Photo-induced fluorescence excitation probes, indazolones and methods of making and using the same

Abstract

The application discloses a light-induced fluorescence excitation probe, an indazolone compound and a preparation method and application thereof, and relates to the field of organic small-molecule fluorescence excitation probes.The light-induced fluorescence excitation probe has a general structure as follows: a preparation method of the light-induced fluorescence excitation probe comprises the following steps: in the presence of a palladium catalyst, potassium acetate and 1,4-dioxane, a first intermediate and a second intermediate are subjected to a first reaction in a protective atmosphere to obtain the light-induced fluorescence excitation probe.The application of the light-induced fluorescence excitation probe is used for fluorescence excitation labeling of proteins.The novel visible light-induced fluorescence excitation probe developed by the application can be used for fluorescence excitation labeling of proteins.Through light regulation of the labeling process, the light-induced fluorescence excitation probe has extremely high space-time resolution, and provides a new tool for biological macromolecule labeling.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic small molecule fluorescence excitation probe, more particularly, to a photo-induced fluorescence excitation probe, an indazolone compound and a preparation method and application thereof. BACKGROUND

[0002] The photo-induced fluorescence excitation probe refers to a weakly fluorescent or non-fluorescent molecule, which can be converted into a corresponding structure capable of producing fluorescence through photochemical reaction by light irradiation, and is an important optical probe for biological imaging. Since they can be used in complex biological systems with high spatial and temporal resolution, and can be used to controllably induce fluorescence probes by regulating the position, energy and light irradiation time of the reaction light source, they provide a powerful tool for high-resolution tracking of biological processes in living cells, tissues and animals.

[0003] The light-induced primary amines and o-nitrobenzyl alcohols cyclization (PANAC) is a new type of photo-click chemistry reaction developed recently. However, the current PANAC reaction uses ultraviolet light as the reaction light source, which has poor penetration and can damage biological macromolecules. When this is applied to protein fluorescence labeling, the fluorescent dye has to be modified, and the unbound fluorescent dye has to be washed away after labeling, which cannot be monitored in real time. The process is complicated and can produce additional background signals, thereby reducing the contrast of imaging. Therefore, it is of great significance to develop a visible light-induced fluorescence excitation probe for the reaction of o-nitrobenzyl alcohol derivatives and primary amines. SUMMARY

[0004] The present application aims to provide a photo-induced fluorescence excitation probe, an indazolone compound and a preparation method and application thereof to solve the above problems.

[0005] According to a first aspect of the present application, a photo-induced fluorescence excitation probe is provided, and the structure general formula is as follows:

[0006]

[0007] wherein R1 and R2 are selected from -NO2 or -CH2OH, R1 and R2 are different; R3 is selected from -CF3 or -O-CH3.

[0008] According to a second aspect of the present application, a preparation method of the photo-induced fluorescence excitation probe is provided, comprising:

[0009] The first intermediate and the second intermediate are subjected to a first reaction in the presence of a palladium catalyst, potassium acetate and 1,4-dioxane in a protective atmosphere to obtain the photo-induced fluorescence excitation probe;

[0010] The first intermediate has a structural formula as follows:

[0011]

[0012] The second intermediate has a structural formula as follows:

[0013]

[0014] Preferably, when the R3 is -CF3, the preparation method of the first intermediate comprises:

[0015] The m-hydroxyphenylpiperazine is subjected to a second reaction with ethyl trifluoroacetylacetate in the presence of sodium bisulfate, and then subjected to a third reaction by adding glacial acetic acid;

[0016] The third intermediate is subjected to a fourth reaction with di-tert-butyl dicarbonate in the presence of N,N-dimethylformamide and triethylamine to obtain a fourth intermediate;

[0017] The fourth intermediate is subjected to a fifth reaction with N-bromosuccinimide to obtain the first intermediate;

[0018] The third intermediate has a structural formula as follows:

[0019]

[0020] The fourth intermediate has a structural formula as follows:

[0021]

[0022] Preferably, the temperature of the first reaction is 80-100°C;

[0023] The temperature of the second reaction and the third reaction is independently 110°C-120°C;

[0024] The temperature of the fourth reaction and the fifth reaction is independently room temperature.

[0025] Preferably, when the R3 is -O-CH3, the preparation method of the first intermediate comprises:

[0026] 2-Hydroxy-4-bromoacetophenone is subjected to a sixth reaction with diethyl carbonate in the presence of sodium hydride to obtain a fifth intermediate;

[0027] The fifth intermediate is subjected to a seventh reaction with trimethyl phosphate in the presence of potassium carbonate to obtain a sixth intermediate;

[0028] The sixth intermediate is subjected to an eighth reaction with 1-Boc-piperazine in the presence of a palladium catalyst, 2,2'-bis(diphenylphosphino)-1,1'-binaphthalene, potassium tert-butoxide and 1,4-dioxane under a protective atmosphere to obtain a seventh intermediate;

[0029] The seventh intermediate is subjected to a ninth reaction with N-bromosuccinimide to obtain the first intermediate;

[0030] The fifth intermediate has the structural formula:

[0031]

[0032] The sixth intermediate has the structural formula:

[0033]

[0034] The seventh intermediate has the structural formula:

[0035]

[0036] Preferably, the temperature of the sixth reaction is 80-100℃, the temperature of the seventh reaction is 130-150℃, the temperature of the eighth reaction is 100-120℃, the temperature of the ninth reaction is room temperature, and the temperature of the first reaction is 80-100℃.

[0037] Preferably, the preparation method of the second intermediate comprises:

[0038] The second intermediate is obtained by reacting 2-nitro-5-bromobenzyl alcohol or 4-bromo-2-nitrobenzyl alcohol with bis(pinacolato)diboron in the presence of a palladium catalyst, tricyclohexylphosphine, potassium acetate and 1,4-dioxane under a protective atmosphere.

[0039] The third aspect of the present application provides an indazolone compound, which has the general structural formula:

[0040]

[0041] wherein R3 is selected from -CF3 or -O-CH3, and R4 is selected from

[0042]

[0043] The fourth aspect of the present application provides a preparation method of the indazolone compound, which comprises:

[0044] The photo-induced fluorescence excitation probe is reacted with benzyloxy carbonyl-protected lysine methyl ester Cbz-lys-OMe under visible light.

[0045] The fifth aspect of the present application provides the use of the light-induced fluorescence excitation probe for fluorescence excitation labeling of proteins.

[0046] According to the technical content disclosed in the present application, the following beneficial effects are achieved:

[0047] The light-induced fluorescence excitation probe provided by the present application is designed to include three parts, i.e., an o-nitrobenzyl alcohol skeleton, a fluorophore and a functional functional group. On the one hand, the introduction of the fluorophore makes the absorption spectrum of the o-nitrobenzyl alcohol skeleton red-shifted, and has a certain absorption in the visible light wavelength range, and undergoes a photo-induced cyclization reaction with a primary amine; on the other hand, the molecule has a nitro group as a strong electron-withdrawing group, and can undergo an intramolecular charge transfer or photo-induced electron transfer process to quench the fluorescence of the fluorophore, and the indazolone product generated after the reaction with the primary amine changes the electronic environment inside the molecule, thereby blocking the intramolecular charge transfer or photo-induced electron transfer process, so that the fluorophore of the indazolone product restores fluorescence.

[0048] The o-nitrobenzyl alcohol derivative synthesized by the present application, as a light-induced fluorescence excitation probe, can react with benzyloxy carbonyl protected lysine methyl ester (Cbz-lys-OMe) to generate an indazolone product with tens of times fluorescence enhancement under the induction of 420 nm blue light in a biocompatible system, and the reaction has a good conversion rate. The novel visible light-induced fluorescence excitation probe developed by the present application can be used for fluorescence excitation labeling of proteins. Through light-regulated labeling process, it has extremely high spatiotemporal resolution, and provides a new tool for biomacromolecule labeling.

[0049] Other features and advantages of the present application will become apparent from the following detailed description of exemplary embodiments thereof, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0050] The accompanying drawings incorporated in and forming a part of the specification, illustrate embodiments of the present application and, together with the description, serve to explain the principles of the application.

[0051] Figure 1 The H spectrum of compound 1 provided for the example is shown in the following table:

[0052] Figure 2 The H spectrum of compound 2 provided for the example is shown in the following table:

[0053] Figure 3 The H spectrum of compound 3 provided for the example is shown in the following table:

[0054] Figure 4 The H spectrum of compound 4 provided for the example is shown in the following table:

[0055] Figure 5H spectrum of compound 1-IP provided for the example;

[0056] Figure 6 H spectrum of compound 2-IP provided for the example;

[0057] Figure 7 H spectrum of compound 3-IP provided for the example;

[0058] Figure 8 H spectrum of compound 4-IP provided for the example;

[0059] Figure 9 UV-Vis absorption spectrum of compounds 1, 2, 3, 4;

[0060] Figure 10 Fluorescence emission spectrum of compounds 1-4 and 1-IP-4-IP. DETAILED DESCRIPTION

[0061] Various exemplary embodiments of the present application will now be described in detail with reference to the figures. It should be noted that the relative arrangements, numerical expressions, and values of components and steps set forth in these embodiments are not limiting to the scope of the present application unless otherwise specifically stated.

[0062] The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the scope of the application its application or uses.

[0063] Techniques, methods, and apparatus known to those of ordinary skill in the relevant art can not be discussed in detail herein. However, where appropriate, such techniques, methods, and apparatus should be considered as being part of the specification.

[0064] In all of the compositions and methods shown and discussed herein, any specific values should be interpreted as merely exemplary, and are not intended to be limiting. Thus, other examples of the exemplary embodiments can have different values.

[0065] It should be noted that like reference numerals and letters in the various figures indicate similar items, and, thus, once an item is defined in one figure, it should not require further discussion in subsequent figures.

[0066] First, the reaction equations of compound 1 and compound 2 obtained from Example 1 and Example 2 are shown in the following two equations, respectively:

[0067]

[0068] Example 1

[0069] This example provides a photo-induced fluorescence excitation probe (compound 1), and the preparation method thereof is as follows:

[0070] Intermediate 1a:

[0071] To a reaction flask was added m-hydroxyphenylpiperazine (1.07 g, 6 mmol), sodium bisulfate monohydrate (828 mg, 6 mmol), ethyl trifluoroacetylacetate 4 mL, and the reaction was stirred at 110 °C for 2 h. The heating was stopped and the reaction was cooled to room temperature. Glacial acetic acid 2 mL was added and the reaction was stirred at 110 °C for another 2 h. After the reaction was complete as monitored by TLC and LC-MS, the heating was stopped and the reaction was cooled to room temperature. The reaction was poured into ice water and a yellow solid precipitated. The solid was collected by filtration and dissolved in dichloromethane:methanol (10:1). The solution was neutralized with saturated sodium bicarbonate solution and washed with saturated sodium chloride solution twice. The organic phase was collected and dried over anhydrous sodium sulfate. The sample was swirled and column chromatography was performed. A yellow solid was obtained (484 mg, 30% yield). MS (ESI): m / z 299.15 [(M+1) + ].

[0072] To a reaction flask was added intermediate 1a (1.9 g, 6.4 mmol), di-tert-butyl dicarbonate (2.1 g, 9.6 mmol), and the mixture was dissolved in N,N-dimethylformamide 10 mL. Triethylamine (1.3 g, 12.8 mmol) was added and the reaction was stirred at room temperature overnight. After the reaction was complete as monitored by TLC and LC-MS, the reaction was extracted with ethyl acetate and washed with saturated sodium chloride solution three times. The organic phase was collected and dried over anhydrous sodium sulfate. The sample was swirled and column chromatography was performed. A yellow solid was obtained (1.8 g, 71% yield). MS (ESI): m / z 399.24 [(M+1) + ].

[0073] To a reaction flask was added intermediate 1b (1.8 g, 4.5 mmol) and the mixture was dissolved in acetonitrile 20 mL. The reaction was stirred at room temperature and N-bromosuccinimide (1 g, 5.4 mmol) was added portionwise. The reaction was stirred. After the reaction was complete as monitored by TLC and LC-MS, the reaction was extracted with ethyl acetate and washed with saturated sodium chloride solution three times. The organic phase was collected and dried over anhydrous sodium sulfate. The sample was swirled and column chromatography was performed. A yellow solid was obtained (1.4 g, 65% yield). MS (ESI): m / z 477.56 [(M+1) + ].

[0074] Intermediate 1d-p: To a two-necked flask was added 2-nitro-5-bromobenzene methanol (696 mg, 3 mmol), bis(pinacolato)diboron (838 mg, 3.3 mmol), tris(dibenzylideneacetone)dipalladium (83 mg, 0.09 mmol), tricyclohexylphosphine (61 mg, 0.22 mmol), potassium acetate (442 mg, 4.5 mmol), 1,4-dioxane 10 mL, stirred at 80 °C under argon protection for 14 h. TLC and LC-MS were used to monitor the reaction completion. The reaction mixture was extracted with ethyl acetate and washed with saturated sodium chloride solution for 3 times. The organic phase was collected and dried over anhydrous sodium sulfate, and the sample was stirred and column chromatographed. 800 mg of light yellow solid was obtained with a yield of 95%. MS (ESI): m / z 280.35 [(M+1) + ]。

[0075] Compound 1: To a two-necked flask was added intermediate 1c (72 mg, 0.15 mmol), intermediate 1d-p (46 mg, 0.17 mmol), 1,1-bis(diphenylphosphino)ferrocene palladium dichloride (7 mg, 0.008 mmol), potassium acetate (45 mg, 0.45 mmol), 1,4-dioxane 5 mL, stirred at 90 °C under argon protection overnight. TLC and LC-MS were used to monitor the reaction completion. The reaction mixture was extracted with ethyl acetate and washed with saturated sodium chloride solution for 3 times. The organic phase was collected and dried over anhydrous sodium sulfate, and the sample was stirred and column chromatographed.

[0076] The structural formula of compound 1 is as follows:

[0077]

[0078] Compound 1, orange yellow solid 40 mg, yield 49%. 1 H NMR (500 MHz, DMSO-d6) δ 8.21 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 1.9 Hz, 1H), 7.70 (dt, J = 8.9, 2.1 Hz, 1H), 7.66 (dd, J = 8.4, 1.9 Hz, 1H), 7.23 (d, J = 9.0 Hz, 1H), 6.87 (s, 1H), 5.58 (q, J = 4.9, 4.4 Hz, 1H), 4.90 (d, J = 5.5 Hz, 2H), 3.14 (t, J = 5.0 Hz, 4H), 2.83 (s, 4H), 1.36 (s, 9H). MS (ESI): m / z 550.45 [(M+1) + ]。

[0079] Example 2

[0080] This example provides a light-induced fluorescence excitation probe (compound 2), and the preparation method thereof is as follows:

[0081] The preparation process of intermediate 1a, intermediate 1b and intermediate 1c is as shown in Example 1.

[0082] Intermediate 1d-m: starting from 4-bromo-2-nitrobenzyl alcohol (4.6 g, 20 mmol), the same method as the synthesis of intermediate 1d-p. Obtained 5.4 g of light yellow solid, yield 97%. MS (ESI): m / z 280.48 [(M+1) + ]。

[0083] The structural formula of compound 2 is as follows:

[0084]

[0085] Compound 2, yellow solid 76 mg, yield 33%. 1 H NMR (500 MHz, DMSO-d6) δ 8.21 (d, J = 1.7 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.92 (dd, J = 8.0, 1.7 Hz, 1H), 7.69 (dt, J = 8.9, 2.1 Hz, 1H), 7.24 (d, J = 9.0 Hz, 1H), 6.88 (s, 1H), 5.62 (t, J = 5.6 Hz, 1H), 4.91 (d, J = 5.6 Hz, 2H), 3.14 (t, J = 5.0 Hz, 4H), 2.83 (t, J = 5.0 Hz, 4H), 1.36 (s, 9H). MS (ESI): m / z 550.56 [(M+1) + ]。

[0086] Example 3

[0087] This example provides a light-induced fluorescence excitation probe (compound 3), and the reaction equation is as follows:

[0088]

[0089] The preparation method is as follows:

[0090] Intermediate 3a: Take 2-hydroxy-4-bromoacetophenone (2.2 g, 10 mmol) dissolved in 40 mL of dimethylsulfoxide, slowly add sodium hydride (1.2 g, 50 mmol) in portions at room temperature, stir until no more bubbles are generated, add diethyl carbonate (1.8 mL, 15 mmol), increase the temperature to 100 °C and continue the reaction for 4 hours, monitor the reaction completion by TLC and LC-MS, cool to room temperature, slowly add saturated ammonium chloride solution to quench, adjust the pH to 1-2 with 6 M hydrochloric acid, extract with ethyl acetate, wash with saturated sodium chloride solution 3 times. Collect the organic phase, dry over anhydrous sodium sulfate, stir the sample, column chromatography. Get yellow solid 1.2 g, yield 51%. MS (ESI): m / z 242.34 [(M+1) + ].

[0091] Intermediate 3b: Take intermediate 3a (1.2 g, 5 mmol), add potassium carbonate (830 mg, 6 mmol), trimethyl phosphate 5 mL, react at 140 °C for 4 hours, monitor the reaction completion by TLC and LC-MS, cool to room temperature, dilute with ethyl acetate and extract, wash with saturated sodium chloride solution 3 times. Collect the organic phase, dry over anhydrous sodium sulfate, stir the sample, column chromatography. Get yellow solid 1.1 g, yield 86%. MS (ESI): m / z 256.75 [(M+1) + ].

[0092] Intermediate 3c: Take intermediate 3b (1.1 g, 4.3 mmol), 1-Boc-piperazine (1.1 g, 6 mmol), tris(dibenzylideneacetone)dipalladium (230 mg, 0.25 mmol), 2,2'-bis(diphenylphosphino)-1,1'-binaphthalene (623 mg, 1 mmol), potassium tert-butoxide (1.5 g, 13.5 mmol) in a two-necked flask, equipped with a reflux condenser, replace argon, add 1,4-dioxane 20 mL, reflux stir under argon protection at 110 °C for 4 hours. Monitor the reaction completion by TLC and LC-MS, cool to room temperature, dilute with ethyl acetate and extract, wash with saturated sodium chloride solution 3 times. Collect the organic phase, dry over anhydrous sodium sulfate, stir the sample, column chromatography. Get red solid 800 mg, yield 52%. MS (ESI): m / z 361.24 [(M+1) + ].

[0093] Intermediate 3d: To a reaction flask was added intermediate 3c (492 mg, 1.36 mmol) dissolved in 6 mL of acetonitrile and stirred at room temperature. N-bromosuccinimide (243 mg, 1.36 mmol) was added in portions and the reaction was continued to stir. TLC and LC-MS monitored the completion of the reaction and extracted with ethyl acetate and washed with saturated sodium chloride solution three times. The organic phase was collected, dried over anhydrous sodium sulfate, swirled and column chromatographed. Obtained 407 mg of light yellow solid with 68% yield. MS (ESI): m / z 440.34 [(M+1) + ].

[0094] Compound 3: To a two necked flask was added intermediate 3d (156 mg, 0.35 mmol), intermediate Id-p (117 mg, 0.42 mmol), l,l-bis(diphenylphosphino)ferrocene palladium dichloride (14 mg, 0.018 mmol), potassium acetate (125 mg, 1.28 mmol), 1,4-dioxane 5 mL and stirred at 90 °C under argon overnight. TLC and LC-MS monitored the completion of the reaction and extracted with ethyl acetate and washed with saturated sodium chloride solution three times. The organic phase was collected, dried over anhydrous sodium sulfate, swirled and column chromatographed.

[0095] The structure of compound 3 is as follows:

[0096]

[0097] Compound 3, orange yellow solid 151 mg, 82% yield. 1 H NMR (500 MHz, DMSO-d6) δ 8.10 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 1.9 Hz, 1H), 7.69 (d, J = 9.0 Hz, 1H), 7.58 (dd, J = 8.4, 2.0 Hz, 1H), 7.02 (dd, J = 9.1, 2.5 Hz, 1H), 6.89 (d, J = 2.4 Hz, 1H), 5.59 (t, J = 5.5 Hz, 1H), 4.87 (d, J = 5.5 Hz, 2H), 3.57 (s, 3H), 3.47 (dd, J = 6.9, 3.6 Hz, 4H), 3.40 (dd, J = 6.8, 3.7 Hz, 4H), 1.43 (s, 9H). MS (ESI): m / z 512.53 [(M+1) + ].

[0098] Example 4

[0099] This example provides a photo-induced fluorescence excitation probe (compound 4) and a method for preparing the same.

[0100] Compound 4 was synthesized using intermediate 3d and Id-m in the same manner as in example 3.

[0101] The structural formula of compound 4 is as follows:

[0102]

[0103] Compound 4, yellow solid 127 mg, yield 69%. 1 H NMR (500 MHz, DMSO-d6) δ 8.11 (d, J = 1.8 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.81 (dd, J = 8.0, 1.8 Hz, 1H), 7.68 (d, J = 9.0 Hz, 1H), 5.60 (t, J = 5.6 Hz, 1H), 4.87 (d, J = 5.6 Hz, 2H), 3.58 (s, 3H), 3.47 (dd, J = 6.9, 3.7 Hz, 4H), 3.39 (dd, J = 6.8, 3.7 Hz, 4H), 1.43 (s, 9H). MS (ESI): m / z 512.39 [(M+1) + ].

[0104] The hydrogen spectrum of compound 1, 2, 3, 4 is shown in Figure 1 、 Figure 2 、 Figure 3 and Figure 4 .

[0105] Example 5

[0106] The present example provides a class of indazolone compounds, including compound 1-IP, compound 2-IP, compound 3-IP, compound 4-IP, and a preparation method thereof is:

[0107] Take the appropriate amount (1.0 eq) of ortho-nitrobenzyl alcohol derivative of example 1 to example 4, dissolve in 5 mL of dimethyl sulfoxide, add Cbz-lys-OMe (2.5 eq), 50 mM PBS / MeOH (1:1, pH 8.0, 50 mL), and irradiate with a 420 nm LED light source while stirring. After monitoring the end of the reaction by LC-MS, extract with ethyl acetate, and wash with water and saturated sodium chloride solution in sequence. Combine the organic phases, dry, concentrate, and purify by column chromatography, preparative liquid chromatography or preparative TLC to obtain the indazolone product.

[0108] Preparation of Buffer solution (50 mM PBS:MeOH = 1:1, pH 8.0): Dissolve 0.6 g of sodium dihydrogen phosphate and 0.71 g of disodium hydrogen phosphate in 200 mL of water to obtain a 50 mM PBS buffer, and then mix with 200 mL of methanol to obtain a uniform mixture. Adjust the pH to 8.0 with 5M sodium hydroxide or 3M hydrochloric acid.

[0109] The general equation of the reaction is:

[0110]

[0111] The structural formula of compound 1-IP is as follows:

[0112]

[0113] Compound 1-IP, yellow solid 21 mg, yield 62%. 1 H NMR (500 MHz, DMSO-d6) δ 8.07 (s, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.74 (s, 1H), 7.66 (dd, J = 9.0, 2.0 Hz, 1H), 7.60 (d, J = 9.0 Hz, 1H), 7.35 (d, J = 3.8 Hz, 5H), 7.30 - 7.27 (m, 2H), 7.21 (d, J = 8.9 Hz, 1H), 6.85 (s, 1H), 5.02 (d, J = 3.9 Hz, 2H), 4.03 (q, J = 3.4 Hz, 1H), 3.61 (s, 3H), 3.13 - 3.10 (m, 4H), 2.90 - 2.82 (m, 4H), 1.87 (h, J = 6.8 Hz, 3H), 1.79 - 1.66 (m, 3H), 1.34 (s, 9H). MS (ESI): m / z 808.65 [(M + 1) + ]。

[0114] The structural formula of compound 2-IP is as follows:

[0115]

[0116] Compound 2-IP, light yellow-green solid 17 mg, yield 54%. 1 H NMR (500 MHz, DMSO-d6) δ 10.33 (s, 1H), 7.73 (d, J = 7.9 Hz, 2H), 7.67 (d, J = 8.7 Hz, 1H), 7.39 - 7.31 (m, 5H), 7.29 (d, J = 6.6 Hz, 1H), 7.20 (t, J = 9.5 Hz, 2H), 6.85 (s, 1H), 5.02 (d, J = 2.1 Hz, 2H), 4.02 (td, J = 8.6, 8.0, 5.6 Hz, 1H), 3.80 (d, J = 39.3 Hz, 2H), 3.60 (s, 3H), 3.13 - 2.80 (m, 8H), 1.71 (m, 6H), 1.34 (s, 9H). MS (ESI): m / z 808.47 [(M + 1) + ]。

[0117] The structural formula of compound 3-IP is as follows:

[0118]

[0119] Compound 3-IP, yellow solid 7 mg, yield 68%. 1 H NMR (500 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 9.0 Hz, 2H), 7.52 (d, J = 9.0 Hz, 1H), 7.39 - 7.26 (m, 6H), 7.22 (d, J = 9.0 Hz, 1H), 7.02 (dd, J = 9.0, 2.4 Hz, 1H), 6.89 (d, J = 2.4 Hz, 1H), 5.02 (d, J = 2.1 Hz, 2H), 4.42 (td, J = 7.0, 3.1 Hz, 2H), 4.01 (ddd, J = 9.2, 7.4, 4.8 Hz, 1H), 3.61 (s, 3H), 3.52 (s, 3H), 3.48 - 3.37 (m, 8H), 1.83 (dt, J = 14.4, 7.2 Hz, 2H), 1.74 - 1.64 (m, 2H), 1.43 (s, 9H), 1.35 (s, 2H). MS (ESI): m / z 770.35 [(M+1) + ]。

[0120] The structural formula of compound 4-IP is as follows:

[0121]

[0122] Compound 4-IP, yellow solid 22 mg, yield 62%. 1 H NMR (500 MHz, DMSO-d6) δ 10.28 (s, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.69 - 7.63 (m, 2H), 7.40 - 7.29 (m, 5H), 7.29 (s, 1H), 7.10 (d, J = 7.9 Hz, 1H), 7.02 (d, J = 9.2 Hz, 1H), 6.88 (s, 1H), 5.02 (s, 2H), 4.01 (td, J = 8.7, 4.9 Hz, 1H), 3.79 (dt, J = 7.6, 3.9 Hz, 2H), 3.60 (s, 3H), 3.52 (s, 3H), 3.38 (dd, J = 6.6, 3.9 Hz, 8H), 1.68 (ddd, J = 23.8, 12.6, 7.1 Hz, 4H), 1.43 (s, 9H), 1.33 (t, J = 7.7 Hz, 2H). MS (ESI): m / z 770.83 [(M+1) + ]。

[0123] The hydrogen spectrum of compounds 1-IP, 2-IP, 3-IP, 4-IP is shown in Figure 5 、 Figure 6 、 Figure 7 and Figure 8 .

[0124] Conversion rate experiment:

[0125] Accurately weigh a proper amount of o-NBA derivative and dissolve it in DMSO to prepare a 10 mM o-NBA stock solution; accurately weigh a proper amount of Cbz-lys-OMe and prepare a 50 mM Cbz-lys-OMe stock solution with the above Buffer; take 50 uL to 1.5 mL into an EP tube with a pipette, then add 400 uL of Buffer and mix; take out 250 uL to 1 mm optical path quartz cuvette, seal with a sealing film, and react for 0.5 h at a distance of 2 cm from a 420 nm LED light source, then take 250 uL of un-irradiated reaction solution and 250 uL of irradiated reaction solution into LC-MS in sequence, and calculate the conversion rate ((A before reaction-A after reaction) / A before reaction x 100%) through the change in chromatographic peak area of o-NBA derivative before and after reaction. The conversion rate of compound 1 is 83%, the conversion rate of compound 2 is 39%, the conversion rate of compound 3 is greater than 98%, and the conversion rate of compound 4 is 34%.

[0126] UV-visible absorption spectrum and fluorescence emission spectrum test:

[0127] Accurately take a proper amount of the test compound, dissolve it in DMSO to prepare a 10 mM stock solution, and dilute 20 uL with Buffer to 4 mL to obtain a test solution with a concentration of 5 x 10 -5 M. The visible light 410 nm is used as the excitation wavelength for determining the fluorescence emission spectrum.

[0128] Blank solution: take 20 uL of DMSO and dilute it to 4 mL with Buffer. Before measuring the UV-visible absorption spectrum, perform baseline correction with the blank solution.

[0129] Figure 9 UV-visible absorption spectrum of compounds 1, 2, 3, and 4; Figure 10 Fluorescence emission spectrum of compounds 1-4 and 1-IP-4-IP.

[0130] Although some specific embodiments of the present application have been described in detail by examples, those skilled in the art should understand that the above examples are only for illustration, but not for limiting the scope of the present application. Those skilled in the art should understand that the above embodiments can be modified without departing from the scope and spirit of the present application. The scope of the present application is defined by the appended claims.

Claims

1. A photo-induced fluorescence excitation probe, characterized in that, A structure general formula of which is: Wherein, R1 and R2 are selected from -NO2 or -CH2OH, R1 and R2 are different; R3 is selected from -CF3 or -O-CH3.

2. A method of preparing the photo-induced fluorescence excitation probe of claim 1, characterized by, Comprise: The first intermediate and the second intermediate are subjected to a first reaction in a protective atmosphere in the presence of a palladium catalyst, potassium acetate and 1,4-dioxane to obtain the photo-induced fluorescence excitation probe; A structure general formula of the first intermediate is: A structure general formula of the second intermediate is:

3. The production method according to claim 2, characterized by, When the R3 is -CF3, the preparation method of the first intermediate comprises: The m-hydroxyphenylpiperazine is subjected to a second reaction with ethyl trifluoroacetylacetate in the presence of sodium bisulfate; then, glacial acetic acid is added to continue a third reaction to obtain a third intermediate; The third intermediate is subjected to a fourth reaction with di-tert-butyl dicarbonate in the presence of N,N-dimethylformamide and triethylamine to obtain a fourth intermediate; The fourth intermediate is subjected to a fifth reaction with N-bromosuccinimide to obtain the first intermediate; A structure formula of the third intermediate is: A structure formula of the fourth intermediate is:

4. The production method according to claim 3, characterized by, The temperature of the first reaction is 80-100 DEG C; The temperature of the second reaction and the third reaction is independently 110 DEG C-120 DEG C; The temperature of the fourth reaction and the fifth reaction is independently room temperature.

5. The preparation method according to claim 2, characterized in that, When the R3 is -O-CH3, the preparation method of the first intermediate comprises: 2-hydroxy-4-bromoacetophenone is subjected to a sixth reaction with diethyl carbonate in the presence of sodium hydride to obtain a fifth intermediate; The fifth intermediate is subjected to a seventh reaction with trimethyl phosphate in the presence of potassium carbonate to obtain a sixth intermediate; The sixth intermediate is subjected to an eighth reaction with 1-Boc-piperazine in the presence of a palladium catalyst, 2,2'-bis(diphenylphosphino)-1,1'-binaphthalene, potassium tert-butoxide and 1,4-dioxane in a protective atmosphere to obtain a seventh intermediate; The seventh intermediate is subjected to a ninth reaction with N-bromosuccinimide to obtain the first intermediate; A structure formula of the fifth intermediate is: A structure formula of the sixth intermediate is: A structure formula of the seventh intermediate is:

6. The preparation method according to claim 5, characterized in that, The temperature of the sixth reaction is 80-100 DEG C, the temperature of the seventh reaction is 130-150 DEG C, the temperature of the eighth reaction is 100-120 DEG C, the temperature of the ninth reaction is room temperature, and the temperature of the first reaction is 80-100 DEG C.

7. The method of any one of claims 2-6, wherein, The preparation method of the second intermediate comprises: 2-nitro-5-bromobenzyl alcohol or 4-bromo-2-nitrobenzyl alcohol is used as a raw material, and the second intermediate is obtained by reacting with pinacol diboron in the presence of a palladium catalyst, tricyclohexylphosphine, potassium acetate and 1,4-dioxane in a protective atmosphere.

8. An indazole ketone compound, characterized in that, A structure general formula of which is: Wherein, R3 is selected from -CF3 or -O-CH3, and R4 is selected from 9. A process for the preparation of the indazolones of claim 8, characterized in that, Comprise: The photo-induced fluorescence excitation probe of claim 1 is reacted with benzyloxy carbonyl protected lysine methyl ester Cbz-lys-OMe under visible light conditions.

10. Use of the photo-induced fluorescence excitation probe according to claim 1, characterized in that, For fluorescence excitation labeling of proteins.

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