A fluorescent probe for detecting threonine tyrosine kinase and its preparation method and application

Abstract

The present invention discloses a fluorescent probe for detecting threonine tyrosine kinase, its preparation method, and application, belonging to the field of organic synthesis technology. The structural formula of the fluorescent probe compound is shown in Formula I: #imgabs0# Based on the scientific problem that threonine tyrosine kinase (TTK) is differentially expressed in normal and cancerous tissues in vivo, but there is a lack of real-time, accurate, and non-invasive detection tools, the present invention designs and synthesizes a fluorescent probe that can detect the concentration of threonine tyrosine kinase in biological tissues and distinguish physiological and pathological cells with temporal and spatial resolution, with broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis, in particular to a fluorescent probe for detecting threonine tyrosine kinase, and a preparation method and application thereof. Background Art

[0002] Malignant tumors seriously threaten people's health and life. Threonine tyrosine kinase (TTK) is a core component of the spindle assembly checkpoint (SAC), which ensures the proper distribution of chromosomes in daughter cells, balances growth and division, and is a protein kinase that ensures mitotic fidelity and genomic stability. SAC is a key monitoring mechanism that delays the progression of mitosis until all chromosomes are properly attached to the spindle microtubules to ensure accurate chromosome segregation and prevent incorrect chromosome division. When TTK is overexpressed, the function of the spindle assembly checkpoint is affected and inactivated, resulting in premature exit from the mitotic point, ultimately leading to chromosomal instability, the formation of aneuploidy, and even cell death. SAC ensures healthy cell growth and accurate cell division. Considering the key role of TTK in cell division, it may become a new biomarker.

[0003] Recent studies have revealed that TTK expression is significantly increased in several malignant tumors, such as pancreatic, lung, and liver cancers. This expression can promote tumor cell proliferation and is associated with tumor invasion and metastasis. In breast cancer, high TTK expression has been shown to induce aneuploidy and confer tolerance to aneuploidy in these cells. In colon cancer, TTK overexpression can lead to increased aneuploidy and, due to decreased SAC function, to carcinogenesis. However, currently, the only methods for measuring TTK concentrations are electrochemiluminescence immunoassays and biodetectors, which are complex and costly. Fluorescent probes, however, offer advantages such as simplicity, low cost, and high sensitivity, and are widely used in biomarker detection. Therefore, in this study, a fluorescent probe for rapid TTK detection was designed and synthesized. This probe exhibits selectivity and sensitivity for TTK, potentially distinguishing cancer cells from normal cells, and potentially providing a tool for early tumor screening. While this method can be used for in situ, real-time monitoring of threonine tyrosine kinases, given the differential expression of TTK in normal and cancerous tissues in vivo, it lacks a real-time, accurate, and non-invasive detection tool.

[0004] Therefore, it is of great significance to design and synthesize a fluorescent probe that can be used to detect threonine tyrosine kinase. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a fluorescent probe for detecting threonine tyrosine kinase, which is selective and responsive to threonine tyrosine kinase and has the potential to detect changes in the cellular microenvironment and TTK response.

[0006] The present invention also provides a method for preparing the fluorescent probe.

[0007] The present invention also provides applications of the fluorescent probe.

[0008] The first aspect of the present invention provides a fluorescent probe for detecting threonine tyrosine kinase, the structural formula of the fluorescent probe is shown in Formula I:

[0009]

[0010] The fluorescent probe for detecting threonine tyrosine kinase according to the embodiment of the present invention has at least the following beneficial effects:

[0011] The fluorescence intensity of the fluorescent probe of the present invention is increased by about 8 times when used to detect TTK, and it has high selectivity and high sensitivity to TTK. The fluorescence intensity increases with the increase of TTK concentration. This is because the fluorescent probe of the present invention is a responsive fluorescent probe based on ICT, and its fluorescent group is directly conjugated with piperazine (electron donor) and (1-ethylpyridyl) as electron acceptors to form a D-π-A type molecule. The push-pull electron system composed of the connection can be used to detect the concentration of threonine tyrosine kinase in organisms.

[0012] The fluorescent probe of the present invention has water solubility and environmental responsiveness, and the fluorescence intensity is a function of pH, with a significant relative change within the pH range of 4.5 to 7.5, and can be used to detect changes in cell microenvironment.

[0013] A second aspect of the present invention provides a method for preparing the above-mentioned fluorescent probe, the preparation method comprising the following steps:

[0014] Compound 8, compound 14, and a condensing agent are added to an organic solvent 1 to react to obtain a compound represented by formula I;

[0015] Wherein, the structural formulas of compound 8 and compound 14 are as follows:

[0016]

[0017] The preparation method provided by the present invention has at least the following beneficial effects:

[0018] The preparation method of the present invention is relatively simple in process, easy to separate and purify, has a high yield, and has good social value and application prospects.

[0019] According to some embodiments of the present invention, the molar ratio of compound 8 to compound 14 is 1:(1.0-1.2).

[0020] The main function of compound 14 is to provide targeting effect and bind to TTK.

[0021] According to some embodiments of the present invention, the organic solvent 1 is selected from at least one of N,N-dimethylformamide and dichloromethane.

[0022] According to some embodiments of the present invention, the reaction is carried out at room temperature for 2 to 6 hours.

[0023] According to some embodiments of the present invention, the condensing agent includes at least one of a carbodiimide condensing agent, an onium salt condensing agent, and an organophosphorus condensing agent.

[0024] According to some embodiments of the present invention, the carbodiimide condensing agent includes at least one of DIC, DCC and EDCI.

[0025] According to some embodiments of the present invention, the onium salt condensing agent includes at least one of HATU, HBTU, HCTU, TBTU, HAPyU, BOP, and PyBOP.

[0026] According to some embodiments of the present invention, the organophosphorus condensing agent includes at least one of DPP-Cl, DPPA, MPTA, BOP-Cl, T3P and T4P.

[0027] Preferably, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole (EDCI).

[0028] According to some embodiments of the present invention, the preparation method of compound 8 comprises the following steps:

[0029] Compound 7 is reacted with a base in an organic solvent 2 to obtain compound 8;

[0030] Wherein, the structural formula of the compound 7 is as follows:

[0031]

[0032] According to some embodiments of the invention, the base comprises sodium hydroxide.

[0033] According to some embodiments of the present invention, the molar ratio of compound 7 to sodium hydroxide is 1:(5-6).

[0034] According to some embodiments of the present invention, the molar concentration of sodium hydroxide in the solution is 0.4M.

[0035] According to some embodiments of the present invention, the organic solvent 2 is selected from at least one of methanol, ethanol and water.

[0036] According to some embodiments of the present invention, the reaction is stirred at room temperature for 4 to 6 hours.

[0037] According to some embodiments of the present invention, the reaction further comprises filtering, washing and concentrating under reduced pressure after completion.

[0038] According to some embodiments of the present invention, the preparation method of compound 7 comprises the following steps:

[0039] Compound 5, compound 6 and a catalyst are refluxed in an organic solvent 3 to obtain compound 7;

[0040] Wherein, the structural formulas of compound 5 and compound 6 are as follows:

[0041]

[0042] According to some embodiments of the present invention, the molar ratio of compound 5, compound 6 and piperidine is 1:(1.2-1.5):0.5.

[0043] According to some embodiments of the present invention, the organic solvent 3 is selected from at least one of acetonitrile and ethanol.

[0044] According to some embodiments of the present invention, the catalyst comprises at least one of a piperidine group, triethylamine, a pyridine group, and DIPEA, wherein the catalysts are all weakly basic.

[0045] Preferably, the catalyst is a piperidine group.

[0046] According to some embodiments of the present invention, the reflux reaction is performed under an inert atmosphere.

[0047] According to some embodiments of the present invention, the inert atmosphere comprises at least one of nitrogen, argon and neon.

[0048] According to some embodiments of the present invention, the preparation method of compound 5 comprises the following steps:

[0049] p-Fluorobenzaldehyde, ethyl 2-(piperazin-1-yl)acetate and an inorganic base are added to the organic solvent 4 for reflux reaction to obtain compound 5.

[0050] According to some embodiments of the present invention, the molar ratio of p-fluorobenzaldehyde, ethyl 2-(piperazin-1-yl)acetate and the inorganic base is 1:(0.8-1):(1.2-2.5).

[0051] According to some embodiments of the present invention, the organic solvent 4 is selected from at least one of acetonitrile and toluene.

[0052] According to some embodiments of the invention, the inorganic base includes at least one of potassium carbonate and sodium carbonate.

[0053] According to some embodiments of the present invention, the temperature of the reflux reaction is 100-120°C.

[0054] According to some embodiments of the present invention, the reflux reaction time is 12 to 24 hours.

[0055] According to some embodiments of the present invention, the reflux reaction further includes separation and purification treatment.

[0056] According to some embodiments of the present invention, the separation and purification step comprises: washing, extraction, and silica gel chromatography purification in sequence.

[0057] According to some embodiments of the present invention, the washing is performed with water.

[0058] According to some embodiments of the invention, the extraction uses at least one of DCM, ethyl acetate and diethyl ether.

[0059] According to some embodiments of the present invention, the eluent for purification by silica gel chromatography is at least one of petroleum ether and ethyl acetate.

[0060] According to some embodiments of the present invention, the volume ratio of petroleum ether to ethyl acetate is (12-20):1.

[0061] According to some embodiments of the present invention, the preparation method of compound 6 comprises the following steps:

[0062] 4-Methylpyridine is reacted with ethane halide in an organic solvent 5 to obtain compound 6.

[0063] According to some embodiments of the invention, the ethane halogenated compound comprises ethyl iodide.

[0064] According to some embodiments of the present invention, the molar ratio of the 4-methylpyridine to the ethyl iodide is 1:(1.5-3).

[0065] According to some embodiments of the present invention, the organic solvent 5 is selected from at least one of acetonitrile, dichloromethane, tetrahydrofuran and DMF.

[0066] According to some embodiments of the present invention, in step S2, the reaction is stirred at room temperature for 20 to 24 hours.

[0067] According to some embodiments of the present invention, the reaction further comprises filtration, concentration and silica gel chromatography purification.

[0068] According to some embodiments of the present invention, the eluent for the silica gel chromatography purification is at least one of PE and EA.

[0069] According to some embodiments of the present invention, the volume ratio of PE to EA is (5-1):1.

[0070] The third aspect of the present invention provides a threonine tyrosine kinase detection kit, comprising a fluorescent probe for threonine tyrosine kinase detection;

[0071] The fluorescent probe for detecting threonine tyrosine kinase is the compound represented by formula I.

[0072] The threonine tyrosine kinase detection kit according to the embodiment of the present invention has at least the following beneficial effects:

[0073] The fluorescent probe detection system obtained by the present invention constructs a method for detecting threonine tyrosine kinase with high sensitivity and accuracy, which is easy to use and popularize and apply.

[0074] According to some embodiments of the present invention, in the kit, the molar concentration of the fluorescent probe for threonine tyrosine kinase detection in the fluorescent probe stock solution is 1 to 10 μM.

[0075] Preferably, in the kit, the molar concentration of the fluorescent probe for threonine tyrosine kinase detection in the fluorescent probe stock solution is 2 μM.

[0076] According to some embodiments of the present invention, the solvent of the threonine tyrosine kinase detection kit is selected from at least one of PBS and dimethyl sulfoxide.

[0077] Preferably, the solvent of the threonine tyrosine kinase detection kit is a mixture of dimethyl sulfoxide and PBS.

[0078] According to some embodiments of the present invention, the mass ratio of the dimethyl sulfoxide to PBS mixture is 0.01 to 0.001.

[0079] According to some embodiments of the present invention, the content of dimethyl sulfoxide is 1%.

[0080] The fourth aspect of the present invention provides an application of the fluorescent probe for detecting threonine tyrosine kinase, including applications in detecting the concentration of threonine tyrosine kinase in an organism or evaluating the microenvironment within an organism cell for purposes other than disease diagnosis or treatment.

[0081] According to some embodiments of the present invention, the method for detecting the concentration of threonine tyrosine kinase in an organism comprises the following steps:

[0082] The fluorescent probe for detecting threonine tyrosine kinase is mixed with a sample to be tested; and the fluorescence ratio of the mixed solution is measured at 580-600 nm.

[0083] Unless otherwise specified, the term “about” in the present invention actually means that the error is allowed to be within the range of ±2%, for example, about 100 actually means 100±2%×100.

[0084] Additional features and advantages of the present invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0086] Figure 1 is the H NMR spectrum of compound 7 in Example 1 of the present invention;

[0087] Figure 2 is the carbon NMR spectrum of compound 7 in Example 1 of the present invention;

[0088] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of compound 8 in Example 1 of the present invention;

[0089] Figure 4 is the carbon NMR spectrum of compound 8 in Example 1 of the present invention;

[0090] Figure 5 is the HPLC chart of Compound Ⅰ in Example 1 of the present invention;

[0091] Figure 6 is the hydrogen nuclear magnetic resonance spectrum of compound I in Example 1 of the present invention;

[0092] Figure 7 This is the carbon NMR spectrum of compound I in Example 1 of the present invention

[0093] Figure 8 is the H NMR spectrum of compound 14 in Example 1 of the present invention;

[0094] Figure 9 is the carbon NMR spectrum of compound 14 in Example 1 of the present invention;

[0095] Figure 10 is the +IDA TOF MS spectrum of compound 14 in Example 1 of the present invention;

[0096] Figure 11 is a fluorescence intensity graph of Example 1 of the present invention in solvents of different polarities;

[0097] Figure 12 is a fluorescence intensity graph of Comparative Example 1 of the present invention in solvents of different polarities;

[0098] Figure 13 This is a graph of fluorescence intensity of Example 1 of the present invention in buffer solutions of different pH values;

[0099] Figure 14 This is a fluorescence intensity graph of Comparative Example 1 of the present invention in buffer solutions of different pH values;

[0100] Figure 15 This is a graph of fluorescence intensity of Example 1 of the present invention in mixed solutions of water and glycerol at different ratios;

[0101] Figure 16 This is a graph of fluorescence intensity of comparative example 1 of the present invention in mixed solutions of water and glycerol at different ratios;

[0102] Figure 17 1 is a fluorescence spectrum diagram of the TTK titration experiment of Comparative Example 1 and Example 1 of the present invention;

[0103] Figure 18 is a comparison diagram of the relationship between the fluorescence intensity and TTK concentration of Example 1 of the present invention and Comparative Example 1;

[0104] Figure 19 is a selective histogram of Example 1 of the present invention;

[0105] Figure 20 1 is a diagram of the cytotoxicity (MTT) test of Example 1 of the present invention and Comparative Example 1;

[0106] Figure 21 These are live cell imaging images of Example 1 of the present invention and Comparative Example 1;

[0107] Figure 22 This is the fluorescence co-localization imaging image of Example 1 of the present invention;

[0108] Figure 23 This is a time-dependent and concentration-dependent fluorescence imaging diagram of Example 1 of the present invention. DETAILED DESCRIPTION

[0109] The embodiments of the present invention are described in detail below. The same or similar reference numerals throughout the embodiments represent the same or similar elements or elements with the same or similar functions. The embodiments described below are exemplary and are only used to explain the present invention and are not to be construed as limiting the present invention.

[0110] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0111] In the description of the present invention, it should be understood that descriptions involving orientation, such as the orientation or positional relationship indicated by up, down, etc., are based on the orientation or positional relationship shown in the embodiment, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0112] The terms "preferably," "more preferably," and the like, used herein refer to embodiments of the present invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the present invention.

[0113] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0114] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of the present invention.

[0115] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0116] The raw materials and instruments used in the examples of the present invention are:

[0117] Instrument model and company:

[0118] Fluorescence spectrometer: Model: FLS1000, EDINBURGH INSTRUMENTS;

[0119] Nuclear magnetic resonance spectrometer: Model: Bruker AVANCE NEO 500, Bruker BioSpin SLS;

[0120] High performance liquid chromatography-tandem mass spectrometry: AB SCIEX / X500R QTOF, SCIEX;

[0121] Fully preparative high performance liquid chromatography: Model: 2545 / 2767 / QDa, WATERS CORPORATION;

[0122] Reagent source and manufacturer:

[0123] 4-Fluorobenzaldehyde: CAS: 459-57-4, Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0124] 2-(piperazin-1-yl)acetic acid ethyl ester, CAS: 40004-08-8, Shanghai Bid Pharmaceutical Technology Co., Ltd.;

[0125] Ethyl iodide: CAS: 75-03-6, Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0126] 4-Methylpyridine: CAS: 108-89-4, Anhui Zesheng Technology Co., Ltd.;

[0127] N,N-dimethylformamide: CAS: 68-12-2, Tianjin Damao Chemical Reagent Factory;

[0128] Potassium carbonate: CAS: 584-08-7, Tianjin Damao Chemical Reagent Factory;

[0129] Piperidine: CAS: 110-89-4, Anhui Zesheng Technology Co., Ltd.;

[0130] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI): San Chemical Technology (Shanghai) Co., Ltd.

[0131] 1-Hydroxybenzotriazole (HOBt): San Chemical Technology (Shanghai) Co., Ltd.;

[0132] Human colon cancer cells (HT-29): Guangzhou Suyan Biotechnology Co., Ltd.

[0133] DAPI:CAS:28718-90-3, Biotime Biotech

[0134] TTK(N1):sc-56968, Gene Biotechnology International Trading (Shanghai) Co., Ltd. Guangzhou Branch

[0135] Example 1

[0136] This embodiment provides a fluorescent probe for detecting threonine tyrosine kinase, the structural formula of which is as follows:

[0137]

[0138] This embodiment also provides a method for preparing the fluorescent probe for detecting threonine tyrosine kinase. The specific steps are as follows:

[0139] (1) To a solution of p-fluorobenzaldehyde (3 g, 24.17 mmol) in N,N-dimethylformamide (10 mL) was added ethyl 2-(piperazin-1-yl)acetate (6.24 g, 36.25 mmol), followed by potassium carbonate (8.35 g, 60.4 mmol). The resulting mixture was stirred at 100°C for 12 h. After completion of the reaction as monitored by TLC, the solution was cooled to room temperature and poured into 50 ml of water. The aqueous phase was extracted with DCM (3 × 50 mL). The organic phases were combined, dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel chromatography using petroleum ether / ethyl acetate (12:1, v / v) as the eluent to obtain compound 5 (3.67 g, 13.28 mmol, yield 55%).

[0140] The test results of the obtained product compound 5 are: 1 H NMR(500MHz,Chloroform-d)δ9.78(s,1H),7.75(d,J=8.9Hz,2H),6.91(d,J=8.9Hz,2H),4.2 1(q,J=7.1Hz,2H),3.51–3.41(m,4H),3.28(s,2H),2.80–2.67(m,4H),1.29(t,J=7.2Hz,3H). 13 C NMR (126MHz, Chloroform-d) δ190.49,170.05,154.95,131.88,127.21,113.62,60.83,59.26,52.52,47.01,14.26.

[0141] From the above spectral data, it can be confirmed that the structure of the obtained compound 5 is as follows:

[0142]

[0143] (2) Iodoethane (1 g, 6.41 mmol) was added to a solution of 4-methylpyridine (1 g, 10.74 mmol) and anhydrous acetonitrile (20 mL). The resulting mixture was stirred at room temperature for 24 h. After filtration and concentration, the mixture was purified by silica gel column chromatography (PE:EA = 1:1) to afford compound 6 (718.58 mg, 5.89 mmol, yield: 91.9%).

[0144] The test results of the obtained product, Compound 6, were as follows: 1H NMR (500 MHz, Chloroform-d) δ (ppm): 8.66 (d, J = 6.3 Hz, 2H), 7.47 (d, J = 6.2 Hz, 2H), 4.34 (q, J = 7.2 Hz, 2H), 2.19 (d, J = 5.4 Hz, 3H), 1.15 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, Chloroform-d) δ (ppm): 158.80, 143.14, 128.75, 56.08, 22.23, 16.86.

[0145] From the above spectral data, it can be confirmed that the structure of the obtained compound 6 is as follows:

[0146]

[0147] (3) Compound 6 (146 mg, 1.2 mmol) was added to a solution of compound 5 (276 mg, 1 mmol) and piperidine (50 uL) in anhydrous ethanol (10 mL). The mixture was refluxed at 80°C for 6 h under nitrogen protection. After the reaction was completed, the reaction solution was cooled to room temperature. An orange-red solid precipitated. The mixture was filtered and the orange-red solid was washed with ether to obtain compound 7 (346.56, 0.912 mmol, yield: 91.2%). The H NMR spectrum and C NMR spectrum of compound 7 are shown in Figure 2. Figure 1 and 2 shown.

[0148] The test results of the obtained product compound 7 are as follows: 1H NMR (500 MHz, DMSO-d6) δ8.85 (d, J = 6.9 Hz, 2H), 8.12 (d, J = 6.9 Hz, 2H), 7.94 (d, J = 16.2 Hz, 1H), 7.62 (d, J = 8.9 Hz, 2H), 7.27 (d, J = 16.2 Hz, 1H), 7.02 (s, 2H), 4.48 (q, J = 7.3 Hz, 2H), 4.10 (q, J = 7.1 Hz, 2H), 3.34–3.31 (m, 4H), 3.30 (s, 2H ),2.69–2.61(m,4H),1.51(t,J=7.3Hz,3H),1.20(t,J=7.1Hz,3H).13CNMR(126MHz,DMSO-d6)δ170.34,153.95,15 2.73,143.96,141.88,130.37,125.17,123.26,119.05,114.90,60.37,58.71,55.29,52.10,47.30,16.65,14.63.

[0149] From the above spectral data, the structure of the obtained compound 7 can be confirmed as follows:

[0150]

[0151] (4) Compound 7 (100 mg, 0.26 mmol) was added to 6 ml of ethanol, and 0.4 M sodium hydroxide (2 ml, 0.8 mmol) was slowly added dropwise to the reaction solution. The mixture was stirred at room temperature for 4 h. After the reaction was completed by TLC monitoring, 0.4 M hydrochloric acid (2 ml, 0.8 mmol) was added dropwise to neutralize the pH of the reaction solution. The mixture was concentrated under reduced pressure and the solid was dissolved with (DCM: MeOH = 20:1). The sodium chloride salt was filtered out and the solvent was concentrated under reduced pressure again. The solid was dissolved with 50 mL of H2O, and the aqueous phase was washed with DCM (3×50 mL). The aqueous phase was concentrated under reduced pressure to precipitate an orange solid, which was filtered and washed with ether to obtain compound 8 (43.44 mg, 0.123 mmol, yield: 46.9%). The H NMR spectrum and C NMR spectrum of compound 8 are shown in the figure. Figure 3 and 4 shown.

[0152] The test results of the obtained product compound 8 are as follows: 1H NMR (500 MHz, DMSO-d6) δ8.84 (d, J = 7.6 Hz, 2H), 8.11 (d, J = 6.9 Hz, 2H), 7.93 (d, J = 16.1 Hz, 1H), 7.61 (d, J = 8.9 Hz, 2H), 7.26 (d, J = 16.1 Hz, 1H), 7.03 (d, J = 8.9 Hz, 2H), 4.47 (q, J = 7.3 Hz, 2H), 3.36–3.32 (m, 4H), 3.20 (s, 2H), 2.71–2.64 (m, 4H), 1.50 (t, J = 7.3 Hz, 3H). 13C NMR (126MHz, DMSO-d6) δ171.49,153.95,152.69,143.96,141.89,130.38,125.18,123.27,119.07,114.90,58.97,55.29,52.14,47.09,16.64.

[0153] From the above spectral data, it can be confirmed that the structure of the obtained compound 8 is as follows:

[0154]

[0155] (5) Compound 8 (26 mg, 0.07 mmol) was added to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (16.9 mg, 0.08 mmol), 1-hydroxybenzotriazole (HOBt) (11.96 mg, 0.08 mmol) and compound 14 (38 mg, 0.07 mmol) (the structure of compound 14 was confirmed as follows Figures 8-10 The mixture was added to a solution of N,N-dimethylformamide (5 mL) and stirred at room temperature for 4 h. After the reaction was completed by TLC monitoring, the mixture was poured into 50 ml of water, and the aqueous phase was extracted with DCM (3×50 mL). The organic phases were combined, dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel chromatography to obtain the compound of formula (I) (20.8 mg, 0.0243 mmol, yield: 33.01%). The structure was confirmed by HPLC. The mobile phase method was as shown in Table 1. The structural confirmation results of the compound of formula (I) are shown in Table 1. Figures 5-7 shown.

[0156] The test results of the obtained product formula (I) compound are as follows: 1H NMR (500 MHz, Methanol-d4) δ8.73 (s, 1H), 8.68 (d, J = 6.7 Hz, 2H), 8.06 (d, J = 6.7 Hz, 2H), 7.90 (d, J = 8.7 Hz, 1H), 7.86 (d, J = 16.1 Hz, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.19 (d, J = 16.1 Hz, 1H), 7.03 (d, J = 8.8 Hz, 2H), 6.75 (d, J = 2.2 Hz, 1H), 6.62 (dd, J = 8.8, 2.3 Hz, 1H), 6.19 (s, 1H), 5.51 (s, 1H), 4.53 (q, J = 7.3 Hz, 2H), 4.0 6(s,1H),3.92(s,3H),3.87–3.83(m,2H),3.83–3.79(m,2H),3.49(s,2H),3. 46–3.40(m,4H),3.30–3.24(m,2H),3.24–3.18(m,2H),2.82–2.77(m,4H),2. 74(d,J=13.1Hz,2H),2.42(s,3H),2.32(q,J=7.6Hz,2H),1.97(d,J=13.6Hz, 2H),1.64(t,J=7.3Hz,6H),1.51(d,J=10.4Hz,2H),1.19(t,J=7.6Hz,3H).13C NMR (126MHz, Methanol-d4) δ175.30,168.20,166.15,164.72,156.37,154.72,152.85,148.78,146.62,142.94,142.17,129.86,125.40,122.92,12 0.98,118.16,117.29,114.60,107.70,107.13,100.76,59.71,55.40,54. 96,50.31,45.32,43.56,41.70,29.71,28.86,22.56,15.76,15.21,9.38.

[0157] From the above spectral data, it can be confirmed that the structure of the compound of formula (I) obtained is as follows:

[0158]

[0159] Table 1 Solvent gradient in HPLC

[0160]

[0161] Comparative Example 1

[0162] Compound 8 prepared in Example 1 was used as this comparative example.

[0163] Test Example 1

[0164] This test example takes Example 1 and Comparative Example 1 of the present invention as examples to detect the effects of different polar environments, different viscosities, and different pH values ​​on fluorescence intensity.

[0165] Different polarity environment testing

[0166] Fluorescence intensity was measured using dioxane, tetrahydrofuran, N,N-dimethylformamide, acetonitrile, methanol, and water as solvents. 10 mM stock solutions (mother solutions) of the compound of Formula I in Example 1 and the compound of Comparative Example 1 were prepared in dimethyl sulfoxide (DMSO). The mother solutions of the compound of Formula I in Example 1 and the compound of Comparative Example 1 were then added to water, dioxane, tetrahydrofuran, N,N-dimethylformamide, acetonitrile, and methanol, respectively, to obtain a final test concentration of 10 μM. In all measurements, the excitation wavelength was 449 nm, the excitation slit width was 1.5 nm, and the emission slit width was 1.5 nm. The results are shown in FIG. Figure 11 and 12 There is no linear relationship between the emission intensity of the compound of Comparative Example 1 and the compound of Example 1 and the solvent polarity.

[0167] Different pH value tests

[0168] The effect of PBS buffer solution with a pH of 3.0 to 11.0 on fluorescence intensity was tested. 10 mM stock solutions of the compound of Comparative Example 1 and the compound of Formula I in Example 1, as well as 1 mM hydrochloric acid solution and 1 mM sodium hydroxide solution were prepared in PBS. A pH detector was used to prepare test solutions of the compound of Comparative Example 1 and the compound of Formula I in Example 1 at pH = 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5 and 11, respectively. The emission spectra of the compound of Formula I in Example 1 and the compound of Comparative Example 1 were measured in a PBS buffer solution with a pH of 3.0-11.0 at room temperature under 396 nm excitation. The pKa values ​​of the compounds were calculated by fitting using Origin software. The pKa values ​​of Example 1 and Comparative Example 1 were 5.73±2% and 7.64±2%, respectively. Figure 13 and 14 As shown, the fluorescence intensity of the compound of Formula I in Example 1 is a function of pH, with significant relative changes within the pH range of 4.5 to 7.5, which can be used to detect changes in the cellular microenvironment. Therefore, the compound in Example 1 is an environmentally responsive molecule that can be further used to detect TTK and changes in the cellular microenvironment.

[0169] Different viscosity tests

[0170] In order to avoid the influence of changes in solvent polarity, two solvents with similar or identical polarity but large differences in viscosity are generally selected. By changing the ratio of the two solvents, solvents with different viscosity values ​​are obtained.

[0171] In this experiment, water (ET(30)=63.1, η20℃=1cP) and glycerol (ET(30)=57, η20℃=1317cP) were used to prepare water-glycerol solutions with different volume ratios, mixed them and set aside. Figure 15 and 16 As shown, the increase in solvent viscosity reduces ineffective collisions between molecules and restricts the rotation of the compound structure, thereby enhancing the molecular rigidity. It is measured that the fluorescence intensity of the compounds in Comparative Example 1 and Example 1 tends to increase with increasing viscosity.

[0172] Test Example 2

[0173] In order to evaluate the selectivity of the compound of formula I in Example 1 for TTK, a 10mM DMSO stock solution was first prepared and then diluted in PBS to finally obtain a 2μM concentration of the compound solution. The compound of Example 1 (2μM) was selected and reacted with threonine tyrosine kinase (3.51μM), bovine serum albumin (8.0μM), glutathione (8.0μM), cellulase (8.0μM), human serum albumin (8.0μM), pepsin (PEP) (8.0μM) and acetylcholinesterase (AchE) (8.0μM) in PBS buffer at 37°C for 10 minutes, and the fluorescence emission spectrum was recorded at an excitation wavelength of 419nm. Compared with the increase in TTK concentration in column 1, the fluorescence intensity did not change significantly. The responsive fluorescence spectra to different concentrations are shown in the figure below. Figure 17 As shown. The excitation slit width is 2.5nm, the emission slit width is 2.5nm, and the results are as follows Figure 18 and 19 As shown, in the presence of various biological enzymes, the fluorescence intensity remained essentially unchanged, while only TTK significantly enhanced the fluorescence intensity of the compound of Example 1 by about 8 times, indicating that the compound of Formula I in Example 1 has a high selectivity for TTK. Figure 18 and 19 Where I represents the fluorescence intensity, I0 is the fluorescence intensity of Example 1 without adding 2 μM protein, and (I-I0) is the increased fluorescence intensity after adding protein, and dividing it by I0 is the multiple of the increased fluorescence intensity.

[0174] Test Example 3

[0175] The cytotoxicity of the compounds of Example 1 and Comparative Example 1 was tested. HT-29 cells were cultured in McCoyS 5A medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were incubated in a 37°C, 5% CO2 incubator. The cytotoxic effects of the compounds of Example 1 and Comparative Example 1 were detected by MTT assay. Logarithmic phase cells were collected, the concentration of the cell suspension was adjusted, 100 μl was added to each well of a 96-well plate, and the plates were plated to adjust the density of the cells to be tested to 4000-8000 / well. Three replicates were plated and incubated at 37°C, 5% CO2 until the cells adhered. Then, different concentrations of the compounds of Example 1 and Comparative Example Compound 1 (0-160 μM) were incubated with HT-29 cells for 24 h. The cells were incubated with the thiazolyl blue solution for 4 h, the culture medium in the wells was discarded, 100 μl of dimethyl sulfoxide was added to each well, and the plates were shaken at low speed for 10 min to fully dissolve the crystals. The absorbance of each well was measured at OD 570nm in an enzyme-linked immunosorbent assay. Figure 20 As shown, the compound of Example 1 had no obvious side effects on cell viability within the active concentration range (0-40 μM).

[0176] Test Example 4

[0177] Live cell imaging and colocalization imaging were performed using the compound of Example 1 and the compound of Comparative Example 1. First, human colon cancer cells HT-29 with high TTK expression were incubated with the compound of Example 1 and the compound of Comparative Example 1 for 4 hours, and fluorescence imaging was performed as shown in FIG. Figure 21 The compound of Example 1 emits obvious fluorescence signal in HT-29 cells, while the compound of Comparative Example 1 does not emit obvious fluorescence signal in HT-29 cells. Figure 21As shown. In contrast, the compound of comparative example 1 showed low fluorescence emission in HT-29 cells. It can be seen that the compound of Example 1 can bind to biological molecules in living cells, and the fluorescence intensity is enhanced. In order to explore whether the molecule bound to Example 1 is TTK molecule, our research group tested TTK cell co-localization imaging. HT-29 cells were seeded on a 15mm confocal culture dish and allowed to adhere for 24 hours. The cell density was optimally 75%-85%. Then, they were incubated with Example 1 (10μM) at 37°C for 4h, the culture medium was discarded, and room temperature PBS was slowly added to the cells and washed 3 times, each time for 5 minutes. The cells were fixed with 4% paraformaldehyde solution at 4°C for 20 minutes and then permeabilized with 1ml 0.1% Triton X-100 at room temperature for 8 minutes. Then, they were washed 3 times with PBS at room temperature, the cells were blocked with 2% BSA for 30 minutes, and washed 3 times with PBS. Subsequently, the cells were treated with TTK antibody (N1) (1:200 dilution) (Santa Cruz sc-56968-200ug / mL) at 4°C, incubated overnight at 4°C in the dark (incubated at room temperature for 1 hour or in a dark humidity chamber at 4°C overnight), and washed three times with PBS for 5 minutes each time. Then the secondary antibody was used with goat anti-mouse IgG-Alexa The cells were incubated with 488 antibody 488 (1:500 dilution) and then washed 3 times with PBS. After further incubation with DAPI (5ug / mL) at room temperature for 20min, the cells were washed 3 times with PBS, and finally the cells were imaged. For fluorescence imaging, a Leica / TCS SP8 laser confocal fluorescence microscope with a 60x objective was used. Example 1 was excited at 488nm, and the emission spectrum was collected at 570-700nm; goat anti-mouse Alexa 488 was excited at 488nm, and the emission spectrum was collected at 500-570nm, and DAPI was excited at 405nm, and the emission spectrum was collected at 425-460nm. In HT-29 cells, the fluorescence imaging of Example 1 highly overlapped with the immunofluorescence imaging signal displayed by the TTK antibody, indicating that the compound of Example 1 specifically targets TTK in the cells. The results are as shown in FIG. Figure 22 shown.

[0178] In addition, the present invention also performed time- and concentration-dependent fluorescence imaging on the compound of Example 1, and the results were as follows: Figure 23 As shown, Figure 23 A shows that the fluorescence intensity of the compound of Example 1 gradually increases from 0.5h to 6h, and B shows that when the concentration of Example 1 increases from 0μM to 40μM, the fluorescence intensity also increases. The results of A and B are shown in Figure 2. Figure 23 As shown in C.

[0179] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A fluorescent probe for detecting threonine tyrosine kinase, characterized in that: The structural formula of the fluorescent probe is shown in Formula I:

2. A method for preparing a fluorescent probe according to claim 1, characterized in that: The preparation method comprises the following steps: Compound 8, compound 14, and a condensing agent are added to an organic solvent 1 to react to obtain a compound represented by formula I; Wherein, the structural formulas of compound 8 and compound 14 are as follows:

3. The preparation method according to claim 2, characterized in that The molar ratio of the compound 8 to the compound 14 is 1:(1.0-1.2).

4. The preparation method according to claim 2, characterized in that The organic solvent 1 is selected from at least one of N,N-dimethylformamide and dichloromethane.

5. The preparation method according to claim 2, characterized in that The reaction is carried out at room temperature for 2 to 6 hours.

6. The preparation method according to claim 2, characterized in that The condensing agent includes at least one of a carbodiimide condensing agent, an onium salt condensing agent and an organic phosphorus condensing agent.

7. The preparation method according to claim 2, characterized in that The preparation method of compound 8 comprises the following steps: Compound 7 is reacted with a base in an organic solvent 2 to obtain compound 8; Wherein, the structural formula of the compound 7 is as follows:

8. The preparation method according to claim 7, characterized in that The base includes sodium hydroxide, and the molar ratio of the compound 7 to sodium hydroxide is 1:(5-6).

9. The preparation method according to claim 7, characterized in that The alkali comprises sodium hydroxide, and the molar concentration of the sodium hydroxide in the solution is 0.2-1M.

10. The preparation method according to claim 7, characterized in that The organic solvent 2 is selected from at least one of methanol, ethanol and water.

11. The preparation method according to claim 7, characterized in that The reaction is stirred at room temperature for 4 to 12 hours.

12. The preparation method according to claim 7, characterized in that The preparation method of compound 7 comprises the following steps: Compound 5, compound 6 and a catalyst are refluxed in an organic solvent 3 to obtain compound 7; Wherein, the structural formulas of compound 5 and compound 6 are as follows: Wherein, the catalyst includes at least one of piperidine, triethylamine, pyridine and DIPEA.

13. The preparation method according to claim 12, characterized in that The catalyst is piperidine.

14. The preparation method according to claim 13, characterized in that The molar ratio of the compound 5, the compound 6 and piperidine is 1:(1.2-1.5):0.

5.

15. The preparation method according to claim 12, characterized in that The organic solvent 3 is selected from at least one of acetonitrile and ethanol.

16. The preparation method according to claim 12, characterized in that The preparation method of compound 5 comprises the following steps: p-Fluorobenzaldehyde, ethyl 2-(piperazin-1-yl)acetate and an inorganic base are added to the organic solvent 4 for reflux reaction to obtain compound 5.

17. The preparation method according to claim 12, characterized in that The preparation method of compound 6 comprises the following steps: 4-Methylpyridine is reacted with ethane halide in an organic solvent 5 to obtain compound 6.

18. The preparation method according to claim 17, characterized in that: The molar ratio of the 4-methylpyridine to the halogenated ethane is 1:(1.5-3).

19. The preparation method according to claim 16, characterized in that The molar ratio of the p-fluorobenzaldehyde, ethyl 2-(piperazin-1-yl)acetate and the inorganic base is 1:(0.8-1):(1.2-2.5).

20. The preparation method according to claim 16, characterized in that The organic solvent 4 is selected from at least one of acetonitrile and toluene.

21. The preparation method according to claim 17, characterized in that The organic solvent 5 is selected from at least one of acetonitrile, dichloromethane, tetrahydrofuran and DMF.

22. A threonine tyrosine kinase detection kit, characterized in that: Includes fluorescent probes for threonine tyrosine kinase detection; The fluorescent probe for detecting threonine tyrosine kinase comprises the compound represented by formula I according to claim 1.

23. Use of the fluorescent probe for detecting threonine tyrosine kinase according to claim 1 in detecting the concentration of threonine tyrosine kinase in an organism or evaluating the intracellular microenvironment of an organism for purposes other than disease diagnosis or treatment.

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