Cytochrome oxidase P450 fluorescent probe based on olefin epoxidation elimination and application of cytochrome oxidase P450 fluorescent probe

Abstract

The invention discloses a cytochrome oxidase P450 fluorescent probe based on olefin epoxidation elimination and application of the cytochrome oxidase P450 fluorescent probe, and belongs to the technical field of biological medicine and analytical chemistry. Aiming at the problems that an existing CYP450 enzyme probe is single in recognition site (only limited to dealkylation and hydroxylation), insufficient in selectivity and the like, a series of CYP450 fluorescent probes which take olefin as recognition units and have different electron donating and withdrawing substituent groups are synthesized, and the CYP450 fluorescent probes have the following general formula. In the general formula (I) of # imgabs0, R1 is a fluorophore and is derived from hydroxyl fluorescent dye, and the hydroxyl fluorescent dye is selected from one of 7-hydroxycoumarin, 4-hydroxynaphthylamide, a heterozygote (hydroxymethyl rhodamine fluorescent dye) of rhodamine and fluorescein dye, resorufin dye, dicyanomethylene-4H-pyran derivative (hydroxyl DCM dye) and hydroxyl hemicyanine lake dye; r2 is selected from one of hydrogen and chlorine atoms; r3 is selected from one of hydrogen, chlorine atoms and methyl; and R4 is selected from one of hydrogen, carboxyl, phenyl and methyl.

Description

Technical Field

[0001] The present invention belongs to the fields of biopharmaceutical technology and analytical chemistry, and particularly relates to a class of cytochrome oxidase P450 fluorescent probes based on olefin epoxidation elimination and their applications. Background Art

[0002] Cytochrome P450 is an important class of monooxygenases (CYP450) in organisms, especially abundant in the liver, mainly located on the endoplasmic reticulum or mitochondrial membrane. They play a key role in various physiological processes such as drug metabolism, hormone synthesis, and toxin clearance. CYP450 is involved in the metabolism of a variety of endogenous substances (such as fatty acids, steroids, etc.) and exogenous substances (drugs, carcinogens, etc.). The change of its activity directly affects the efficacy and toxicity of related drugs, and further affects the treatment effects of diseases such as inflammation, liver injury, and cancer. Therefore, studying the enzyme activity changes and action mechanisms of CYP450 in these diseases has important value for drug development, disease prevention and control, and early diagnosis.

[0003] CYP450 is a large supergene family, containing multiple subtypes, and there are significant differences among different subtypes in terms of substrate preference, expression distribution, genetic polymorphism, etc. It has been found that there are more than 50 subtypes of enzymes in humans, mainly including three families: CYP1, CYP2, and CYP3. Among them, CYP1A2, 2A6, 2C9, 2C19, 2E1, 2D6, and 3A4 are important drug-metabolizing enzymes mainly expressed in the liver, small intestine, lung, placenta, and kidney, accounting for about 80% of drug metabolism. The functional activation of CYP450 depends on the synergistic action of coenzyme NADPH and oxygen (O2). This enzyme mainly participates in oxidation reactions during the biotransformation process of drugs, involving electron loss, dehydrogenation, and oxidation reactions. A large number of studies have found that CYP450 can catalyze many types of chemical reactions, including hydroxylation of carbon atoms, dealkylation of N-, O-, and S atoms, sulfur oxidation, epoxidation, deamidation, desulfurization, dehalogenation, peroxidation, N-oxidation reduction, C-C bond cleavage, etc. In addition, different subtypes of CYP450 can selectively catalyze different types of chemical reactions according to the structural characteristics of the enzyme catalytic cavity and substrate preference. Therefore, developing tools that can specifically detect the activities of different CYP450 subtypes is of great significance for drug research and development, disease diagnosis, and treatment monitoring.

[0004] Currently, there are some methods for detecting CYP450 activity, such as traditional chromatography, mass spectrometry, spectroscopy, etc. However, these methods have disadvantages such as complex operation, low sensitivity, the need for a large experimental volume, and high enzyme concentration, and are not suitable for high-throughput screening and in vivo or in vitro detection applications. Fluorescence imaging technology has been widely used in modern biomedical research due to its advantages of high sensitivity, non-invasiveness, and real-time monitoring. As a practical tool for fluorescence imaging, small molecule fluorescent probes can monitor the activity of target enzymes in real time in complex biological systems, and play an important role in understanding the metabolic dynamics of CYP450 in vivo and evaluating the interaction between each CYP450 subtype and drugs.

[0005] However, there are still some problems with existing CYP450 fluorescent probes. For example, the probe recognition unit is single, and the recognition reaction mainly focuses on dealkylation and hydroxylation, and the unique catalytic characteristics of different CYP450 subtypes cannot be fully utilized, resulting in the problem of insufficient probe specificity; at the same time, it also limits the detection objects of the probe, mainly focusing on subfamilies such as CYP3A and CYP1A. There are still many important CYP450 subtypes without specific probes, such as CYP2C19. The lack of specific probes for specific subtypes limits the in-depth understanding of the functions and mechanisms of these CYP450 subtype enzymes. Therefore, it is urgent to develop new CYP450 fluorescent probes with higher specificity and wider applicability based on the catalytic characteristics and spatial structures of different CYP450 subtypes. Summary of the Invention

[0006] Aiming at the deficiencies of the existing CYP450 probe recognition reaction being single and the detection of CYP450 subtypes being limited, the purpose of the present invention is to provide a class of cytochrome oxidase P450 fluorescent probes based on olefin epoxidation elimination and their applications. This class of fluorescent probes can utilize the epoxidation activity of different CYP450 subtype enzymes to achieve highly sensitive and specific detection of the activities of different CYP450 subtype enzymes; such new fluorescent probes can be applied to the screening of specific inhibitors of CYP450 subtype enzymes and the imaging of CYP450 enzyme activities in cells / tissues, etc.; the present invention provides a new analysis tool for the research of CYP450-related biomedical processes and the research and development of disease drugs, and has good application prospects in the field of biochemical analysis.

[0007] The present invention provides a class of cytochrome oxidase P450 fluorescent probes based on olefin epoxidation elimination, and its general structural formula is shown in (I):

[0008]

[0009] In general formula (I): R1 is a fluorophore, which is derived from a hydroxy fluorescent dye, and the hydroxy fluorescent dye is selected from the group consisting of 7-hydroxycoumarin, 4-hydroxynaphthamide, rhodamine, a hybrid of fluorescein dye (hydroxymethylrhodamine fluor dye), resorufin dye, dicyanomethylene-4H-pyran derivative (hydroxy DCM dye), and hydroxy hemicyanine Hubei dye;

[0010] R2 is selected from the group consisting of hydrogen and chlorine atom;

[0011] R3 is selected from the group consisting of hydrogen, chlorine atom, and methyl;

[0012] R4 is selected from the group consisting of hydrogen, carboxyl, phenyl, and methyl.

[0013] In a preferred embodiment, the structural formula of R1 is:

[0014]

[0015] In a further preferred embodiment, the structural formula of the fluorescent probe is:

[0016]

[0017] In a preferred embodiment, R2 and R3 are chlorine atoms, and R4 is a hydrogen atom, and the fluorescent probe includes any one of the following structural formulas:

[0018]

[0019] In a preferred embodiment, R2, R3, and R4 are all hydrogen atoms, and the fluorescent probe includes any one of the following structural formulas:

[0020]

[0021] In a preferred embodiment, the fluorophore may also be an organelle-targeted substituted fluorescent dye.

[0022] In addition, the inventors characterized the product by means of 1H NMR, 13C NMR, mass spectrometry, ultraviolet spectroscopy, etc., indicating that the cytochrome P450 fluorescent probe based on olefin epoxidation elimination was successfully synthesized.

[0023] The present invention also provides the application of the fluorescent probe in the detection of CYP450 different subtype enzyme activities.

[0024] Specifically, the present invention provides the application of the fluorescent probe in the detection of CYP450 different subtype enzyme activities in vitro, in cells or in vivo.

[0025] Specifically, the present invention provides the application of the fluorescent probe in the detection of CYP450 different subtype enzyme activities in live small animals.

[0026] Specifically, the present invention provides the application of the fluorescent probe in the preparation of a kit or test strip for detecting the activities of different subtypes of CYP450 enzymes.

[0027] Specifically, the present invention provides the application of the fluorescent probe as a specific fluorescent probe substrate for CYP2C family enzyme CYP2C19. As a specific substrate for CYP2C19, this substrate undergoes an olefin epoxidation hydrolysis reaction. The activity of CYP2C19 in biological samples such as enzyme or cell preparation solutions and cells is determined by quantitatively detecting the amount of de-olefination hydrolysis products generated per unit time. The specific measurement method is as follows:

[0028] In the system, a derivative with O-chloroethylene as the recognition unit is used as the specific probe substrate for CYP2C19. In 100 mM potassium phosphate buffer, the reaction temperature is between 20 °C and 45 °C, the incubation pH environment is between 5.5 - 10.5, preferably pH 7.4 is the optimal reaction value, the reaction time is 0 - 120 min. Ensure that the de-olefination products of the above substrate reach the quantification limit to terminate the reaction. The substrate and products can be rapidly and sensitively detected simultaneously using an ultraviolet spectrophotometer and a fluorescence detector, which is used as an evaluation index for CYP2C19 enzyme activity.

[0029] Specifically, the present invention provides the application of the fluorescent probe as a specific fluorescent probe substrate for CYP1B family enzyme CYP1B1. As a specific substrate for CYP1B1, this substrate undergoes an olefin epoxidation hydrolysis reaction. The activity of CYP1B1 in biological samples such as enzyme or cell preparation solutions and cells is determined by quantitatively detecting the amount of de-olefination hydrolysis products generated per unit time. The specific measurement method is as follows:

[0030] In the system, a derivative with O-ethylene as the recognition unit is used as the specific probe substrate for CYP1B1. In 100 mM potassium phosphate buffer, the reaction temperature is between 20 °C and 45 °C, the incubation pH environment is between 5.5 - 10.5, preferably pH 7.4 is the optimal reaction value, the reaction time is 0 - 120 min. Ensure that the de-olefination products of the above substrate reach the quantification limit to terminate the reaction. The substrate and products can be rapidly and sensitively detected simultaneously using an ultraviolet spectrophotometer and a fluorescence detector. The fluorescence detection conditions are: the excitation wavelength is 405 nm, and the maximum emission wavelengths are 451 nm and 564 nm respectively, which is used as an evaluation index for CYP1B1 enzyme activity.

[0031] The cytochrome oxidase P450 fluorescent probe based on olefin epoxidation elimination provided by the present invention. The olefin epoxidation elimination specific probe reaction can be used for the quantitative determination of the activities of enzymes such as CYP2C19 and CYP1B1 in recombinant single enzymes, human and animal tissue preparation solutions, and various tissue cells, and can also be used for the rapid screening of inhibitors of enzymes such as CYP2C19 and CYP1B1 and the quantitative evaluation of their inhibitory abilities.

[0032] The investigation was carried out using a recombinant CYP450 monoenzyme and a liver microsome incubation system. Through various evidence such as correlation analysis, specific inhibition experiments, recombinant monoenzyme metabolic reactions, and enzyme reaction kinetics, it was demonstrated that O-alkene derivatives can be specifically metabolized by enzymes such as CYP2C19 and CYP1B1 to generate corresponding hydrolysis products.

[0033] As a class of novel highly specific fluorescent probe substrates for CYP450 enzymes, these compounds can be used to detect the activities of enzymes such as CYP2C19 and CYP1B1. They are particularly suitable for the determination of the enzyme activities of CYP450 recombinant enzymes produced by bacterial, insect cell, mammalian cell, and yeast cloning expression systems, as well as the calibration of the activities of CYP450 in preparations such as tissue microsomes derived from various mammalian tissue organs.

[0034] The cytochrome oxidase P450 fluorescent probe designed in the present invention is based on olefin epoxidation elimination. Utilizing the epoxidation catalytic activity of CYP450 enzymes, an olefin recognition unit is introduced into the probe molecule. This unit can be catalytically epoxidized and eliminated by CYP450 enzymes to achieve fluorescence signal turning on or ratio detection and imaging.

[0035] Compared with the prior art, the beneficial technical effects are as follows:

[0036] 1) Inexpensive and easily available: It can be obtained through simple chemical synthesis. The synthetic raw materials are inexpensive and easily available, the synthetic process is simple and feasible, the cost is low, the economic and technical effects are obvious, and it is easy to promote.

[0037] 2) Novel recognition reaction mechanism: The present invention uses the olefin epoxidation elimination reaction as the response mechanism of the probe, which is different from the dealkylation and hydroxylation reactions of traditional CYP450 fluorescent probes. It can better utilize the catalytic characteristics of different CYP450 subtypes, is expected to improve the selectivity of the probe, and can be used for the detection of the activities of CYP450 subtype enzymes.

[0038] 3) High specificity and high sensitivity: By reasonably designing the olefin recognition unit and elimination group, it can be optimized for specific CYP450 subtype enzymes to improve the subtype selectivity of the probe for CYP450. The probe can utilize the olefin epoxidation elimination reaction of CYP450 subtype enzymes to cause significant changes in the fluorescent reporter group, thereby achieving high-sensitivity detection of CYP450 activity. The detection limits for CYP2C19 and CYP1B1 are 0.077 nM and 0.1 nM respectively.

[0039] 4) Wide application: This type of probe can be applied to multiple fields such as in vitro detection of CYP450 subtype enzyme activities, drug screening and metabolism, live cell and tissue and in vivo imaging, and disease monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the 1 1 H NMR spectrum of the fluorescent probe 1.1, Nath-2ClVe in Example 1;

[0041] Figure 2 is the 13 13 C NMR spectrum of the fluorescent probe 1.1, Nath-2ClVe in Example 1;

[0042] Figure 3 is the 1 1 HNMR spectrum of the fluorescent probe 1.2, Nath-Ve in Example 2;

[0043] Figure 4 is the 13 13 C NMR spectrum of the fluorescent probe 1.2, Nath-Ve in Example 2;

[0044] Figure 5 is the 1 1 H NMR spectrum of the fluorescent probe 3.1, ER-Nath-Ve in Example 3;

[0045] Figure 6 is the 1 1 H NMR spectrum of the fluorescent probe 2.7, HDS-2ClVe in Example 6;

[0046] Figure 7 is the fluorescence response spectra of the fluorescent probe 1.1, Nath-2ClVe (a) and the fluorescent probe 1.2, Nath-Ve (b) to different concentrations of rat liver microsomes in Example 7;

[0047] Figure 8 :(a) Fluorescence spectrum of the fluorescent probe 1.1, Nath-2ClVe in Example 8 in response to different concentrations of recombinant protein CYP2C19; (b) Relationship diagram between the fluorescence intensity ratio of the fluorescent probe 1.1, Nath-2ClVe and different concentrations of CYP2C19 in Example 8; (c) Selectivity of the fluorescent probe 1.1, Nath-2ClVe for different CYP450 subtype recombinant proteins in Example 9; (d) Enzyme activity kinetics test of the fluorescent probe 1.1, Nath-2ClVe for CYP2C19 in Example 10;

[0048] Figure 9: (a) Fluorescence spectra of the fluorescent probe 1.2, Nath-Ve in Example 8 in response to different concentrations of recombinant protein CYP1B1; (b) Relationship diagram between the fluorescence intensity ratio of the fluorescent probe 1.2, Nath-Ve in Example 8 and different concentrations of CYP1B1; (c) Selectivity of the fluorescent probe 1.2, Nath-Ve for recombinant proteins of different CYP450 subtypes in Example 9; (d) Enzyme activity kinetics test of the fluorescent probe 1.2, Nath-Ve for CYP1B1 in Example 10;

[0049] Figure 10 In vitro spectral screening of different candidate inhibitors of CYP2C19 by the fluorescent probe 1.1, Nath-2ClVe and determination of its half-inhibitor concentration in Example 11;

[0050] Figure 11 Fluorescent imaging of CYP2C19 enzyme activity in hepatocytes under the action of the hepatic drug enzyme inducer rifampicin (RIF) by the fluorescent probe 1.1, Nath-2ClVe in Example 12;

[0051] Figure 12 In vivo fluorescent imaging of changes in CYP2C19 enzyme activity in the liver of mice by the fluorescent probe 1.1, Nath-2ClVe in Example 13;

[0052] Figure 13 Fluorescent imaging of the activity of endogenous CYP1B1 in the endoplasmic reticulum of subcellular organelles in living cells by the fluorescent probe 3.1, ER-Nath-Ve in Example 14;

[0053] Figure 14 Ratio fluorescent imaging of the activity of endogenous CYP1B1 in mouse tumor tissues by the fluorescent probe 1.2, Nath-Ve in Example 15;

[0054] Figure 15 Schematic diagram of the specific detection and imaging principle of the CYP450 enzyme activity in biological samples by the CYP450 fluorescent probe for olefin epoxidation elimination. Detailed implementation manners

[0055] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0056] The present invention also provides a preparation method of the fluorescent probe 1.1, including:

[0057] Add the hydroxy fluorescent dye (R1-OH) and sodium hydroxide to dimethyl sulfoxide as the solvent, stir at 30-60 °C for 10-30 min, then add trichloroethylene and heat under reflux for 1-8 h, cool and extract, spin-dry the solvent and perform column chromatography separation to obtain the fluorescent probe 1.1;

[0058] Among them, the molar ratio of R1-OH, sodium hydroxide, and trichloroethylene is 1:(1-3):(1-3);

[0059] The molar volume ratio of R1-OH to dimethyl sulfoxide is 1:(5-7) mmol / mL.

[0060] The reaction process is as follows:

[0061]

[0062] The preparation methods of fluorescent probes 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, and 2.7 are similar to the preparation method of fluorescent probe 1.1.

[0063] The present invention also provides preparation methods of fluorescent probes 1.2, 3.2, 3.3, and 3.4, including:

[0064] Mix the hydroxy fluorescent dye (R1-OH) with ethanol and dissolve it by stirring at room temperature for 10-20 min, then add potassium carbonate and activate it at 60-80 °C for 30 min. Subsequently, add dibromoethane to the reaction solution and react for 8-12 h. After the reaction is completed, first filter to remove the alkali and undissolved R1-OH, and then use a CH2Cl2 / MeOH mixed solvent to purify the compound by gradient to obtain fluorescent probes 1.2, 3.2, 3.3, and 3.4.

[0065] Among them, the molar ratio of R1-OH, dibromoethane, and potassium carbonate is 1:(1-3):(1-3);

[0066] The molar volume ratio of R1-OH to ethanol is 1:(5-7) mmol / mL;

[0067] The molar ratio of compound 1 to potassium tert-butoxide is 1:(1-3); the molar volume ratio of compound 1 to dimethyl sulfoxide is 1:(5-7) mmol / mL.

[0068] The reaction process is as follows:

[0069]

[0070] The present invention also provides a preparation method of fluorescent probe 3.1, including:

[0071] Hydroxy fluorescent dye (R1-OH) and copper acetate were added to a mixed solvent of acetonitrile and N,N-dimethylformamide as the solvent, and then tetraethyltin was added. The reaction was stirred at room temperature for 8-16 h. After the reaction was completed, the reaction solution was filtered to remove insoluble solids, and the filtrate was distilled under reduced pressure to obtain a solid, which was separated by column chromatography to obtain probe 3.1;

[0072] Among them, the molar ratio of R1-OH, copper acetate, and tetraethyltin is 1:(1-3):(1-3);

[0073] The volume ratio of R1-OH to the mixed solution of acetonitrile and N,N-dimethylformamide is 9:1, and the molar volume ratio is 1:(5-7) mmol / mL;

[0074] The reaction process is as follows:

[0075]

[0076] The present invention also provides a preparation method of fluorescent probe 1.4, including:

[0077] Hydroxy fluorescent dye (R1-OH) and sodium hydroxide were dissolved in absolute ethanol, stirred at 30-60 °C for 10-30 min, then ethyl (Z)-3-iodoacrylate was added and heated under reflux for 1-4 h, cooled and extracted, and the solvent was evaporated to dryness and separated by column chromatography to obtain probe 1.4;

[0078] Among them, the molar ratio of R1-OH, sodium hydroxide, and ethyl (Z)-3-iodoacrylate is 1:(1-3):(1-3);

[0079] The molar volume ratio of R1-OH to ethanol is 1:(5-7) mmol / mL.

[0080] The reaction process is as follows:

[0081]

[0082] The present invention also provides a preparation method of fluorescent probe 1.5, including:

[0083] Dissolve the hydroxy fluorescent dye (R1-OH), 1-bromo-2-methyl-2-propanol, and potassium carbonate in absolute ethanol and heat under reflux for 6 - 12 h. Filter by suction to obtain the filtrate, rotary evaporate the solvent, and perform column chromatography separation to obtain Compound 2. Then, add a mixed solution of diethyl ether and 1,4-dioxane (volume ratio 3:1) as the solvent to Compound 2 and zinc chloride, place the reaction solution in an ice-water bath at 0 °C and stir, then dropwise add thionyl chloride, and react the reaction solution in the ice-water bath for 1 - 2 h. After the reaction is completed, dilute the mixture with diethyl ether, neutralize the reaction solution with saturated sodium bicarbonate in the ice-water bath, then filter by suction to obtain a yellow solid mixture, and perform column chromatography purification and separation to obtain Compound 3. Dissolve Compound 3 in dimethyl sulfoxide solution, slowly add potassium tert-butoxide under ice bath conditions, then transfer the reaction solution to room temperature and react for 2 - 4 h. After the reaction is completed, add water to quench the reaction, extract the reaction solution with ethyl acetate, collect the organic phase, and perform column chromatography separation and purification to obtain Probe 1.5;

[0084] Among them, the molar ratio of R1-OH, 1-bromo-2-methyl-2-propanol, and potassium carbonate is 1:(1 - 3):(1 - 3), and the molar volume ratio of R1-OH to absolute ethanol is 1:(2 - 5) mmol / mL;

[0085] The molar ratio of Compound 2 to zinc chloride and thionyl chloride is 1:(1 - 3):(1 - 3); the molar volume ratio of Compound 2 to the mixed solution of diethyl ether and 1,4-dioxane is 1:(2 - 5) mmol / mL;

[0086] The molar ratio of Compound 3 to potassium tert-butoxide is 1:(1 - 4); the molar volume ratio of Compound 1 to dimethyl sulfoxide is 1:(5 - 7) mmol / mL;

[0087] The reaction process is as follows:

[0088]

[0089] The present invention also provides a preparation method of Fluorescent Probe 1.6, including:

[0090] Dissolve the hydroxy fluorescent dye (R1-OH), copper iodide, 2-(2-pyridyl)-benzimidazole, and cesium carbonate in absolute ethanol, stir at 30 - 60 °C for 10 - 30 min, then add styryl bromide and heat under reflux for 8 - 16 h, cool and extract, rotary evaporate the solvent, and perform column chromatography separation to obtain Probe 1.6;

[0091] Among them, the molar ratio of R1-OH, copper iodide, 2-(2-pyridyl)-benzimidazole, cesium carbonate, and styryl bromide is 1:(0.1 - 0.5):(0.1 - 0.5):(1 - 3):(1 - 5);

[0092] The molar volume ratio of R1-OH to ethanol is 1:(5-7) mmol / mL;

[0093] The reaction process is as follows:

[0094]

[0095] In the present invention, after the substrate containing the fluorescent probe is mixed with the biological sample containing CYP450 enzyme, an enzymatic reaction is carried out, and it can be specifically catalyzed by CYP450 enzyme to eliminate olefin epoxidation to generate the corresponding hydroxyl product. The enzyme activity of CYP450 in different biological systems is quantitatively determined by quantitatively detecting the substrate elimination rate per unit time or the generation rate of its de-olefination product. The specific measurement methods and conditions are as follows:

[0096] A. In the system, a derivative containing an olefin recognition unit is used as the probe substrate; the substrate concentration is selected to be 1-30 μM;

[0097] B. In PBS buffer, the reaction temperature is between 20 °C and 45 °C, and the pH of the incubation system is between 5.5 and 10.5;

[0098] C. The reaction time is 5-120 minutes, and the reaction is terminated when the corresponding O-de-olefin product of the above substrate reaches the limit of quantification and the substrate conversion rate does not exceed 20%;

[0099] D. Measuring the decrease in substrate amount or the generation amount of O-de-olefination product per unit time as an evaluation index of CYP450 activity.

[0100] Furthermore, when performing single-point measurement, the substrate concentration is preferably 10 μM; the reaction temperature is preferably 37 °C; and preferably pH 7.4 is the optimal reaction pH value.

[0101] Furthermore, based on the fluorescence signals of the probe substrate and its de-olefination product, different detection wavelengths can be used for detection (in the emission range of 400-750 nm), or a single excitation wavelength and the ratio of double fluorescence emission wavelengths can be used for ratio fluorescence detection.

[0102] Furthermore, the biological system is any one of recombinant expressed CYP450 enzyme, liver microsomes, cells or tissue preparations of human or animal tissues.

[0103] The substrate containing the fluorescent probe can also be used for rapid screening of CYP450 inhibitors and quantitative evaluation of inhibitory ability in vitro, in cells or in vivo.

[0104] The substrate containing the fluorescent probe can also be used for real-time dynamic monitoring of CYP450 function in living cell tissues and in vivo, and evaluating the individual and species differences of the endogenous metabolic enzyme CYP450.

[0105] CYP450 enzyme activity assay procedure: Select the epoxidation elimination reaction of O-alkene derivatives as the probe reaction, and measure the actual activity of CYP450 enzymes in various biological samples by quantitatively detecting the production amount of its hydrolyzed metabolite hydroxyfluorophore per unit time. Further, by changing the structure of the fluorophore, CYP450 enzyme fluorescent probes with multiple emission wavelengths in the visible light to near-infrared light regions are obtained for imaging and detection of the enzyme activities in living cell organelles, tissues, and in vivo. The probe described in the present invention can not only be used for detecting the activity of CYP450 enzymes in biological samples, but also for rapid screening of CYP450 inhibitors and inducers in vitro and for studying drug interactions at the in vivo level.

[0106] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings.

[0107] Example 1 Synthesis of Fluorescent Probe 1.1, Nath-2ClVe

[0108] Take compound FP-1 (200 mg, 0.738 mmol) and sodium hydroxide (59.0 mg, 1.476 mmol) in a 25 mL round-bottom flask, add 5 mL of dimethyl sulfoxide as the solvent, stir at 45 °C for 20 min, then add trichloroethylene (193.4 mg, 1.476 mmol) and react for 2 h. After the reaction is completed, extract the reaction solution with a large amount of ethyl acetate, collect the organic phase and distill it under reduced pressure to obtain a solid, and then purify the compound by gradient with a DCM / MeOH mixed solvent to obtain the fluorescent probe 1.1, Nath-2ClVe (solid, 144.9 mg, yield 54%).

[0109] The structural formula of compound FP-1 is:

[0110]

[0111] The relevant characterization data of the fluorescent probe 1.1, Nath-2ClVe are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.68 (d, J = 7.3 Hz, 1H), 8.59 (t, J = 7.2 Hz, 2H), 7.82 (t, J = 7.7 Hz, 1H), 7.29 (s, 1H), 6.24–6.21 (m, 1H), 4.45 (t, J = 5.0 Hz, 2H), 3.73 (t, J = 5.1 Hz, 2H), 3.37 (s, 3H).

[0112] 1313C NMR (100 MHz, Chloroform-d) δ 163.61, 154.38, 138.71, 132.39, 132.24, 129.70, 128.10, 127.13, 123.30, 122.63, 118.64, 110.28, 106.76, 69.64, 58.84, 39.30.

[0113] Example 2 Synthesis of Fluorescent Probe 1.2, Nath-Ve

[0114] Weigh (200 mg, 0.738 mmol) of compound FP-1 into a 50 mL round-bottom flask, add 15 mL of ethanol as the solvent, stir at room temperature for 10 min to dissolve the compound. Subsequently, weigh potassium carbonate (508 mg, 3.69 mmol) and add it to the reaction system. Heat the temperature to 80 °C, stir for 30 min, then add dibromoethane (230 mg, 1.476 mmol) to the reaction solution, and react for 8 h. After the reaction is completed, first filter off the alkali and undissolved FP-1 by suction filtration, and then use a CH2Cl2 / MeOH mixed solvent to purify the compound by gradient to obtain the fluorescent probe 1.2, Nath-Ve (solid, 147.4 mg, 53%).

[0115] The relevant characterization data of the fluorescent probe 1.2, Nath-Ve are as follows: 1 1H NMR (400 MHz, Chloroform-d) δ 8.71–8.46 (m, 3H), 7.79 (d, J = 7.9 Hz, 2H), 7.25 (d, J = 7.8 Hz, 2H), 7.04 (d, J = 8.3 Hz, 1H), 5.77 (s, 1H), 4.64 (t, J = 5.8 Hz, 2H), 4.20 (t, J = 6.1 Hz, 2H), 3.88 (t, J = 5.8 Hz, 2H), 2.94 (d, J = 6.2 Hz, 2H), 2.37 (s, 3H), 1.94 (s, 2H).

[0116] 13 13C NMR (100 MHz, Chloroform-d) δ 164.78, 164.15, 159.47, 143.02, 137.32, 133.62, 132.02, 129.58, 129.37, 129.12, 127.06, 126.27, 123.41, 121.86, 115.10, 106.11, 68.52, 40.00, 36.94, 28.53, 28.01, 21.48.

[0117] Example 3 Synthesis of Endoplasmic Reticulum-Targeted Fluorescent Probe 3.1, ER-Nath-Ve

[0118] Weigh FP-7 (170.0 mg, 0.401 mmol) and copper acetate (159.2 mg, 0.802 mmol) into a 25 mL round-bottom flask, add 10 mL of a mixed solvent of acetonitrile and N,N-dimethylformamide (9:1) as the solvent, then add tetraethyltin (91 mg, 0.802 mmol). Stir the reaction at room temperature for 12 h. After the reaction is completed, filter the reaction solution to remove insoluble solids, distill the filtrate under reduced pressure to obtain a solid, and then purify the compound by gradient elution with a mixed solvent of DCM / MeOH to obtain the fluorescent probe 3.1, ER-Nath-Ve (solid, 77.0 mg, yield 43%).

[0119]

[0120] The relevant characterization data of the fluorescent probe 3.1, ER-Nath-Ve are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.67–8.52 (m, 3H), 7.83–7.76 (m, 3H), 7.32–7.23 (m, 3H), 6.91 (dd, J = 13.6, 5.8 Hz, 1H), 5.72 (s, 1H), 5.21 (dd, J = 13.6, 2.1 Hz, 1H), 4.89 (dd, J = 5.8, 2.2 Hz, 1H), 4.22 (t, J = 6.0 Hz, 2H), 2.95 (d, J = 6.1 Hz, 2H), 2.39 (d, J = 3.3 Hz, 3H), 1.95 (s, 2H).

[0121] 13 C NMR (100 MHz, Chloroform-d) δ 164.73, 164.11, 158.19, 145.81, 143.02, 137.37, 133.26, 132.18, 129.57, 128.94, 127.08, 126.55, 123.39, 122.01, 116.40, 109.33, 100.25, 39.96, 36.99, 28.02, 21.48. MS (ESI): calcd for C24H22N2O5S [M+] 450.12 found 451.36.

[0122] Example 4 Synthesis of the fluorescent probe 2.4, Res-2ClVe

[0123] Weigh the compound tryptanthrin (200 mg, 0.851 mmol) and sodium hydroxide (68.0 mg, 1.702 mmol) into a 25 mL round-bottom flask. Evacuate the flask with an oil pump and protect it with nitrogen. Then, add 5 mL of N,N-dimethylformamide as a solvent using a syringe. Stir the mixture at 110 °C for 20 min, and then add trichloroethylene (223.0 mg, 1.476 mmol) and react for 12 h. After the reaction, extract the reaction solution with a large amount of ethyl acetate. Collect the organic phase and distill it under reduced pressure to obtain a solid. Then, purify the compound by gradient elution with a DCM / MeOH mixed solvent to obtain the fluorescent probe 2.4, Res-2ClVe (solid, 65.39 mg, yield 25%).

[0124] The relevant characterization data of the fluorescent probe 2.4, Res-2ClVe are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 7.81 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 9.8 Hz, 1H), 7.09 (dd, J = 8.8, 2.7 Hz, 1H), 7.02 (d, J = 2.7 Hz, 1H), 6.86 (dd, J = 9.9, 2.0 Hz, 1H), 6.34 (d, J = 2.0 Hz, 1H), 6.13 (s, 1H).

[0125] 13 C NMR (100 MHz, Chloroform-d) δ 186.31, 145.11, 135.00, 134.81, 131.90, 114.47, 107.27, 106.32, 104.01. MS (ESI): calcd for C14H7Cl2NO3 [M+] 308.11, found 308.02.

[0126] Synthesis of the fluorescent probe 2.1, Cou-2ClVe, the fluorescent probe 2.2, Rh-2ClVe, and the fluorescent probe 2.5, DCM-2ClVe in Example 5

[0127] Adopt a synthesis method similar to that of the fluorescent probe 2.4, Res-2ClVe;

[0128] The fluorescent probe 2.1, Cou-2ClVe (solid, 70.8 mg, yield 88%) is synthesized. The relevant characterization data of the fluorescent probe 2.1, Cou-2ClVe are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 7.65 (d, J = 9.5 Hz, 1H), 7.07 (dd, J = 4.6, 2.3 Hz, 2H), 6.28 (s, 1H), 6.13 (s, 1H), 2.47 (s, 3H).13 C NMR (100 MHz, Chloroform-d) δ 160.51, 156.25, 154.77, 152.00, 138.94, 126.09, 116.67, 113.87, 113.20, 105.83, 104.98, 18.76.

[0129] The fluorescent probe 2.2, Rh-2ClVe was synthesized (solid, 14.5 mg, yield: 32%). The relevant characterization data of the fluorescent probe 2.2, Rh-2ClVe are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 7.40 (d, J = 5.9 Hz, 2H), 7.32 (d, J = 4.8 Hz, 1H), 7.03–6.94 (m, 3H), 6.84–6.73 (m, 2H), 6.50–6.41 (m, 2H), 6.03 (d, J = 6.1 Hz, 1H), 5.32 (q, J = 2.9 Hz, 2H), 3.40 (t, J = 6.7 Hz, 4H), 1.21 (q, J = 6.7 Hz, 6H). 13 C NMR (100 MHz, Chloroform-d) δ 153.99, 151.89, 151.85, 148.82, 144.66, 139.72, 139.63, 130.45, 129.60, 128.32, 128.06, 124.09, 121.47, 120.67, 112.05, 111.10, 108.45, 104.60, 104.50, 97.61, 83.66, 71.73, 44.50, 12.62. MS (ESI): calcd for C26H23Cl2NO3 [M+] 467.10, found 468.16.

[0130] The fluorescent probe 2.5, DCM-2ClVe was synthesized (solid, 23.6 mg, yield: 64.5%). The relevant characterization data of the fluorescent probe 2.5, DCM-2ClVe are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.92 (d, J = 8.3 Hz, 1H), 7.75 (t, J = 7.9 Hz, 1H), 7.60 (dd, J = 11.1, 6.4 Hz, 4H), 7.46 (t, J = 7.9 Hz, 1H), 7.14 (d, J = 8.0 Hz, 2H), 6.88 (s, 1H), 6.76 (d, J = 15.7 Hz, 1H), 6.05 (s, 1H).

[0131] Example 6 Synthesis of Fluorescent Probe 2.7, HDS-2ClVe

[0132] p-Hydroxybenzyl alcohol (200 mg, 1.61 mmol), trichloroethylene (416 mg, 3.22 mmol), and potassium carbonate (445 mg, 3.22 mmol) were dissolved in N,N-dimethylformamide (10 mL), and then the mixture was stirred at 45 °C for 6 hours. After the reaction was completed, the reaction solution was poured into ice water, and then extracted three times with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure to collect the solid residue. The obtained solid was purified by column chromatography using a gradient of DCM / MeOH mixed solvent to obtain Compound 1 (oil, 300 mg, yield 73.5%);

[0133] The obtained Compound 1 (300 mg, 1.38 mmol) was mixed with dichloromethane (5 mL), and then phosphorus tribromide (1.107 g, 4.14 mmol) was added dropwise in an ice bath. After one hour, the reaction was completed. The reaction solution was poured into ice water and extracted three times with ethyl acetate to collect the organic phase. The obtained organic phase was dried over anhydrous sodium sulfate and then the solvent was removed by distillation under pressure to collect the obtained solid. Then, it was quickly passed through a column using a PE / EA mixed solvent to obtain Compound 2 (oil, 288 mg, yield 75%);

[0134] HDS (30 mg, 0.0493 mmol) and Cs2CO3 (14.6 mg, 0.106 mmol) were mixed and then N,N-dimethylformamide (5 mL) was added as a solvent. Subsequently, the reaction system was activated at 55 °C for 30 minutes, and then Compound 2 (27.53 mg, 0.0986 mmol) dissolved in N,N-dimethylformamide was added dropwise. After 10 hours, the dye had completely reacted. After removing N,N-dimethylformamide by freeze-drying, the obtained solid was purified by column chromatography to obtain the fluorescent probe 2.7, HDS-2ClVE (10.32 mg, dark purple solid, yield 25.5%). The reaction process is shown as follows:

[0135]

[0136] The relevant characterization data of the fluorescent probe 2.7, HDS-2ClVe are as follows:

[0137] 11H NMR (400 MHz, Methanol-d4) δ 8.73 (d, J = 14.8 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 6.7 Hz, 2H), 7.50 (d, J = 8.2 Hz, 1H), 7.46–7.39 (m, 2H), 7.33 (s, 1H), 7.13–7.06 (m, 2H), 7.02 (d, J = 8.6 Hz, 1H), 6.69 (d, J = 14.6 Hz, 1H), 6.30 (s, 1H), 5.23 (s, 1H), 4.59 (s, 2H), 4.55 (t, J = 8.0 Hz, 2H), 2.98 (t, J = 6.6 Hz, 2H), 2.74 (q, J = 5.8 Hz, 4H), 2.34–2.25 (m, 2H), 1.90 (t, J = 6.1 Hz, 2H), 1.80 (s, 6H).

[0138] In summary, characterization by means of 1H NMR, 13C NMR and other methods indicated that the CYP450 fluorescent probe for olefin epoxidation elimination was successfully synthesized.

[0139] Example 7 Detection of CYP450 in vitro hepatic microsomes using the fluorescent probe

[0140] The fluorescent probes 1.1, Nath-2ClVe obtained in Example 1 and 1.2, Nath-Ve obtained in Example 2 were respectively prepared into DMSO stock solutions with a concentration of 1 mM. To a test system with a probe concentration of 10 μM, 0.005–0.2 mg / mL of rat liver microsomes (RLM) was added. After pre-incubation for 3 min, the NADPH generating system (containing NADP + , G-6-P (glucose-6-phosphate), MgCl2 and G-6-PD (glucose-6-phosphate dehydrogenase)) was added, and the purchased Solution A and Solution B were added to the system in a volume ratio of 5:1 to immediately generate NADPH) to trigger the reaction. The reaction was carried out at a constant temperature of 37 °C for 45 min, and then ice acetonitrile (200 μL) was added to terminate the reaction. After high-speed centrifugation at 4 °C and 20,000 x g for 20 min, the supernatant was taken for fluorescence detection (excitation wavelengths were 405 nm / 450 nm), as Figure 7 shown.

[0141] Example 8 Detection of the activity of recombinant expressed CYP450 enzyme in vitro using the fluorescent probes 1.1, Nath-2ClVe and 1.2, Nath-Ve

[0142] The total volume of the incubation mixture was 200 μL and consisted of 100 mM potassium phosphate buffer (pH 7.4), an NADPH generating system (containing NADP+, G-6-P (glucose-6-phosphate), MgCl2 and G-6-PD (glucose-6-phosphate dehydrogenase), and adding the commercially available Solution A and Solution B to the system at a volume ratio of 5:1 could immediately generate NADPH), and CYP2C19 or CYP1B1. A certain concentration of CYP2C19 or CYP1B1 (0 - 35 nM) was added to the incubation system dissolved with 10 μM probe. After pre-incubating at 37 °C for 3 min, the NADPH generating system was added to trigger the reaction, and further incubation was carried out at 37 °C for 45 min. After the reaction ended, ice acetonitrile (200 μL) was added to terminate the reaction. After high-speed centrifugation at 4 °C and 20,000 x g for 20 min using a high-speed refrigerated centrifuge, the supernatant was taken for fluorescence detection. According to the change in the fluorescence intensity value, a corresponding fitting curve could be made, as shown in Figure 8 (a~b), 9(a~b).

[0143] Example 9 Selectivity of Recombinant Expressed CYP450 Subtype Enzymes

[0144] In an incubation system with a total volume of 100 μL of the incubation mixture, potassium phosphate buffer solution, an NADPH generating system (containing NADP+, G-6-P (glucose-6-phosphate), MgCl2 and G-6-PD (glucose-6-phosphate dehydrogenase), and adding the commercially available Solution A and Solution B to the system at a volume ratio of 5:1 could immediately generate NADPH), and CYP single enzyme (CYP1A1, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4A11, CYP4F2, CYP4F12, or CYP4F3B) (final concentration 20 nM) were added respectively. This type of probe (final concentration 10 μM) was added and incubated for 60 min. Then 100 μL of ice acetonitrile was added to terminate the reaction, and after mixing, centrifugation was carried out at 20,000 x g for 20 min to take the supernatant. Detection was carried out using an enzyme-labeling instrument, and a corresponding bar chart was made, as shown in Figure 8 c, Figure 9 as shown in c.

[0145] Example 10 Fluorescent Probe for CYP450 Enzyme Activity Kinetics Test

[0146] The total volume of the incubation system is 200 μL, which consists of 100 mM potassium phosphate buffer (pH 7.4), NADPH generating system, and CYP2C19 or CYP1B1. A certain concentration of CYP2C19 or CYP1B1 (0 - 35 nM) was added to the incubation system containing 10 μM probe. After pre - incubating at 37 °C for 3 min, the NADPH generating system was added to trigger the reaction, and further incubation was carried out at 37 °C for 0 - 100 min. After the reaction ended, ice - acetonitrile (200 μL) was added to terminate the reaction. The supernatant was taken after high - speed centrifugation at 4 °C and 20,000 xg for 20 min using a high - speed refrigerated centrifuge, and fluorescence detection was performed. In addition, the concentration of CYP450 enzyme was fixed, and the concentration of the substrate probe was changed to measure the enzyme reaction rate. The Michaelis constant Km and the reaction rate Vmax of the CYP450 - catalyzed reaction were determined by the double - reciprocal plot method.

[0147] 1) Fluorescent probe 1.1, Nath - 2ClVe has a highly sensitive and rapid response to CYP2C19 in PBS (100 mM, pH = 7.4). As Figure 8 shown in d, after adding 0 - 10 nM CYP2C19, the fluorescence emission intensity at 550 nm gradually increased and reached saturation. The lowest detection limit was calculated to be 0.077 nM through the linear relationship between its fluorescence intensity and CYP2C19. In addition, with the concentration of CYP2C19 fixed (10 nM), the concentration of the enzyme - catalyzed substrate probe 1.1, Nath - 2ClVe (0.25, 0.5, 1, 2, 4 μM) was changed for fluorescence testing, and the Michaelis constant (Km) of CYP2C19 - catalyzed oxidation of the probe substrate 1.1, Nath - 2ClVe was calculated to be 0.81 μM by plotting, and the maximum reaction rate (Vmax) was 163.1 μM / s, indicating that the probe 1.1, Nath - 2ClVe has a good affinity with the enzyme as a catalytic substrate of CYP2C19.

[0148] 2) Fluorescent probe 1.2, Nath - Ve has a highly sensitive and rapid response to CYP1B1 in PBS (100 mM, pH = 7.4). As Figure 9 shown in d, after adding 0 - 15 nM CYP1B1, the fluorescence emission intensity at 550 nm gradually increased, while the fluorescence intensity at 450 nm gradually decreased and reached saturation. The lowest detection limit was calculated to be 0.1 nM through the linear relationship between the ratio signal I 550 / I 460 of the fluorescence intensities of its two emission peaks and CYP1B1.

[0149] Example 11 Fluorescent probe 1.1, Nath - 2ClVe is used for CYP450 inhibitor screening test

[0150] First, place the mixture of CYP2C19 (5 nM), CYP450 inhibitor or potential inhibitory drug, and NADPH generating system in PBS (pH 7.4) solution and pre-incubate at 37 °C for 45 min. Then add the fluorescent probe 1.1,Nath-2ClVe (10 mM) and incubate for another 45 min. By measuring the inhibitory effects of inhibitors omeprazole (0 - 200 μM) or ticlopidine (0 - 200 μM) or potential inhibitory drugs at different concentrations on the enzyme, the half-inhibitory concentration value of the drug is determined. The inhibition is expressed as the percentage decrease in fluorescence intensity relative to the control group. Use GraphPad to plot the dose-response curve and calculate the concentration of the drug corresponding to 50% inhibition of the analyte activity using the non-linear fitting method. This value is the half-inhibitor concentration (IC 50 ) of the drug on the analyte. As Figure 10 shown, when CYP2C19 is pre-incubated with omeprazole and ticlopidine for 45 min, the fluorescence intensity of the fluorescent probe 1.1,Nath-2ClVe decreases and decreases with the increase in the concentration of the inhibitor drug, indicating a significant decrease in the enzyme activity of CYP2C19, further indicating that the fluorescence change of the fluorescent probe 1.1,Nath-2ClVe is caused by the specific recognition response of CYP2C19.

[0151] In addition, 18 related drugs metabolized by CYP2C19 or drugs with similar structures were selected for inhibitor screening. As Figure 10 shown, for the reported CYP2C19 inhibitors omeprazole (D-3), ticlopidine (D-12), and the CYP450 broad-spectrum inhibitor 1-aminobenzotriazole (D-14), the inhibitory effects of these drugs on CYP2C19 can be detected by observing the fluorescence spectrum changes of the fluorescent probe 1.1,Nath-2ClVe. In addition, other drugs resveratrol (D-10), lansoprazole (D-13), and flavonoid 1 (D-17) also have certain inhibitory effects on the enzyme activity of CYP2C19. However, after pre-incubation of the drugs scutellarin (D-1), thalidomide (D-2), rifampicin (D-4), dexamethasone (D-8), and warfarin (D-9) with CYP2C19, an increase in the activity of CYP2C19 can be detected by the fluorescent probe 1.1,Nath-2ClVe, which is an obvious induction effect. The experimental results prove that the probe 1.1,Nath-2ClVe has great application potential for screening CYP2C19 inhibitors or inducers to reduce drug interactions and adverse reactions.

[0152] Example 12 Fluorescent probe 1.1,Nath-2ClVe is used for imaging the endogenous CYP2C19 enzyme activity in hepatocytes HepG2

[0153] Hepatoma HepG-29 cells were cultured in DMEM medium (containing 10% FBS). When the cells reached 70% confluence in the culture flask, they were digested and seeded into confocal dishes at a concentration of 1×10 5 cells / well, and then incubated overnight at 37 °C in an incubator containing 5% CO2. Rifampicin (RIF) is a strong hepatic enzyme inducer. The drug rifampicin (0 - 100 μM) was selected as the inducer of CYP2C19. Rifampicin was pre-added and incubated for 6 - 24 h, and then 30 μM of the fluorescent probe 1.1,Nath-2ClVe was added and incubated for 30 min. After incubation, the cells were washed three times with DPBS at 37 °C, and then observed under a Nikon confocal microscope. The excitation wavelength was 543 nm, and the fluorescence collection range was 570 - 620 nm. As Figure 11 shown, among them, the fluorescence intensity in the red channel of HepG2 cell imaging after RIF (100 μM) stimulation for different times (12, 24 h) increased significantly. The increase in fluorescence intensity was positively correlated with the pre-incubation time of RIF, indicating that the fluorescent probe 1.1,Nath-2ClVe can monitor the changes in endogenous CYP2C19 activity in living cells.

[0154] Example 13 Fluorescent Probe 2.7,HDS-2ClVe and Imaging of Endogenous CYP2C19 Activity in Mouse Liver

[0155] The fluorescent probe 2.7,HDS-2ClVe obtained in Example 6 was dissolved in DMSO to prepare a 10 mM stock solution, and then this stock solution was dissolved in PBS buffer at pH = 7.4 to prepare a working solution with a concentration of 100 μM. After fasting mice injected subcutaneously with acetaminophen (APAP) at different time intervals, the working solution of the probe was injected through the tail vein. Immediately after the mice were injected with the probe, real-time imaging was performed on the mice to observe the fluorescence changes in their livers. After 4 hours of real-time imaging, the mice were sacrificed by the method required by animal ethics, and then different organs were taken as the basis for in vitro imaging. The imaging results are as Figure 12 shown.

[0156] Example 14 Fluorescent Imaging Diagram of Endogenous CYP1B1 Activity in the Endoplasmic Reticulum of Hepatoma Cells Detected by Fluorescent Probe 3.1,ER-Nath-Ve

[0157] HepG2 live cells were inoculated in a confocal dish and incubated in the staining solution at 37 °C for 2 hours, washed three times with DPBS, and then imaged with a fluorescence microscope. The excitation wavelength was 405 nm, and the emission collection band was 425 - 475 nm. At the same time, localization experiments were carried out with commercial subcellular organelle localization reagents (ER-tracker Green for the endoplasmic reticulum, Lyso-tracker Green for lysosomes, and Mito-tracker Deep Red for mitochondria). Among them, the excitation wavelength of ER-tracker Green and Lyso-tracker Green was 488 nm, and the emission band was 500 - 550 nm. The excitation wavelength of Mito-tracker Deep Red was 640 nm, and the emission band was 663 - 738 nm. The co-localization images obtained are as follows Figure 13 shown. The first row: the co-localization map of the probe and ER-tracker Red for the endoplasmic reticulum and its superimposed map; the second row: the co-localization map of the probe and Lyso-tracker Red for lysosomes and its superimposed map; the third row: the co-localization map of the probe and Mito-tracker Deep Red for mitochondria and its superimposed map. By comparing the three commercial co-localization reagents, it can be found that probe 3.1, ER-Nath-Ve is mainly localized in the subcellular organelle endoplasmic reticulum.

[0158] Example 15 Fluorescence ratio imaging map of fluorescence probe 1.2, Nath-Ve for detecting endogenous CYP1B1 activity in mouse tissues

[0159] The fluorescence probe 1.2, Nath-Ve was dissolved in DMSO to prepare a stock solution of 10 mM, and diluted with DPBS to a final concentration of 40 μM for staining. Tumor tissues and normal mammary gland tissues were incubated in the staining solution at 37 °C for 4 hours, washed three times with DPBS, the tissues were placed on slides, and then single / double photon imaging was performed with a fluorescence microscope. The single / double photon excitation wavelengths were 405 nm respectively, and the emission collection bands were: 425 - 475 nm for the green channel and 500 - 550 nm for the red channel. As shown in Figure 14 shown, in tumor tissues and normal tissues, the fluorescence ratio of the red channel to the green channel (F 红 / F 绿 ) in tumor tissues was significantly higher than that in normal tissues. In addition, after pre-incubating tumor tissue sections with the inhibitor TMS, the probe could further detect the decrease in the fluorescence ratio signal caused by the inhibitor. It can be seen that the activity of CYP1B1 in tumor tissues is much higher than that in normal tissues, demonstrating the possibility of monitoring the dynamic drug metabolism during tumor treatment.

[0160] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A class of cytochrome P450 fluorescent probes based on olefin epoxidation elimination, characterized in that, Its structural general formula is shown as (I): In general formula (I): R1 is a fluorophore, derived from a hydroxy-fluorescent dye, and the hydroxy-fluorescent dye is selected from one of 7-hydroxycoumarin, 4-hydroxynaphthamide, rhodamine, a hybrid of fluorescein dye (hydroxymethylrhodamine fluor dye), resorufin dye, dicyanomethylene-4H-pyran derivative (hydroxy-DCM dye), and hydroxy-semicyanine Hubei dye; R2 is selected from one of hydrogen and chlorine atom; R3 is selected from one of hydrogen, chlorine atom, and methyl; R4 is selected from one of hydrogen, carboxyl, phenyl, and methyl.

2. The cytochrome P450 fluorescent probe based on olefin epoxidation elimination according to claim 1, wherein The structural formula of R1 is: The structural formula of this fluorescent probe is:

3. The cytochrome P450 fluorescent probe based on olefin epoxidation elimination according to claim 1, wherein When R2 and R3 are chlorine atoms and R4 is a hydrogen atom, this fluorescent probe includes any one of the following structural formulas:

4. The cytochrome P450 fluorescent probe based on olefin epoxidation elimination according to claim 1, wherein When R2, R3, and R4 are all hydrogen atoms, this fluorescent probe includes any one of the following structural formulas:

5. The cytochrome P450 fluorescent probe based on olefin epoxidation elimination according to claim 1, wherein The fluorophore may also be an organelle-targeted substituted fluorescent dye.

6. Use of the fluorescent probe according to any one of claims 1 to 5 in the detection of the activities of different subtypes of CYP450 enzymes.

7. Use of the fluorescent probe according to claim 6, characterized in that, Apply the fluorescent probe to the detection of the activities of different subtypes of CYP450 enzymes in vitro, in cells or in vivo.

8. The application of the fluorescent probe according to claim 6, wherein Apply the fluorescent probe in the process of preparing a kit or test strip for detecting the activities of different subtypes of CYP450 enzymes.

9. The application of the fluorescent probe according to claim 6, wherein, Apply the fluorescent probe to a specific fluorescent probe substrate for the CYP2C family enzyme CYP2C19.

10. The application of the fluorescent probe according to claim 6, wherein, Apply the fluorescent probe to a specific fluorescent probe substrate for the CYP1B family enzyme CYP1B1.

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