm. The sections were washed with PBS to remove the edge OCT gel. After antigen repair, the sections were blocked in a blocking solution containing 10% NGS and 0.1% Triton X-100 at room temperature for 1 hour, and the primary antibody was diluted with a primary antibody diluent (10% NGS; PBS) at a certain ratio (1:100), and incubated at 4°C overnight; washed with PBS 3 times, 10 minutes each time; incubated with secondary antibody, the secondary antibody was diluted with a secondary antibody diluent (3% NGS; PBS) at a certain ratio (1:200), incubated at room temperature in the dark for 1 hour, washed with PBS to remove the secondary antibody, incubated with cold fluorine control molecules and ThT (10 μM) at room temperature in the dark for 1 hour, washed with PBS to remove the cold fluorine probe, incubated with DAPI at room temperature for 10 minutes, and then washed with PBS to remove excess dyes, sealed, dried in the dark, and observed the staining 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 show strong fluorescence effect in the brain of AD mice. Figure 6 As shown, 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 F, dynamic PET imaging was performed on AD mice and WT mice to investigate the thermal fluorine 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 (10% EtOH) with physiological saline 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 the 3D-OSEM algorithm based on the Monte Carlo model and then processed by the osirix software. Dynamic PET / CT fusion images of the brain were drawn. The experimental results are shown in 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 It has the structural formula shown in Formula I: 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, acyl, trifluoromethyl, n=0 or 1.

2. A β-amyloid protein probe according to claim 1, characterized in that: The R2 is selected from any one of the following groups:

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

4. A β-amyloid protein probe according to claim 3, characterized in that: The amyloid β probe is selected from the following compounds:

5. A β-amyloid protein probe according to any one of claims 1 to 4, characterized in that: The fluorine substitution of the amyloid β probe is 18 F.

6. A method for preparing a β-amyloid protein probe, characterized in that: The following steps are involved: Under the protection of an inert gas, after 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 a β-amyloid protein probe; or In 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 to obtain a bioorthogonal reaction with a dienophile. After the reaction, a β-amyloid protein probe is obtained; Formula II: Formula III: Wherein, R1 in formula II is selected from N,N'-dimethylaryl, methylaminoaryl, N,N'-dimethylnaphthyl, N-azetidinylaryl, 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.

7. The method for preparing a β-amyloid protein probe according to claim 6, characterized in that: The R3 is a substituted pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrazinyl, wherein the substituent is a C1-C2 alkyl, acyl, trifluoromethyl, hydroxyethyl, protected hydroxyethyl, or butynyl.

8. The method for preparing a β-amyloid protein probe according to claim 6, 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.

9. The method for preparing a β-amyloid protein probe according to claim 6, 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.

10. Use of a β-amyloid protein probe according to any one of claims 1 to 5 in labeling Aβ protein, or in preparing an imaging molecular probe for labeling Aβ protein.

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