Specific chemiluminescent probe for determining CYP3A, preparation method and application

Abstract

The invention relates to a specific chemiluminescent probe for determining CYP3A in the technical field of medicines, which is structurally characterized by being shown in the following formula: # imgabs0 #. The probe structurally comprises a high-specificity reaction response group of CYP3A, a chemiluminescent group, a self-hyperchromic group and a self-leaving linking group connected with the high-specificity reaction response group, the chemiluminescent group and the self-hyperchromic group, the chemiluminescent probe does not need to be excited by an external light source, is free of phototoxicity, has extremely high signal-to-noise ratio and sensitivity, can be used for cell and living body imaging, has the advantages of being free of interference of biological matrixes, different in detection mode and the like compared with a traditional fluorescent probe, guarantees the accuracy of experiments, has the characteristics of detection convenience and high-throughput detection, and is suitable for large-scale popularization and application. The potential application value is realized in new drug research and development and individualized drug treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and relates to a chemiluminescent probe for enzymes, a synthesis method and application thereof, and specifically to a specific chemiluminescent probe for measuring CYP3A, a preparation method and application thereof. Background Art

[0002] Cytochrome P450s enzymes (CYP) are an important superfamily of monooxygenases containing heme. These enzymes are mainly located in subcellular organelles such as the endoplasmic reticulum. P450 enzymes are involved in a variety of catalytic reactions, including hydroxylation, epoxidation, N-dealkylation, and O-dealkylation. CYP3A, as the most important phase I drug metabolizing enzyme of P450s, is involved in the metabolism of the most extensive endogenous and exogenous substances. These substances include most drugs and toxins, which are essential for maintaining normal physiological functions of the human body. Therefore, understanding and studying the function and regulatory mechanism of CYP3A enzymes is of great significance for drug safety and personalized treatment.

[0003] However, due to the influence of medication, genetic polymorphism and environmental factors, there are large differences in the activity of P450 enzymes between individuals, which may lead to a variety of adverse effects. For example, CYP3A is affected by genetic polymorphism and may cause drug-induced liver injury (DILI). In addition, the activity of CYP3A can be inhibited or activated to cause drug-drug interactions (DDI). For example, rifampicin upregulates the activity of P4503A enzymes, causing patients to produce toxic products when using antiretroviral drugs containing ritonavir, thereby causing oxidative stress and liver cell damage. Changes in CYP3A activity are the most common cause of drug-drug interactions. Therefore, it is of great significance to select appropriate detection methods to accurately evaluate the activity of CYP3A enzymes, so as to better understand the characteristics of individuals in drug metabolism and interactions, thereby using drugs more safely and reducing the risk of adverse reactions.

[0004] Among the current detection methods, the detection methods for CYP3A mainly include ultraviolet spectroscopy, proteomics, immunoblotting and fluorescent probe detection. Ultraviolet spectroscopy can determine the enzyme activity based on the change in absorbance value after CYP3A oxygen dealkylation and hydroxylation of the substrate, but the sensitivity is extremely poor and it is more susceptible to interference from biological matrices. Proteomics uses liquid chromatography-mass spectrometry (LC-MS) to identify and quantify CYP3A, but the instruments and equipment are expensive, the maintenance cost is high, the sample preparation process is complicated, and the required analytical data is huge. The immunoblotting method can detect and quantify CYP3A by utilizing the principle of specific antibodies binding to target proteins, but the use of immunoblotting is limited by the specificity and affinity of antibodies, and can only achieve semi-quantification. Although the fluorescent probe method improves the sensitivity of CYP3A detection and is easier to operate, it can only play an advantage in in vitro detection. Due to the interference of many matrices in complex biological systems, the excitation of external light sources will produce many irremovable background signals that interfere with real-time imaging.

[0005] As mentioned in patent CN101608212B, schisandrin is used as a specific probe for CYP3A. This method evaluates the activity of CYP3A by mass spectrometry, specifically by measuring the reduction of schisandrin substrate or the production of schisandrin A product. However, this method has high requirements for sample preparation, is expensive, and is technically complex; at the same time, the detection process is affected by differences in ionization efficiency and matrix effects, and has certain limitations. For example, patent CN104975066B mentions that the reduction of the substrate oligosaccharide ester compound Tenuifoliside A or the production of its corresponding hydroxylation product is detected by ultraviolet absorption to quantify CYP3A4 in different biological samples. However, this method is susceptible to interference from substances with similar ultraviolet absorption wavelengths in complex biological samples, resulting in poor discrimination; in addition, the ultraviolet absorption detection method has limited sensitivity to low-concentration samples, and the signal-to-noise ratio is also low, which greatly limits its scope of application. Patent CN114105979A proposes a 4-hydroxylation reaction based on a naphthylimide fluorophore as a probe reaction for detecting CYP3A activity, and can be used for rapid in vitro screening of reversible and irreversible inhibitors, activators and inducers of CYP3A. However, the excitation wavelength of the fluorescent probe method is short, which easily causes high background fluorescence. In contrast, the CYP3A probe detection method based on chemiluminescence activation relies on the self-luminous system of the emissive excited state formed by the chemical reaction, and does not require the irradiation of an external excitation light source. Therefore, there is no self-luminescence and light scattering caused by the external excitation light source, and zero interference of the biological matrix can be achieved, which greatly improves the sensitivity and signal-to-noise ratio of the detection, and realizes real-time imaging in complex biological systems. Despite this, there is currently no report on a chemiluminescent human CYP3A probe substrate. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a CYP3A chemiluminescent probe that does not require external light source excitation and combines a new CYP3A oxidative dealkylation response group. Compared with traditional fluorescent probes, the probe has an ultra-high signal-to-noise ratio and sensitivity and can also achieve zero interference with biological matrices. The probe can be used to measure and locate the enzyme activity detection and distribution of CYP3A in complex biological systems.

[0007] One of the purposes of the present invention is to provide a chemiluminescent probe LP, characterized in that its structural features are shown in the following formula:

[0008]

[0009] Specifically, it includes a selective response group R1, a self-leaving group R2, a self-color enhancement group R3 and a chemiluminescent group R4; R1 is selected from: 1-ethyl-3-fluorobenzene, ethylbenzene, methyl, ethyl, chloroethyl or bromoethyl; R2 is selected from: hydrogen, fluorine, chlorine, bromine or iodine; R3 is selected from: tricyanofuran, cyano, acetic acid, methyl acetate, quinoline nitrile, pyran nitrile, semicyanine or methylquinoline nitrile; R4 is selected from: hydrogen, fluorine, chlorine, bromine or iodine.

[0010] Further, when R1 is 1-ethyl-3-fluorobenzene, R2 is cyano, R3 is chlorine, and R4 is chlorine, the chemiluminescent probe LP is named 3-(3-chloro-2-((3-chloro-4-((3-fluorobenzyl)oxy)benzyl)oxy)-4-methoxyspiro[adamantane-2,3'-[1,2]dioxetane]-4'-yl)phenyl)acrylonitrile (LP-01).

[0011] The second object of the present invention is to provide a method for preparing a chemiluminescent probe LP. The synthetic route is as follows:

[0012]

[0013] The trigger group LH was linked to the chemiluminescent matrix LC by using the Mitsunobu reaction to obtain the CYP3A-specific chemiluminescent probe LP.

[0014] Further, the synthetic route of LH is:

[0015] The self-leaving group R2 is connected to the response group R1 through a benzylation nucleophilic reaction, and then reduced by sodium borohydride to obtain the trigger group LH.

[0016] Further, the synthetic route of LC is:

[0017]

[0018] R4,-3-hydroxybenzaldehyde is converted to the chemiluminescent matrix LM through 6 steps, which are the acetal reaction of R4,-3-hydroxybenzaldehyde with trimethyl orthoformate and tetrabutylammonium tribromide, the silyl ether protection of the phenolic hydroxyl group of 2-chloro-3-dimethoxycresol, followed by the phosphation reaction with trimethyl phosphite under the catalysis of titanium tetrachloride, and then further nucleophilic addition with adamantane ketone under the catalysis of lithium diisopropylamide, and then the silyl ether bond protection of the hydroxyl group is removed with hydrochloric acid, and finally the aldehyde reaction with polyformaldehyde is carried out, and then the chemiluminescent matrix LM is modified by Wittig reaction, Mohr reaction and nucleophilic addition of aldehyde group to obtain the chemiluminescent matrix LC modified with different chromophores.

[0019] The present invention introduces the Schaap-dioxetane (LM) chemiluminescent system into the CYP3A detection system for the first time. LM has a larger molecular structure. The inventors have modified the color-enhancing group with corresponding basic groups, such as cyano and pyranonitrile, in order to obtain a chemiluminescent matrix (LC) that effectively recognizes the CYP3A active site. This design fully considers the characteristics of the CYP3A active cavity, including its preference for hydrophobicity, neutral alkalinity and larger substrate molecules.

[0020] At the trigger end of LC, the inventors designed a series of CYP3A-recognizable trigger groups (LH), including methoxy, trifluoromethyl and diisopropyl acetal. Among them, a trigger structure that has not appeared in previous CYP3A probes was introduced for the first time: 1-chloro-2-(3-fluorophenyl)methoxy (LH-01). This group was inspired by the CYP3A drug substrate Lapatinib and showed high selectivity for CYP3A.

[0021] In addition, in order to facilitate the effective entry of the CYP3A trigger group LH into the CYP3A active cavity, the inventors constructed a linker group between LC and LH. After catalysis by CYP3A, the trigger group LH will cause the linker group to undergo electron rearrangement and self-leave, releasing the high-energy unstable intermediate state of dioxetane of the phenoxy anion. Through the electron exchange luminescence mechanism, it will eventually decompose to generate benzoic acid ester and produce a significant chemiluminescent signal.

[0022] This patent provides a newly designed and invented enzyme-catalyzed chemiluminescent substrate, which consists of four parts: CYP3A specific trigger group, linker group, self-color enhancement group and luminescent group. The new CYP3A detection method developed by chemiluminescence activation has the advantages of convenience, anti-interference and high-throughput determination of CYP3A activity in complex biological systems. In addition, this method provides an effective tool for in-depth research on the diversity and specificity of CYP3A metabolic catalysis, and has important application value for new drug development, individual diagnosis and individual medication.

[0023] Compared with the probes in the prior art, the present invention constructs a combination of chemiluminescent LC and a new CYP3A trigger group LH, which creates a new detection method for CYP3A, fundamentally improves the disadvantages of traditional CYP3A probes, has ultra-high specificity, sensitivity and zero background matrix interference, provides more accurate and reliable detection results, and has broad application prospects, especially in the fields of biomedical testing and new drug screening.

[0024] The third object of the present invention is to provide the application of chemiluminescent probe LP in determining CYP3A activity.

[0025] Furthermore, the probe is mixed with a biological sample containing CYP3A and then subjected to an enzymatic reaction. The activity of CYP3A in different biological systems is quantitatively determined by quantitatively collecting and detecting the chemiluminescent signal generated per unit time or the generation rate of the corresponding product benzoate.

[0026] Furthermore, the specific steps of the detection include the following:

[0027] A. Set the reaction temperature between 20 and 60°C and the incubation system pH between 5.5 and 10.5;

[0028] B. After adding the prepared 0.1M phosphate buffer to the reaction vessel, add the probe, the substrate concentration is 1.0-50μM, add the biological sample containing CYP3A at a concentration of 0.02mg / mL and pre-incubate at 37℃ for 3 minutes;

[0029] C. Add NADPH to start the chemiluminescent reaction for 1 hour to ensure that the above substrates reach the quantitative limit in terms of O-dealkylation products and the conversion rate of the substrate does not exceed 10%;

[0030] D. Determine the maximum chemiluminescence signal value within 3 hours as an evaluation index for determining CYP3A activity in different complex biological systems.

[0031] Preferably, the biological sample containing CYP3A includes any one of human liver microsomes, S9, animal or human tissue preparation fluid, and corresponding tissue cells and preparations thereof of CYP3A.

[0032] Preferably, the substrate concentration in a single point assay is 20 μM.

[0033] Preferably, the pH of the incubation system is 7.4.

[0034] The chemiluminescent probe LP of the present invention releases a high-energy unstable intermediate state of a phenoxy anion under the O-dealkylation action of CYP3A, and then decomposes through a chemical reaction triggering an electron transfer luminescence process. The released chemiluminescent signal is detected by an enzyme reader and a full-wavelength scan is performed to obtain a maximum luminescent signal value. The corresponding benzoate product obtained can also be directly detected by an enzyme reader and used to determine the CYP3A enzyme activity.

[0035] The chemiluminescent probe LP can be used to quantitatively detect the activity of CYP3A in cell or tissue samples from different species.

[0036] This chemiluminescent probe LP can be used to rapidly screen CYP3A enzyme inhibitors and quantitatively evaluate their inhibitory ability.

[0037] The chemiluminescent probe LP can also be used for CYP3A enzyme detection in experimental animals, and can evaluate the differences in metabolic enzyme CYP3A between individuals and species.

[0038] The present invention provides an innovative application, using the chemiluminescent probe reaction of CYP3A. The chemiluminescent probe LP does not require external light source excitation under the enzymatic reaction, so it does not produce spontaneous luminescence, avoiding interference with enzyme activity detection. On the contrary, it can use a chemiluminescent detector to achieve rapid and sensitive detection of the product. In addition, the chemiluminescent probe LP can perform full wavelength scanning detection after undergoing the O-dealkylation reaction of CYP3A.

[0039] The chemiluminescent probe LP provided by the present invention has excellent anti-interference ability of biological system matrix and impurities in the process of CYP3A activity detection, which enables it to be reliably used for quantitative determination of CYP3A activity in various human liver microsomes, human and animal tissue preparations, and various tissue cells. At the same time, the chemiluminescent probe LP can also be widely used in CYP3A research in animals as a whole to evaluate the differences in metabolic enzyme CYP3A between individuals and species.

[0040] The chemiluminescent probe LP of the present invention has significant advantages in detecting the in vitro activity of CYP3A, including the following aspects: (1) high sensitivity: the detection of the chemiluminescent probe substrate does not require excitation from an external light source, and therefore does not have background interference; (2) mature technology: the parent can be obtained by chemical synthesis and can be prepared in large quantities; (3) it has the convenience of high-throughput detection: it can be easily tested in batches on a common laboratory fluorescence microplate reader. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The synthetic route of the chemiluminescent matrix LM is shown in Figure 1.

[0042] Figure 2It is the synthetic route map of CYP3A trigger gene LH;

[0043] Figure 3 The synthesis diagram of chemiluminescent matrix LC modified with different chromophores;

[0044] Figure 4 For the synthesis of CYP3A probe substrate LP;

[0045] Figure 5 For LP-01 1 H-NMR spectrum;

[0046] Figure 6 For LP-01 13 C-NMR spectrum;

[0047] Figure 7 For LP-02 1 H-NMR spectrum;

[0048] Figure 8 For LP-02 13 C-NMR spectrum;

[0049] Fig. 9 The chemiluminescent response mechanism diagram of LP-01 for detecting CYP3A;

[0050] Fig.10 This is a selectivity test diagram of the human CYP recombinant single enzyme of LP-01;

[0051] Fig.11 The figure shows the HiHep cytotoxicity and HepRG evaluation of LP-01;

[0052] Fig.12 This is the enzymatic kinetic curve of LP-01 dealkylation catalyzed by CYP3A;

[0053] Fig.13 This is the detection limit diagram of LP-01 for detecting CYP3A5;

[0054] Fig.14 This is the detection limit diagram of LP-01 for detecting CYP3A4;

[0055] Fig.15 This is the enzymatic kinetic curve of LP-01 dealkylation catalyzed by CYP3A;

[0056] Fig.16 This is a curve diagram for quantitative evaluation of CYP3A activity in human tissue microsomes. DETAILED DESCRIPTION

[0057] The following is further described in detail through specific implementation methods:

[0058] The chemiluminescent probe LP of the present invention is as follows:

[0059]

[0060] Specifically, it includes a selective response group R1, a self-leaving group R2, a self-color enhancement group R3 and a chemiluminescent group R4; R1 is selected from: 1-ethyl-3-fluorobenzene, ethylbenzene, methyl, ethyl, chloroethyl or bromoethyl; R2 is selected from: hydrogen, fluorine, chlorine, bromine or iodine; R3 is selected from: tricyanofuran, cyano, acetic acid, methyl acetate, quinoline nitrile, pyran nitrile, semicyanine or methylquinoline nitrile; R4 is selected from: hydrogen, fluorine, chlorine, bromine or iodine.

[0061] Preferably, when R1 is 1-ethyl-3-fluorobenzene, R2 is cyano, R3 is chlorine, and R4 is chlorine, the chemiluminescent probe LP is named 3-(3-chloro-2-((3-chloro-4-((3-fluorobenzyl)oxy)benzyl)oxy)-4-methoxyspiro[adamantane-2,3'-[1,2]dioxetane]-4'-yl)phenyl)acrylonitrile LP-01.

[0062] Example 1

[0063] The synthetic routes of chemiluminescent matrix LM-01, LM-02 and LM-03 are as follows: Figure 1 Shown

[0064] Synthesis of LM-01:

[0065] Compound 1b

[0066] Compound 1a (1.6 g, 12.8 mmol) was dissolved in 12 mL of methanol, and tetra-n-butylammonium tribromide (155.0 mg, 0.32 mmol) and trimethyl orthoformate (2.2 mL, 20.4 mmol) were added in sequence. The mixture was stirred at room temperature for 30-45 min and the reaction was monitored by TLC (P / E=4 / 1, v / v). After the reaction was completed, the reaction solution was extracted with 50 mL of ethyl acetate and 0.01 M sodium bicarbonate aqueous solution, the organic phase was separated and dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure to obtain a yellow oil (crude product yield (2.0 g, yield was 92.3%).

[0067] Compound 1c

[0068] Compound 1b (2.0 g, 12.2 mmol) and imidazole (1.66 g, 24.4 mmol) were added to 10.0 mL of dichloromethane to dissolve, and then tert-butyldimethylsilyl chloride (TBS-Cl) (312 mg, 14.6 mmol) was added to the reaction solution and reacted at room temperature for about 2 h. After the reaction was completed, the reaction was monitored by TLC plate (P / E=5 / 1, v / v), and the white precipitate was removed by filtration. The obtained filtrate was washed with 40 mL of ethyl acetate and 0.01 M sodium bicarbonate solution, and the organic phase was separated and dried with anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure to obtain a yellow oil to obtain a crude product (4.3 g, crude product yield was 126.5%).

[0069] Compound 2d

[0070] Compound 1c (calculated according to 100% product content, 4.4 g, 15.9 mmol) and trimethyl phosphite (2.8 mL, 20.8 mmol) were dissolved in 15 mL of dichloromethane, and the reaction temperature was cooled to 0°C, and then titanium tetrachloride (1 mol / L dissolved in dichloromethane) (19.2 mL, 19.2 mmol) was added dropwise. After reacting for about 2.5 h at this temperature, the reaction was monitored by TLC plate (P / E=1 / 3, v / v). Subsequently, the reaction solution was poured into an appropriate amount of saturated sodium bicarbonate aqueous solution at 0°C until white bubbles disappeared and 60 mL of dichloromethane was used to extract the reaction solution, and then the organic phase was separated and dried over anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure to obtain a colorless oily crude product, which was separated by silica gel column chromatography (P / E=1 / 3, v / v) to obtain a colorless oily compound 1d (2.6 g, crude product yield was 45.5%).

[0071] Compound 1f

[0072] Compound 1d (1.8 g, 5.0 mmol) was added to 10 mL of anhydrous tetrahydrofuran (THF), and the reaction temperature was cooled to -78 °C. A 2.0 M THF solution (3.7 mL, 7.5 mmol) of lithium diisopropylamide (LDA) was slowly added dropwise under nitrogen and continued to stir for 20 min. Then, a 10.0 mL THF solution of adamantane ketone (751.1 mg, 5.0 mmol) was slowly added to the reaction solution, and stirring was continued at -78 °C for 15 min before warming to room temperature for reaction. After the reaction, 100 mL of ethyl acetate and 100 mL of saturated brine were added, and the organic phase was dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The crude product was then dissolved in THF, and 5 mL of a 1.0 M THF solution of tetrabutylammonium fluoride was added and reacted for 2 h. After the reaction was complete at room temperature, it was extracted with ethyl acetate and 1 M hydrochloric acid. After the organic phase was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure and the crude product was separated by silica gel column chromatography (petroleum ether ethyl acetate = 8 / 1, v / v) to obtain a white solid compound 1f (0.4 g, yield 35.1%).

[0073] Compound LM-01

[0074] In a flask, compound 1f (811.1 mg, 3.0 mmol) was dissolved in 5 mL of anhydrous acetonitrile, and magnesium chloride (891.0 mg, 9.4 mmol) and triethylamine (0.9 ml, 6.2 mmol) were added in sequence, followed by addition of paraformaldehyde (328.0 mg, 10.9 mmol) and heating the reaction to reflux overnight. After the reaction was completed, the mixture was returned to room temperature, washed with dichloromethane and water, and the pH was adjusted to 6.0 with hydrochloric acid solution (1 M). The organic phase was separated and the product was dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and the crude product was separated by silica gel column chromatography to obtain a light yellow solid compound LM-01 (502.0 mg, yield 56.1%).

[0075] Synthesis of LM-02:

[0076] Compound 2b

[0077] Compound 2a (2 g, 12.8 mmol) was dissolved in 12 mL of methanol, and tetra-n-butylammonium tribromide (155.0 mg, 0.32 mmol) and trimethyl orthoformate (2.2 mL, 20.4 mmol) were added in sequence. The mixture was stirred at room temperature for 30-45 min and the reaction was monitored by TLC (P / E=5 / 1, v / v). After the reaction was completed, the reaction solution was extracted with 50 mL of ethyl acetate and 0.01 M sodium bicarbonate aqueous solution, the organic phase was separated and dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure to obtain a yellow oil (crude product yield (2.4 g, yield was 95%).

[0078] Compound 2c

[0079] Compound 2b (2.4 g, 12.2 mmol) and imidazole (1.66 g, 24.4 mmol) were added to 10.0 mL of dichloromethane to dissolve, and then tert-butyldimethylsilyl chloride (TBS-Cl) (312 mg, 14.6 mmol) was added to the reaction solution and reacted at room temperature for about 2 h. After the reaction was completed, the reaction was monitored by TLC plate (P / E=4 / 1, v / v), and the white precipitate was removed by filtration. The obtained filtrate was washed with 40 mL of ethyl acetate and 0.01 M sodium bicarbonate solution, and the organic phase was separated and dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure to obtain a yellow oil to obtain a crude product (5.1 g, crude product yield was 131.0%).

[0080] Compound 2d

[0081] Compound 2c (calculated according to 100% product content, 5.08g, 15.9mmol) and trimethyl phosphite (2.8mL, 20.8mmol) were dissolved in 15mL dichloromethane, and the reaction temperature was cooled to 0°C, and then titanium tetrachloride (1mol / L dissolved in dichloromethane) (19.2mL, 19.2mmol) was added dropwise, and the reaction was monitored by TLC plate (P / E=2 / 3, v / v) after reacting for about 2.5h at this temperature. Subsequently, the reaction solution was poured into an appropriate amount of saturated sodium bicarbonate aqueous solution at 0°C until white bubbles disappeared and 60mL dichloromethane was used to extract the reaction solution, and then the organic phase was separated and dried over anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure to obtain a colorless oily crude product, which was separated by silica gel column chromatography (P / E=2 / 3, v / v) to obtain a colorless oily compound 2d (1.9g, crude product yield was 31%).

[0082] Compound 2f

[0083] Compound 2d (1.9 g, 5 mmol) was added to 10 mL of anhydrous tetrahydrofuran (THF), and the reaction temperature was cooled to -78 °C. A 2.0 M THF solution (3.7 mL, 7.5 mmol) of lithium diisopropylamide (LDA) was slowly added dropwise under nitrogen and stirred for 20 min. Then, a 10.0 mL THF solution of adamantane ketone (751.1 mg, 5.0 mmol) was slowly added to the reaction solution, and stirring was continued at -78 °C for 15 min before warming to room temperature for reaction. After the reaction, 100 mL of ethyl acetate and 100 mL of saturated brine were added, and the organic phase was dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The crude product was then dissolved in THF, and 5 mL of a 1.0 M THF solution of tetrabutylammonium fluoride was added and reacted for 2 h. After the reaction was complete at room temperature, it was extracted with ethyl acetate and 1 M hydrochloric acid. After the organic phase was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure and the crude product was separated by silica gel column chromatography (petroleum ether ethyl acetate = 5 / 1, v / v) to obtain a white solid compound 2f (0.6 g, yield 40.0%).

[0084] Compound LM-02

[0085] Compound 2f (950 mg, 3.1 mmol) was dissolved in 5 mL of anhydrous acetonitrile in a flask, and magnesium chloride (891.0 mg, 9.4 mmol) and triethylamine (0.9 ml, 6.2 mmol) were added in sequence, followed by the addition of paraformaldehyde (328.0 mg, 10.9 mmol) and the reaction was heated to reflux overnight. After the reaction was completed, the mixture was returned to room temperature, washed with dichloromethane and water, and the pH was adjusted to 6.0 with hydrochloric acid solution (1 M). The organic phase was separated and the product was dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and the crude product was separated by silica gel column chromatography to obtain a light yellow solid compound LM-02 (620.0 mg, yield 60.0%).

[0086] Synthesis of LM-03:

[0087] Compound 3b

[0088] Compound 3a (2.5 g, 12.8 mmol) was dissolved in 12 mL of methanol, and tetra-n-butylammonium tribromide (155.0 mg, 0.32 mmol) and trimethyl orthoformate (2.2 mL, 20.4 mmol) were added in sequence. The mixture was stirred at room temperature for 30-45 min and the reaction was monitored by TLC (P / E=10 / 1, v / v). After the reaction was completed, the reaction solution was extracted with 50 mL of ethyl acetate and 0.01 M sodium bicarbonate aqueous solution, the organic phase was separated and dried over anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure to obtain a light yellow oil (crude product yield (3.1 g, yield was 96.5%).

[0089] Compound 3c

[0090] Compound 3b (3.0 g, 12.2 mmol) and imidazole (1.66 g, 24.4 mmol) were added to 10.0 mL of dichloromethane to dissolve, and then tert-butyldimethylsilyl chloride (TBS-Cl) (312 mg, 14.6 mmol) was added to the reaction solution and reacted at room temperature for about 2 h. After the reaction was completed, the reaction was monitored by TLC plate (P / E=4 / 1, v / v), and the white precipitate was removed by filtration. The obtained filtrate was washed with 50 mL of ethyl acetate and 0.01 M sodium bicarbonate solution, and the organic phase was separated and dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure to obtain a yellow oil to obtain a crude product (5.6 g, crude product yield was 127.2%).

[0091] Compound 3d

[0092] Compound 3c (calculated according to 100% product content, 5.6 g, 15.5 mmol) and trimethyl phosphite (2.8 mL, 20.8 mmol) were dissolved in 15 mL of dichloromethane, and the reaction temperature was cooled to 0°C, and then titanium tetrachloride (1 mol / L dissolved in dichloromethane) (19.2 mL, 19.2 mmol) was added dropwise. After reacting for about 2.5 h at this temperature, the reaction was monitored by TLC plate (P / E=2 / 3, v / v). Subsequently, the reaction solution was poured into an appropriate amount of saturated sodium bicarbonate aqueous solution at 0°C until white bubbles disappeared and 60 mL of dichloromethane was used to extract the reaction solution, and then the organic phase was separated and dried over anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure to obtain a colorless oily crude product, which was separated by silica gel column chromatography (P / E=1 / 4, v / v) to obtain a powdery white oily compound 3d (1.9 g, crude product yield was 27.6%).

[0093] Compound 3f

[0094] Compound 3d (1.9 g, 4.2 mmol) was added to 10 mL of anhydrous tetrahydrofuran (THF), and the reaction temperature was cooled to -78 °C. A 2.0 M THF solution (2.6 mL, 5.0 mmol) of lithium diisopropylamide (LDA) was slowly added dropwise under nitrogen and stirred for 20 min. Then, a 10.0 mL THF solution of adamantane ketone (751.1 mg, 5.0 mmol) was slowly added to the reaction solution, and stirring was continued at -78 °C for 15 min before warming to room temperature for reaction. After the reaction, 100 mL of ethyl acetate and 100 mL of saturated brine were added, and the organic phase was dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The crude product was then dissolved in THF, and 5 mL of a 1.0 M THF solution of tetrabutylammonium fluoride was added and reacted for 2 h. After the reaction was complete at room temperature, it was extracted with ethyl acetate and 1 M hydrochloric acid. After the organic phase was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure and the crude product was separated by silica gel column chromatography (petroleum ether ethyl acetate = / 1, v / v) to obtain a pale white solid compound 3f (0.6 g, yield 37.6%).

[0095] Compound LM-03

[0096] Compound 3f (2.0 g, 5.7 mmol) was dissolved in 5 mL of anhydrous acetonitrile in a flask, and magnesium chloride (6.5 g, 70.5 mmol) and triethylamine (4.5 ml, 31.0 mmol) were added in sequence, followed by the addition of paraformaldehyde (1.6 g, 54.5 mmol) and the reaction was heated to reflux overnight. After the reaction was completed, the mixture was returned to room temperature, washed with dichloromethane and water, and the pH was adjusted to 6.0 with hydrochloric acid solution (1 M). The organic phase was separated and the product was dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and the crude product was separated by silica gel column chromatography to obtain yellow solid compound LM-03 (907.6 mg, yield 42.2%).

[0097] Example 2

[0098] Synthesis of CYP3A trigger groups LH-01, LH-02, and LH-03 Figure 2 Shown

[0099] Synthesis of LH-01:

[0100] Compound 4b

[0101] In a flask, compound 4a (1134.0 mg, 6.0 mmol) was dissolved in 10 mL of anhydrous acetonitrile, and potassium carbonate (829.3 mg, 6.0 mmol) was added in sequence, and finally 3-chloro-4-hydroxybenzaldehyde (470.0 mg, 3.0 mmol) was added and heated under reflux for 3 h. After the reaction was completed, the mixture was extracted with ethyl acetate and water and the solvent was removed by distillation under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a white solid compound (619.4 mg, yield 78.0%).

[0102] Compound LH-01

[0103] Compound 4b (264.7 mg, 1.0 mmol) was dissolved in 5 mL of anhydrous methanol in a flask, and sodium borohydride (56.7 mg, 1.5 mmol) was slowly added. After reacting for 3 h in an ice bath, the mixture was extracted with ethyl acetate and water and the solvent was removed by distillation under reduced pressure. The crude product was separated by silica gel column chromatography to finally give compound LH-01 (215.4 mg, 95.5%) as a white solid.

[0104] Synthesis of LH-02:

[0105] Compound 5b

[0106] Compound 5a (1134.0 mg, 6.0 mmol) was dissolved in 10 mL of anhydrous acetonitrile in a flask, and potassium carbonate (829.3 mg, 6.0 mmol) and bromoethane (447.7 μL, 6.0 mmol) were added in sequence and heated under reflux for 3 h. After the reaction was completed, the mixture was extracted with ethyl acetate and water and the solvent was removed by distillation under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a white transparent liquid compound (802.8 mg, yield 91.1%).

[0107] Compound LH-02

[0108] Compound 5b (150.2 mg, 1.0 mmol) was dissolved in 5 mL of anhydrous methanol in a flask, and sodium borohydride (56.7 mg, 1.5 mmol) was slowly added. After reacting for 3 h in an ice bath, the mixture was extracted with ethyl acetate and water and the solvent was removed by distillation under reduced pressure. The crude product was separated by silica gel column chromatography to finally give white solid compound LH-02 (144.7 mg, 95.1%).

[0109] Synthesis of LH-03:

[0110] Compound 6b

[0111] In a flask, compound 6a (1134.0 mg, 6.0 mmol) was dissolved in 10 mL of anhydrous acetonitrile, and (829.0 mg, 6.0 mmol) was added in sequence, and finally 3-chloro-4-hydroxybenzaldehyde (470.0 mg, 3.0 mmol) was added and heated under reflux for 3 h. After the reaction was completed, the mixture was extracted with ethyl acetate and water and the solvent was removed by distillation under reduced pressure. The crude product was separated by silica gel column chromatography to obtain a light white solid compound (619.4 mg, yield: 78.3%).

[0112] Compound LH-03

[0113] Compound 6b (212.2 mg, 1.0 mmol) was dissolved in 5 mL of anhydrous methanol in a flask, and sodium borohydride (56.7 mg, 1.5 mmol) was slowly added. After reacting for 3 h in an ice bath, the mixture was extracted with ethyl acetate and water and the solvent was removed by distillation under reduced pressure. The crude product was separated by silica gel column chromatography to finally give white solid compound LH-03 (206.1 mg, 96.2%).

[0114] Example 3

[0115] The synthesis of chemiluminescent precursors LC-01, LC-02, and LC-03 modified with different chromophores is as follows: Figure 3 Shown

[0116] Compound LC-01

[0117] In a flask, compound LM-02 (266.7 mg, 1.0 mmol) was dissolved in 5 mL of tetrahydrofuran, and triphenylphosphine (PPh3) (314.0 mg, 2.0 mmol) and potassium tert-butoxide (102.0 mg, 0.9 mmol) were added in sequence. After stirring at room temperature for 30 min, the mixture was extracted with ethyl acetate and water and the solvent was removed by distillation under reduced pressure. The crude product was separated by silica gel column chromatography to finally give a white solid product LC-01 (292.5 mg, yield 82.2%).

[0118] Compound LC-02

[0119] In a flask, compound LM-02 (266.7 mg, 1.0 mmol) was dissolved in 5 mL of tetrahydrofuran, and piperidine (Pi) (99.0 μL, 2.0 mmol) and pyranonitrile (252.2 mg, 1.2 mmol) were added in sequence, and the reaction was heated to reflux overnight. After the reaction was completed, the temperature was restored to room temperature, and the mixture was extracted with dichloromethane and water, and separated and purified by silica gel column chromatography to obtain a red product LC-02 (299.2 mg, yield 57.0%).

[0120] Compound LC-03

[0121] In a flask, compound LM-02 (266.7 mg, 1.0 mmol) was dissolved in 5 mL of tetrahydrofuran, and piperidine (Pi) (99.0 μL, 2.0 mmol) and tricyanofuran (239.1 mg, 1.2 mmol) were added in sequence. The reaction was heated to reflux overnight. After the reaction was completed, the temperature was restored to room temperature, and the mixture was extracted with dichloromethane and water. The mixture was separated and purified by silica gel column chromatography to obtain an orange-yellow product LC-03 (387.1 mg, yield 75.3%).

[0122] Example 4

[0123] The synthesis of chemiluminescent probes LP-01, LP-02, and LP-03 is as follows Figure 4 Shown

[0124] LP-01

[0125] Compound LH-01 (266.7 mg, 1.0 mmol) was dissolved in 5 mL of tetrahydrofuran in a flask, and diethyl azodicarboxylate (DEAD) (348.3 mg, 2.0 mmol) and PPh3 (786.9 mg, 3.0 mmol) were added in turn under ice bath conditions, and the mixture was stirred for 30 min before adding compound LC-01 (348.3 mg, 0.8 mmol). After stirring at room temperature overnight, a catalytic amount of methylene blue was added, and the solution was irradiated with yellow light and oxygen was introduced into the solution. The reaction was monitored by preparative liquid phase. After the reaction was completed, the solvent was concentrated under reduced pressure, and the product LP-01 (461.9 mg, yield was 69.1%) was obtained by separation and purification using preparative liquid phase. 1 H-NMR spectra and 13 C-NMR spectrum Figure 5 , Figure 6 shown.

[0126] LP-02

[0127] Compound LH-01 (266.7 mg, 1.0 mmol) was dissolved in 5 mL of tetrahydrofuran in a flask, and diethyl azodicarboxylate (DEAD) (420.0 mg, 2.0 mmol) and PPh3 (786.9 mg, 3.0 mmol) were added in turn under ice bath conditions, and the mixture was stirred for 30 min before adding compound LC-02 (355.9 mg, 0.8 mmol). After stirring at room temperature overnight, a catalytic amount of methylene blue was added, and the solution was irradiated with yellow light and oxygen was introduced into the solution. The reaction was monitored by preparative liquid phase chromatography. After the reaction was completed, the solvent was concentrated under reduced pressure, and the product LP-02 (404.0 mg, yield was 72.3%) was obtained by separation and purification using preparative liquid phase chromatography. 1 H-NMR spectra and 13 C-NMR spectrum Figure 7 , Figure 8shown.

[0128] LP-03

[0129] Compound LH-03 (266.7 mg, 1.0 mmol) was dissolved in 5 mL of tetrahydrofuran in a flask, and diethyl azodicarboxylate (DEAD) (348.3 mg, 2.0 mmol) and PPh3 (786.9 mg, 3.0 mmol) were added in turn under ice bath conditions, and the mixture was stirred for 30 min before adding compound LC-03 (411.3 mg, 0.8 mmol). After stirring at room temperature overnight, a catalytic amount of methylene blue was added, and the solution was irradiated with yellow light and oxygen was introduced into the solution. The reaction was monitored by preparative liquid phase. After the reaction was completed, the solvent was concentrated under reduced pressure, and the product LP-03 (461.9 mg, yield 69.1%) was obtained by separation and purification using preparative liquid phase.

[0130] Example 5

[0131] In vitro determination of single enzyme selectivity of human recombinant CYP3A

[0132] The following zymogens were used in the single enzyme screening: CYP450 single enzymes (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2A13, CYP2J2, CYP3A4 or CYP3A5). The reaction system used the same enzyme amount, mainly including 2.0 μL enzyme (final concentration 5 nM), 2.0 μL LP-01 probe substrate (final concentration 20.0 μM), 177.0 μL PBS buffer (pH = 7.4) and NADPH solution (final concentration 1.0 mM). The reaction process is to first add 2.0 μL of different single enzymes to 177.0 μL PBS buffer and 1 μL LP-01 probe substrate and incubate at 37°C for 3 minutes, then add 20 μL NADPH solution to start the reaction, and finally immediately put it into the microplate reader to detect the chemiluminescence signal, scan the full wavelength, detect once every 1 minute, and continue to detect for 30 minutes, and take the highest value as comparison. Three groups of parallel experiments were set up for each experiment, and the average was calculated. The probe was only specifically dealkylated by the recombinant human CYP3A single enzyme, and other single enzymes had almost no dealkylation reaction, such as Fig. 9 As shown, LP-01 releases adamantane ketone and emits an optical signal after obtaining a high-energy intermediate under the action of oxygen dealkylation of CYP3A. The corresponding single enzyme selectivity results are shown in Fig.10 As shown, the selectivity for CYP3A5 and CYP3A4 is at least 1250 and 875 times that of other CYP isozymes.

[0133] Example 6

[0134] Cytotoxicity assay of LP-01

[0135] In the in vitro evaluation, LP-01 became a specific substrate of CYP3A with its extremely high specificity and sensitivity. Next, the detection ability of LP-01 in living cells was further evaluated. The inventors first selected cell lines from different species that have been confirmed to contain CYP3A activity, the HepRG cell line (mouse hippocampal neuronal cells) and the hiHep cell line (human malignant glioblastoma cells) as model cells for the LP-01 chemiluminescent imaging experiment. Before imaging, the inventors used the CCK-8 detection method to evaluate the possible toxicity of LP-01 to cells. The results showed that the two cell lines have high biocompatibility and cell viability is not affected within a small dose range ( Fig.11 ).

[0136] Example 7

[0137] CYP3A time kinetic curve determination

[0138] The reaction system mainly includes 2μL CYP3A4 / 5 single enzyme (final concentration 0.2nM), 1μL LP-01 probe substrate (final concentration 20μM), 177μL PBS buffer (0.1M, pH=7.4) and NADPH solution (final concentration 1mM). The reaction process is: first, add 2μL CYP3A4 / 5 single enzyme to 177μL PBS buffer, then incubate 1μL LP-01 probe substrate at 37℃ for 3 minutes, then add 20μL NADPH solution to start the reaction, and finally immediately put it into the microplate reader to detect the luminescence signal, scan the full wavelength, detect once every 1 minute, and continue to detect for 30 minutes. Take the highest value when the reaction is parallel as a comparison, measure the metabolite generation-time curve, and determine the linear reaction time range. Three parallel groups were set up for each experiment, as shown in the following example. Fig.12 As shown, the peak time is 25 minutes.

[0139] Example 8

[0140] In vitro detection limit of CYP3A

[0141] The reaction system mainly includes 5 μL enzyme (final concentrations are 0.001, 0.002, 0.005, 0.010, 0.025, 0.050, 0.100, 0.500, 1.000 nM), 1 μL LP-01 probe substrate (final concentration 20 μM), 177 μL PBS buffer (0.1M, pH = 7.4). The reaction process is to first add 2 μL enzyme to 177 μL PBS buffer, then incubate 1 μL LP-01 probe substrate at 37 ° C for 3 minutes, then add 20 μL NADPH solution to start the reaction, react for 30 minutes, and finally immediately put it into the microplate reader to detect the luminescent signal, detect once every 1 minute, and continue to detect for 30 minutes. The highest value is taken as a comparison, and the metabolite generation-time curve is measured to determine the linear reaction enzyme concentration range. Three parallel groups were set up for each experiment. The average value of each group was compared with the control group without CYP3A4 / 5, and the detection limit of the probe in vitro CYP3A4 / 5 was determined to be 0.001 nM. Fig.13 , Fig.14 shown.

[0142] Example 9

[0143] Enzyme kinetics testing of CYP3A and human liver microsomes

[0144] The experiment was carried out on a microplate reader using a 96-well plate, 1 μL substrate LP-01 (final concentration of 1-100 μM), 2 μL CYP3A4 / 5 single enzyme (final concentration of 0.2 nM), 2 μL human liver microsomes (HLM) (final concentration of 0.02 mg / mL), 177 μL PBS buffer (pH 7.4, final concentration of 100 mM), 20 μL NADPH solution (final concentration of 1 mM) was added to start the reaction, the total volume was 200 μL, incubated at 37°C for 3 minutes, analyzed by microplate reader for 30 minutes, and measured every 1 minute. Detection conditions: full wavelength scan. The obtained fluorescence intensity was substituted into the standard curve to obtain the Vmax and Km of HLM, CYP3A4 / 5 for LP-01, such as Fig.15 As shown, Km is 4.32±0.67μM; Vmax is 1.52*10 6 ±3.99*10 4 .

[0145] Example 10

[0146] Quantitative detection of CYP3A activity in human liver microsomes (HLM)

[0147] The experiment was carried out on a microplate reader using a 96-well plate. The reaction system mainly included 1 μL of LP-01 probe substrate (final concentration was 20 μM), 2 μL of CYP3A single enzyme (final concentrations were 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.01, 0.2, and 0.3 mg / mL, respectively), and 177 μL of PBS buffer (0.1 M, pH = 7.4). The reaction process was as follows: first, 2 μL of enzyme was added to 177 μL of PBS buffer and incubated at 37°C for 3 min. Then, 1 μL of LP-01 probe substrate was added, and then 20 μL of NADPH solution was added to initiate the reaction. The microplate reader was immediately placed to detect the luminescent signal. The detection was performed once every 1 minute for 30 min. The highest value was taken as a comparison. The metabolite generation amount-time curve was determined to determine the linear reaction range of the enzyme activity. Fig.16 As shown, there is a good linear relationship between the chemiluminescence intensity and the enzyme concentration (R 2 =0.9511, p<0.0001).

[0148] Embodiment 11

[0149] CYP3A inhibitor screening

[0150] Four natural compounds were selected to test the inhibitory effect on the hydrolysis of two probes mediated by CYP3A. The final concentration of the inhibitor was set to 1μM, 10μM, and 100μM. The final concentration of the two probe substrates was 20μM, and the final concentration of the enzyme was 0.02mg / mL. The experiment was measured using a 96-well plate on an ELISA instrument. First, the enzyme, inhibitors of various concentrations, and buffer were pre-incubated at 37°C for 3min, and then the probe substrate was added to start the reaction. Then 20μL NADPH solution was added to start the reaction, and then the ELISA instrument was immediately placed for detection for 30min, with measurements every 1 minute. Detection conditions: full wavelength scanning. Three sets of data were set in parallel for each group of experiments.

[0151] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A chemiluminescent probe LP, characterized in that: Its structural features are shown below: Specifically, it includes a selective response group R1, a self-leaving group R2, a self-color enhancement group R3 and a chemiluminescent group R4; R1 is selected from: 1-ethyl-3-fluorobenzene, ethylbenzene, methyl, ethyl, chloroethyl or bromoethyl; R2 is selected from: hydrogen, fluorine, chlorine, bromine or iodine; R3 is selected from: tricyanofuran, cyano, acetic acid, methyl acetate, quinoline nitrile, pyran nitrile, semicyanine or methylquinoline nitrile; R4 is selected from: hydrogen, fluorine, chlorine, bromine or iodine.

2. A chemiluminescent probe LP according to claim 1, characterized in that: When R1 is 1-ethyl-3-fluorobenzene, R2 is cyano, R3 is chlorine, and R4 is chlorine, the chemiluminescent probe LP is named 3-(3-chloro-2-((3-chloro-4-((3-fluorobenzyl)oxy)benzyl)oxy)-4-methoxyspiro[adamantane-2,3'-[1,2]dioxetane]-4'-yl)phenyl)acrylonitrile.

3. The method for preparing the chemiluminescent probe LP according to claim 1, characterized in that: The synthetic route is as follows: The trigger group H is connected to the chemiluminescent matrix C by using the photoluminescent reaction to obtain the chemiluminescent probe LP.

4. The method for preparing the chemiluminescent probe LP according to claim 3, characterized in that: The synthetic route of LH is: The self-leaving group R2 is connected to the response group R1 through a benzylation nucleophilic reaction, and then reduced by sodium borohydride to obtain the trigger group LH.

5. The method for preparing the chemiluminescent probe LP according to claim 1, characterized in that: The synthetic route of LC is: 2-R4,-3-Hydroxybenzaldehyde is converted to the chemiluminescent matrix LM through 6 steps, which are the acetal reaction of R4,-3-hydroxybenzaldehyde with trimethyl orthoformate and tetrabutylammonium tribromide, the silyl ether protection of the phenolic hydroxyl group of 2-chloro-3-dimethoxycresol, followed by the phosphation reaction with trimethyl phosphite under the catalysis of titanium tetrachloride, and then further nucleophilic addition with adamantane ketone under the catalysis of lithium diisopropylamide, and then the silyl ether bond protection of the hydroxyl group is removed with hydrochloric acid, and finally the aldehyde reaction with polyformaldehyde occurs, and then the chemiluminescent matrix LM is modified by Wittig reaction, Mohr reaction and nucleophilic addition of aldehyde group to obtain the chemiluminescent matrix LC modified with different chromophores.

6. Use of the chemiluminescent probe LP as claimed in claim 1 in determining CYP3A activity.

7. The use according to claim 6, characterized in that: The probe is mixed with a biological sample containing CYP3A to carry out an enzymatic reaction, and the activity of CYP3A in different biological systems is quantitatively determined by quantitatively collecting and detecting the chemiluminescent signal generated per unit time or the generation rate of the corresponding product benzoate.

8. The use according to claim 7, characterized in that: The specific steps of the test include the following: A. Set the reaction temperature between 20 and 60°C and the incubation system pH between 5.5 and 10.5; B. After adding the prepared 0.1M phosphate buffer to the reaction vessel, add the probe at a concentration of 1.0 to 50μM, add the biological sample containing CYP3A at a concentration of 0.02mg / mL and pre-incubate at 37°C for 3 minutes; C. Add NADPH to start the chemiluminescent reaction for 1 hour, ensuring that the above substrates reach the quantitative limit in terms of O-dealkylation products and the conversion rate of the substrates does not exceed 10%; D. Determine the maximum chemiluminescence signal value within 3 hours as an evaluation index for determining CYP3A activity in different complex biological systems.

9. The use according to claim 8, characterized in that: The biological sample containing CYP3A includes any one of human liver microsomes, S9, animal or human tissue preparation fluid, and corresponding tissue cells and preparations thereof of CYP3A.

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