Specific chemiluminescent probe for determining cyp3a, preparation method and application thereof

Abstract

The application relates to a specific chemiluminescent probe for measuring CYP3A in the field of medicine, and the structural features of the probe are shown in the following formula: the probe structure comprises a high-specificity reaction response group of CYP3A, a chemiluminescent group, a self-coloring group and a self-leaving connecting group connected with the three groups, the chemiluminescent probe of the application does not need external light source excitation, has no light toxicity, has very high signal-to-noise ratio and sensitivity, can be used for cell and living body imaging, compared with traditional fluorescent probes, has the advantages of not being interfered by biological matrix, different detection modes and the like, and ensures the accuracy of experiments, the probe has the characteristics of convenient detection, high-throughput detection, and has potential application value in new drug research and development and individualized drug treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a chemiluminescent probe for enzymes, a synthesis method and application thereof, in particular, a specific chemiluminescent probe for determining CYP3A, a preparation method and application thereof. BACKGROUND

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

[0003] However, due to the influence of taking drugs, genetic polymorphism and environmental factors, there is a large difference in P450 enzyme activity between individuals, which may lead to various adverse effects, for example, CYP3A is affected by genetic polymorphism, which may cause drug-induced liver injury (DILI), in addition, the activity of CYP3A may also be inhibited or activated to cause drug-drug interactions (DDI), for example, rifampicin can cause the production of toxic products in patients using antiretroviral drugs containing ritonavir by up-regulating the activity of P4503A enzyme, thereby causing oxidative stress and hepatocyte damage, the change of CYP3A activity is the most common cause of drug-drug interactions. Therefore, it is of great significance to choose appropriate detection methods to accurately evaluate the activity of CYP3A enzyme, which can better understand the characteristics of individuals in drug metabolism and interaction, so as to use drugs more safely and reduce the risk of adverse reactions.

[0004] Currently, the detection methods for CYP3A mainly include ultraviolet spectroscopy, proteomics, immunoblotting, and fluorescent probe detection. Ultraviolet spectroscopy can determine enzyme activity based on the absorbance value changes of CYP3A after oxygen dealkylation and hydroxylation of the substrate, but the sensitivity is extremely poor and is easily disturbed by biological matrix. Proteomics uses liquid chromatography-mass spectrometry (LC-MS) to identify and quantify CYP3A, but the instrument is expensive, the maintenance cost is high, the sample preparation process is complex, the required analysis data is large, and the use of immunoblotting is limited by the specificity and affinity of the antibody. Moreover, immunoblotting can only achieve semi-quantification. Although the fluorescent probe method improves the sensitivity of CYP3A detection and is easier to operate, it can only be used in in vitro detection. Due to the interference of many matrixes in complex biological systems, external light excitation will generate many unremovable background signal interference real-time images.

[0005] As mentioned in patent CN101608212B, schisantherin A is used as a specific probe for CYP3A. This method evaluates the activity of CYP3A by mass spectrometry, specifically by measuring the reduction of the schisantherin A substrate or the generation of schisanol A product. However, this method has high requirements for sample preparation, is expensive, and has complex technology. At the same time, the detection process is affected by ionization efficiency differences and matrix effects, which has certain limitations. As mentioned in patent CN104975066B, the reduction of the substrate oligosaccharide ester compound Tenuifoliside A or the generation of its corresponding hydroxylated product is detected by ultraviolet absorption method to quantify CYP3A4 in different biological samples. However, this method is easily disturbed by substances with similar ultraviolet absorption wavelengths in complex biological samples, resulting in poor discrimination. In addition, the sensitivity of the ultraviolet absorption detection method for low concentration samples is limited, and the signal-to-noise ratio is also low, which greatly limits its application range. Patent CN114105979A proposes a 4-position hydroxylation reaction based on naphthalimide fluorescent probes as a probe reaction for detecting CYP3A activity, and can be used for in vitro rapid screening of reversible and irreversible inhibitors, activators, and inducers of CYP3A. However, the excitation wavelength of this fluorescent probe method is short, which can easily cause high background fluorescence. In contrast, the CYP3A probe detection method based on chemiluminescence activation relies on the self-luminous system of the emission excited state formed by chemical reaction, and does not require external excitation light source irradiation. Therefore, there is no self-luminous and light scattering caused by external excitation light source, which can realize zero interference of biological matrix, greatly improve the sensitivity and signal-to-noise ratio of detection, and realize real-time imaging in complex biological systems. However, there is currently no report on chemiluminescent human CYP3A probe substrates. SUMMARY

[0006] The present application aims at the deficiencies of the prior art, and provides a CYP3A chemiluminescent probe without external light source excitation, which combines a brand-new CYP3A oxidative dealkylation response group, has super-high signal-to-noise ratio and sensitivity compared with a conventional fluorescent probe, and can realize zero interference of a biological matrix, and can be used for measuring and positioning the enzyme activity and distribution of CYP3A in a complex biological system.

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

[0008]

[0009] Specifically, the selective response group R1, the self-leaving group R2, the self-color developing group R3 and the chemiluminescent group R4 are included; the R1 is selected from 1-ethyl-3-fluorobenzene, ethylbenzene, methyl, ethyl, chloroethyl or bromoethyl; the R2 is selected from hydrogen, fluorine, chlorine, bromine or iodine; the R3 is selected from tricyano furan, cyano, acetic acid, methyl acetate, quinoline nitrile, pyran nitrile, hemicyanine or methyl quinoline nitrile; and the R4 is selected from hydrogen, fluorine, chlorine, bromine or iodine.

[0010] Further, when the R1 is 1-ethyl-3-fluorobenzene, the R2 is cyano, the R3 is chlorine and the R4 is chlorine, the chemiluminescent probe LP is named as 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 purpose of the present application is to provide a preparation method of the chemiluminescent probe LP, and a synthesis route is as shown below:

[0012]

[0013] The trigger group LH is connected with the chemiluminescent parent LC by a photo-induced reaction to obtain the CYP3A specific chemiluminescent probe LP.

[0014] Further, a synthesis route of the LH is as follows:

[0015] The self-leaving group R2 is connected with the response group R1 by a benzylation nucleophilic reaction, and then the trigger group LH is obtained by reduction of sodium borohydride.

[0016] Further, a synthesis route of the LC is as follows:

[0017]

[0018] R4, 3-hydroxybenzaldehyde to chemiluminescence parent LM through 6-step reactions, which are 3-hydroxybenzaldehyde and trimethyl orthoformate and tetrabutylammonium tribromide acetal reaction, silicon ether protection of phenolic hydroxyl group of 2-chloro-3-dimethoxy methyl phenol, followed by phosphoric acid esterification with trimethyl phosphite under the catalysis of titanium tetrachloride, then further nucleophilic addition with adamantone under the catalysis of diisopropyl amino lithium, then removing the silicon ether bond protection of hydroxyl group with hydrochloric acid, and finally aldehyde group reaction with polyformal, then through Wittig reaction, Morita-Baylis-Hillman reaction and aldehyde nucleophilic addition to modify the chemiluminescence parent LM to obtain the chemiluminescence parent LC modified by different color enhancer groups.

[0019] The present application first introduces the Schaap-dioxetane (LM) chemiluminescence system into the CYP3A detection system, LM has a large molecular structure, the inventors modify the color enhancer group part with corresponding basic groups, such as cyano and pyran nitrile, in order to obtain a chemiluminescence parent (LC) which can effectively recognize the active site of CYP3A, and the design fully considers the characteristics of the active cavity of CYP3A, including its preference for hydrophobicity, medium basicity and large substrate molecules.

[0020] In the triggering end of LC, the inventors design a series of triggering groups (LH) that can be recognized by CYP3A, including methoxy, trifluoromethyl and diisopropyl acetal, etc., among which the triggering structure 1-chloro-2-(3-fluorophenyl) methoxy (LH-01) is first introduced, which is derived from the inspiration of CYP3A drug substrate Lapatinib, and shows high selectivity to CYP3A.

[0021] In addition, in order to facilitate the effective entry of CYP3A triggering group LH into the active cavity of CYP3A, the inventors construct a linking group between LC and LH, after catalysis by CYP3A, the triggering group LH will cause the electronic rearrangement of the linking group and self-leave, releasing the high-energy unstable intermediate state of dioxetane of phenoxy anion. Through the mechanism of electron exchange luminescence, benzyl benzoate is finally decomposed, and a significant chemiluminescence signal is generated.

[0022] The present application provides a novel design and invented enzyme-catalyzed chemiluminescence substrate, which is composed of CYP3A specific triggering group, linking group, self-coloring group and luminescent group, and a new CYP3A detection method developed by chemiluminescence activation, which has the advantages of convenience, anti-interference and high-throughput determination of CYP3A activity in biological complex system. In addition, the method provides an effective tool for in-depth study of the diversity and specificity mechanism of CYP3A metabolic catalysis, and has important application value for new drug research and development, individual diagnosis and individual medication.

[0023] Compared with the prior art probe, the application constructs a combination chemiluminescence body LC and a new trigger group LH of CYP3A, creates a new detection method of CYP3A, fundamentally improves the disadvantages of the traditional CYP3A probe, has ultra-high specificity, sensitivity and zero background matrix interference, provides more accurate and reliable detection results, has a wide application prospect, and is especially in the fields of biomedical detection and new drug screening.

[0024] The third object of the application is to provide an application of the chemiluminescence probe LP in the determination of CYP3A activity.

[0025] Further, after mixing the probe with the biological sample containing CYP3A, the enzymatic reaction is carried out, and the activity of CYP3A in different biological systems is quantitatively determined by quantitatively collecting the chemiluminescence signal generated in a unit time or the generation rate of the corresponding product benzoate.

[0026] Further, the specific steps in the detection include the following:

[0027] A. The reaction temperature is set to be between 20-60 DEG C, and the incubation system pH is between 5.5-10.5;

[0028] B. After adding the prepared 0.1M phosphate buffer into the reaction container, the probe is added, the substrate concentration is 1.0-50 μM, the biological sample containing CYP3A with a concentration of 0.02 mg / mL is added, and pre-incubation is carried out at 37 DEG C for 3 minutes;

[0029] C. NADPH is added to start the chemiluminescence reaction, the reaction time is 1 hour, and it is ensured that the above substrate reaches the quantitative limit in the O-dealkylation product and the conversion rate of the substrate is not more than 10%;

[0030] D. The maximum signal value of the chemiluminescence in 3 hours is determined as an evaluation index of the CYP3A activity in different complex biological systems.

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

[0032] Preferably, the substrate concentration is 20 μM in single-point determination.

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

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

[0035] The chemiluminescent probe LP can be used for quantitative detection of the activity of CYP3A in cell or tissue samples of different species.

[0036] The chemiluminescent probe LP can be used for rapid screening of CYP3A enzyme inhibitors and quantitative evaluation of their inhibition ability.

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

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

[0039] The chemiluminescent probe LP provided by the present application 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 preparation liquids, and various tissue cells. At the same time, the chemiluminescent probe LP can also be widely used in CYP3A research in animal whole body, to evaluate the differences in metabolic enzyme CYP3A between individuals and species.

[0040] The chemiluminescent probe LP of the present application has significant advantages in detecting CYP3A in vitro activity, including the following aspects: (1) high sensitivity: the detection of the chemiluminescent probe substrate does not require external light excitation, so it does not have background interference; (2) mature technology: the parent compound can be obtained by chemical synthesis and can be prepared in large quantities; (3) convenience of high-throughput detection: batch detection can be conveniently performed on common laboratory fluorescent enzyme label meters. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The synthesis route of the chemiluminescent parent compound LM is shown in the following figure:

[0042] Figure 2A synthetic route for the CYP3A triggering gene LH;

[0043] Figure 3 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] Figure 9 The chemiluminescent response mechanism diagram of LP-01 for detecting CYP3A;

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

[0051] Figure 11 The figure shows the evaluation of LP-01's HiHep cytotoxicity and HepRG cytotoxicity;

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

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

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

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

[0056] Figure 16 This is a quantitative evaluation curve for 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 application is shown as follows:

[0059]

[0060] Specifically, it comprises a selective response group R1, a self-leaving group R2, a self-color developing group R3, and a chemiluminescent group R4; the R1 is selected from 1-ethyl-3-fluorobenzene, ethylbenzene, methyl, ethyl, chloroethyl, or bromoethyl; the R2 is selected from hydrogen, fluorine, chlorine, bromine, or iodine; the R3 is selected from tricyano furan, cyano, acetic acid, methyl acetate, quinoline nitrile, pyran nitrile, hemicyanine, or methyl quinoline nitrile; and the R4 is selected from hydrogen, fluorine, chlorine, bromine, or iodine.

[0061] Preferably, when the R1 is 1-ethyl-3-fluorobenzene, the R2 is cyano, the R3 is chlorine, and the R4 is chlorine, the chemiluminescent probe LP is named as 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 route of the chemiluminescent parent substance LM-01, LM-02, and LM-03 is shown as follows Figure 1

[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. After the mixture was stirred at room temperature for 30-45 min, 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 aqueous sodium bicarbonate solution, the organic phase was separated and dried with anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure to obtain a yellow oil (crude product, crude yield (2.0 g, yield 92.3%).

[0067] Compound 1c

[0068] ​Compound 1b (2.0 g, 12.2 mmol) and imidazole (1.66 g, 24.4 mmol) were sequentially dissolved in 10.0 mL of dichloromethane, 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 using a TLC plate (P / E = 5 / 1, v / v), and a white precipitate was removed by filtration to obtain a filtrate. The obtained filtrate was washed with 40 mL of ethyl acetate and 0.01 M sodium bicarbonate solution, and then the organic phase was separated and dried with anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure to obtain a yellow oily crude product (4.3 g, crude product yield 126.5%).

[0069] Compound 2d

[0070] Compound 1c (4.4 g, 15.9 mmol, calculated as 100% product content) 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 the reaction was performed at this temperature for about 2.5 h, the reaction was monitored using a TLC plate (P / E = 1 / 3, v / v). Subsequently, the reaction solution was poured into an appropriate amount of saturated aqueous sodium bicarbonate solution at 0°C until white bubbles disappeared, and then the reaction solution was washed with 60 mL of dichloromethane, and then the organic phase was separated and dried with anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure to obtain a colorless oily crude product, and then the colorless oily compound 1d (2.6 g, crude product yield 45.5%) was separated by silica gel column chromatography (P / E = 1 / 3, v / v).

[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, and a 2.0 M THF solution of lithium diisopropylamide (LDA) (3.7 mL, 7.5 mmol) was slowly added dropwise under nitrogen and stirring was continued for 20 min, and then a 10.0 mL THF solution of adamantane ketone (751.1 mg, 5.0 mmol) was slowly added to the reaction solution, and after stirring at -78°C for 15 min, the reaction was allowed to proceed at room temperature. After the reaction was completed, 100 mL of ethyl acetate and 100 mL of saturated brine were added, and the organic phase was dried with anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure, and then the crude product was dissolved in THF, and 5 mL of a 1.0 M THF solution of tetrabutylammonium fluoride was added, and the reaction was allowed to proceed for 2 h, and after the reaction was completed at room temperature, it was washed with ethyl acetate and 1 M hydrochloric acid, and the organic phase was dried with anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure, and the crude product was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 8 / 1, v / v) to obtain compound 1f (0.4 g, yield 35.1%) as a white solid.

[0073] Compound LM-01

[0074] Compound 1f (811.1 mg, 3.0 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 sequentially added, and then paraformaldehyde (328.0 mg, 10.9 mmol) was added and the reaction was heated to reflux overnight, and after the reaction was completed, it was returned to room temperature, extracted with dichloromethane and water, and the pH was adjusted to 6.0 with a hydrochloric acid solution (1 M), and the organic phase was separated and the product was dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure, and the crude product was separated by silica gel column chromatography to obtain compound LM-01 (502.0 mg, yield 56.1%) as a light yellow solid.

[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 sequentially added, and 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 aqueous sodium bicarbonate solution, and the organic phase was separated and dried with anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure to obtain a yellow oil (crude product, crude yield (2.4 g, yield 95%).

[0078] Compound 2c

[0079] Compound 2b (2.4 g, 12.2 mmol) and imidazole (1.66 g, 24.4 mmol) were sequentially dissolved in 10.0 mL of dichloromethane, 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 a white precipitate was removed by filtration, and the obtained filtrate was washed with 40 mL of ethyl acetate and 0.01 M sodium bicarbonate solution, and then the organic phase was separated and dried with anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure to obtain a yellow oily crude product (5.1 g, crude product yield 131.0%).

[0080] Compound 2d

[0081] Compound 2c (5.08 g, 15.9 mmol, calculated as 100% product content) 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, and after the reaction was performed at this temperature for about 2.5 h, 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 aqueous sodium bicarbonate solution at 0°C until the white bubbles disappeared, and 60 mL of dichloromethane was added to wash the reaction solution, and then the organic phase was separated and dried with anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure to obtain a colorless oily crude product, and then the colorless oily compound 2d (1.9 g, crude product yield 31%) was separated by silica gel column chromatography (P / E = 2 / 3, v / v).

[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, 2.0 M THF solution of lithium diisopropylamide (LDA) (3.7 mL, 7.5 mmol) was slowly added dropwise under nitrogen and stirring was continued for 20 min, then 10.0 mL of THF solution of adamantane ketone (751.1 mg, 5.0 mmol) was slowly added to the reaction solution and stirring was continued for 15 min at -78 °C and then the reaction was allowed to proceed at room temperature. After the reaction was completed, 100 mL of ethyl acetate and 100 mL of saturated brine were added, the organic phase was dried with anhydrous sodium sulfate, then the solvent was removed by distillation under reduced pressure, then the crude product was dissolved in THF and 5 mL of 1.0 M THF solution of tetrabutylammonium fluoride was added and the reaction was allowed to proceed for 2 h, after the reaction was completed at room temperature, it was washed with ethyl acetate and 1 M hydrochloric acid, the organic phase was dried with anhydrous sodium sulfate, then the solvent was removed by distillation under reduced pressure and the crude product was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 1, v / v) to obtain compound 2f (0.6 g, yield 40.0%) as a white solid.

[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 sequentially added, 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, it was returned to room temperature, extracted with dichloromethane and water, and the pH was adjusted to 6.0 with a hydrochloric acid solution (1 M), the organic phase was separated and the product was dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure to obtain compound LM-02 (620.0 mg, yield 60.0%) as a light yellow solid.

[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 sequentially added, and 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 aqueous sodium bicarbonate solution, the organic phase was separated and dried with anhydrous sodium sulfate, then the solvent was removed by distillation under reduced pressure to obtain a light yellow oil (crude product, crude yield (3.1 g, yield 96.5%).

[0089] Compound 3c

[0090] Compound 3b (3.0 g, 12.2 mmol) and imidazole (1.66 g, 24.4 mmol) were sequentially dissolved in 10.0 mL of dichloromethane, 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 a white precipitate was removed by filtration, and the obtained filtrate was washed with 50 mL of ethyl acetate and 0.01 M sodium bicarbonate solution, and then the organic phase was separated and dried with anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure to obtain a yellow oily crude product (5.6 g, crude product yield 127.2%).

[0091] Compound 3d

[0092] Compound 3c (5.6 g, 15.5 mmol, calculated as 100% product content) 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, and after the reaction was performed at this temperature for about 2.5 h, 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 aqueous sodium bicarbonate solution at 0°C until the white bubbles disappeared, and 60 mL of dichloromethane was added to wash the reaction solution, and then the organic phase was separated and dried with anhydrous sodium sulfate. The solvent was removed by distillation 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 white powdery oily compound 3d (1.9 g, crude product yield 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, and 2.0 M THF solution (2.6 mL, 5.0 mmol) of diisopropylamino lithium (LDA) was slowly added dropwise under nitrogen and stirring was continued for 20 min, and then 10.0 mL of THF solution of adamantane ketone (751.1 mg, 5.0 mmol) was slowly added to the reaction solution, and after stirring at -78°C for 15 min, the reaction was allowed to proceed at room temperature. After the reaction was completed, 100 mL of ethyl acetate and 100 mL of saturated brine were added, and the organic phase was dried with anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure, and then the crude product was dissolved in THF, and 5 mL of 1.0 M THF solution of tetrabutylammonium fluoride was added, and the reaction was allowed to proceed for 2 h, and after the reaction was completed at room temperature, it was washed with ethyl acetate and 1 M hydrochloric acid, and the organic phase was dried with anhydrous sodium sulfate, and then the solvent was removed by distillation under reduced pressure, and the crude product was separated by silica gel column chromatography (petroleum ether / ethyl acetate = / 1, v / v) to obtain compound 3f (0.6 g, yield 37.6%) as a light white solid.

[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 sequentially added, and then paraformaldehyde (1.6 g, 54.5 mmol) was added and the reaction was heated to reflux overnight, and after the reaction was completed, it was returned to room temperature, extracted with dichloromethane and water, and the pH was adjusted to 6.0 with a hydrochloric acid solution (1 M), and the organic phase was separated and the product was dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure, and the obtained crude product was separated by silica gel column chromatography to obtain compound LM-03 (907.6 mg, yield 42.2%) as a yellow solid.

[0097] Example 2

[0098] Synthesis of CYP3A trigger group LH-01, LH-02, LH-03 as Figure 2 indicated

[0099] Synthesis of LH-01:

[0100] Compound 4b

[0101] Compound 4a (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) was added sequentially, 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 in an ice bath for 3 h, 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 sequentially 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 in an ice bath for 3 h, 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-02 (144.7 mg, 95.1%) as a white solid.

[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 sequentially, and finally 3-chloro-4-hydroxybenzaldehyde (470.0 mg, 3.0 mmol) was added and heated to reflux for 3 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the obtained 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] In a flask, compound 6b (212.2 mg, 1.0 mmol) was dissolved in 5 mL of anhydrous methanol, and sodium borohydride (56.7 mg, 1.5 mmol) was slowly added. After the reaction was completed at an ice bath for 3 h, the solvent was removed by distillation under reduced pressure, and the obtained crude product was separated by silica gel column chromatography to finally obtain a white solid compound LH-03 (206.1 mg, 96.2%).

[0114] Example 3

[0115] The synthesis of chemiluminescent parent compounds LC-01, LC-02, LC-03 modified with different color enhancer groups as shown in Figure 3

[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 sequentially added, and stirring was continued at room temperature for 30 min. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the obtained crude product was separated by silica gel column chromatography to finally obtain 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 pyridine cyanide (252.2 mg, 1.2 mmol) were sequentially added, and the reaction was heated to reflux overnight. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the obtained crude product was separated by silica gel column chromatography to finally obtain a red product LC-02 (299.2 mg, yield 57.0%).

[0120] Compound LC-03

[0121] ​Compound LM-02 (266.7 mg, 1.0 mmol) was dissolved in 5 mL of tetrahydrofuran in a flask, and piperidine (Pi) (99.0 μL, 2.0 mmol) and tricyano furan (239.1 mg, 1.2 mmol) were sequentially added, and the reaction was heated to reflux overnight. After the reaction was completed, it was returned to room temperature, washed with dichloromethane and water, and separated and purified by silica gel column chromatography to obtain an orange 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 shown in Figure 4

[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) (420.0 mg, 2.0 mmol) and PPh3 (786.9 mg, 3.0 mmol) were sequentially added under ice bath conditions, and after stirring for 30 min, compound LC-02 (355.9 mg, 0.8 mmol) was added. After stirring at room temperature overnight, a catalytic amount of methylene blue was added, the solution was irradiated with yellow light, and oxygen was introduced into the solution. The reaction was monitored by preparative liquid chromatography, and after the reaction was completed, the solvent was concentrated under reduced pressure, and the product LP-02 (404.0 mg, yield 72.3%) was separated and purified by preparative liquid chromatography, 1 H-NMR spectrum and 13 C-NMR spectrum as shown in Figure 5 Figure 6

[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 sequentially added under ice bath conditions, and after stirring for 30 min, compound LC-02 (355.9 mg, 0.8 mmol) was added. After stirring at room temperature overnight, a catalytic amount of methylene blue was added, the solution was irradiated with yellow light, and oxygen was introduced into the solution. The reaction was monitored by preparative liquid chromatography, and after the reaction was completed, the solvent was concentrated under reduced pressure, and the product LP-02 (404.0 mg, yield 72.3%) was separated and purified by preparative liquid chromatography, 1 H-NMR spectrum and 13 C-NMR spectrum as shown in Figure 7 Figure 8 ​​​​shown.

[0128] LP-03

[0129] In a flask, compound LH-03 (266.7 mg, 1.0 mmol) was dissolved in 5 mL of tetrahydrofuran, and diethyl azodicarboxylate (DEAD) (348.3 mg, 2.0 mmol) and PPh3 (786.9 mg, 3.0 mmol) were added successively under ice bath conditions, and after stirring for 30 min, compound LC-03 (411.3 mg, 0.8 mmol) was added, and after stirring overnight at room temperature, a catalytic amount of methylene blue was added, and the solution was irradiated with yellow light and oxygen was bubbled into the solution, and the reaction was monitored by preparative liquid chromatography, and after the reaction was completed, the solvent was concentrated under reduced pressure, and the product LP-03 (461.9 mg, yield 69.1%) was separated and purified by preparative liquid chromatography.

[0130] Example 5

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

[0132] The following enzyme origins 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 amount of enzyme, mainly including 2.0 μL of enzyme (5 nM final concentration), 2.0 μL of LP-01 probe substrate (20.0 μM final concentration), 177.0 μL of PBS buffer (pH = 7.4) and NADPH solution (1.0 mM final concentration). The reaction process was as follows: first, 2.0 μL of different single enzymes were added to 177.0 μL of PBS buffer and 1 μL of LP-01 probe substrate, and incubated at 37°C for 3 min, then 20 μL of NADPH solution was added to start the reaction, and finally it was immediately placed into a microplate reader to detect the chemiluminescence signal, full wavelength scanning, 1 min detection, continuous detection for 30 min, and the highest value was taken as the comparison. Each test was set up with 3 parallel tests, and the average value was taken for calculation. The probe was only specifically dealkylated by the recombinant human CYP3A single enzyme, and there was almost no dealkylation by other single enzymes, as shown in Figure 9 As shown, LP-01 releases adamantane ketone and emits an optical signal after obtaining a high-energy intermediate under the action of CYP3A oxygen dealkylation, and the corresponding single enzyme selectivity results are as shown in Figure 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] Cell toxicity experiment of LP-01

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

[0136] Example 7

[0137] CYP3A time kinetic curve determination

[0138] The reaction system mainly includes 2 μL CYP3A4 / 5 single enzyme (final concentration 0.2 nM), 1 μL LP-01 probe substrate (final concentration 20 μM), 177 μL PBS buffer (0.1 M, pH = 7.4) and NADPH solution (final concentration 1 mM). The reaction process is as follows: first, 2 μL CYP3A4 / 5 single enzyme is added to 177 μL PBS buffer, then 1 μL LP-01 probe substrate is incubated at 37°C for 3 minutes, then 20 μL NADPH solution is added to start the reaction, and finally it is immediately placed into the enzyme marker for detection of luminescence signal, full wavelength scanning, 1 minute detection, continuous detection for 30 min, taking the highest value of parallel reaction as comparison, determining the generation amount-time curve of metabolite, and determining the linear reaction time range. Each experiment sets 3 groups of parallel, as shown in Figure 12 The peak time is 25 minutes.

[0139] Example 8

[0140] Determination of the detection lower limit of CYP3A in vitro

[0141] The reaction system mainly includes 5 μL of enzyme (final concentration is 0.001, 0.002, 0.005, 0.010, 0.025, 0.050, 0.100, 0.500, 1.000 nM respectively), 1 μL of LP-01 probe substrate (final concentration 20 μM), 177 μL of PBS buffer (0.1 M, pH = 7.4). The reaction process is as follows: first, 2 μL of enzyme is added to 177 μL of PBS buffer, then 1 μL of LP-01 probe substrate is incubated at 37°C for 3 min, then 20 μL of NADPH solution is added to start the reaction, the reaction is carried out for 30 min, finally it is immediately placed into the enzyme label instrument to detect the luminescence signal, 1 min detection once, continuous detection for 30 min, the highest value is taken as comparison, the metabolic product generation-time curve is determined, and the linear reaction enzyme concentration range is determined. Each experiment sets 3 groups in parallel. The average value of each group is compared with the control group without CYP3A4 / 5, and the detection lower limit of the probe in vitro CYP3A4 / 5 is determined to be 0.001 nM, as shown in Figure 13 、 Figure 14

[0142] Example 9

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

[0144] The experiment is determined on the enzyme label instrument using a 96-well plate, 1 μL of substrate LP-01 (final concentration 1-100 μM), 2 μL of CYP3A4 / 5 single enzyme (final concentration 0.2 nM), 2 μL of human liver microsomes (HLM) (final concentration 0.02 mg / mL), 177 μL of PBS buffer (pH 7.4, final concentration 100 mM), 20 μL of NADPH solution (final concentration 1 mM) is added to start the reaction, the total volume is 200 μL, incubated at 37°C for 3 min, and analyzed by the enzyme label instrument for 30 min, 1 min detection once. The detection condition is full wavelength scanning. The obtained fluorescence intensity is substituted into the standard curve to obtain the Vmax and Km of HLM, CYP3A4 / 5 to LP-01, as shown in Figure 15 , the Km is 4.32 ± 0.67 μM; the 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 determined on an enzyme marker using a 96-well plate, and the reaction system mainly included 1 μL of LP-01 probe substrate (a final concentration of 20 μM), 2 μL of CYP3A single enzyme (a final concentration of 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.01, 0.2, 0.3 mg / mL), 177 μL of PBS buffer (0.1 M, pH = 7.4), and the reaction process was as follows: 2 μL of enzyme was first added to 177 μL of PBS buffer, 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 start the reaction, and then it was immediately placed into an enzyme marker to detect the luminescence signal, which was detected once every 1 min, and the detection was continued for 30 min, the highest value was taken as the comparison, the generation amount-time curve of the metabolic product was determined, and the linear reaction range of the enzyme activity was determined, as shown in Figure 16 2 = 0.9511, p < 0.0001).

[0148] Example 11

[0149] CYP3A inhibitor primary screening

[0150] Four natural compounds were selected to test the inhibition of CYP3A-mediated hydrolysis of two probes, and the final concentration of the inhibitor was set to 1 μM, 10 μM, and 100 μM, the final concentration of the two probe substrates was 20 μM, and the final concentration of the enzyme was 0.02 mg / mL, the experiment was determined on an enzyme marker using a 96-well plate, first, the enzyme, each concentration of inhibitor, and buffer were pre-incubated at 37°C for 3 min, then the probe substrate was added to start the reaction, then 20 μL of NADPH solution was added to start the reaction, and then it was immediately placed into an enzyme marker to detect for 30 min, every 1 min, and the detection condition was full wavelength scanning. Each group of experiments set 3 groups of data in parallel.

[0151] The above is only an embodiment of the present application, and the specific structure and characteristics of the scheme are not described in detail. It should be noted that for those skilled in the art, without departing from the structure of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope claimed in this application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain the content of the claims.​

Claims

1. A chemiluminescent probe LP, characterized in that Its structural characteristics are shown below: or .

2. The method for preparing the chemiluminescent probe LP according to claim 1, characterized in that: The synthetic route is as follows: ； The trigger group LH is linked to the chemiluminescent matrix LC using the Mitsunobu reaction to obtain the chemiluminescent probe LP-01 or LP-02.

3. The method for preparing the chemiluminescent probe LP according to claim 2, 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.

4. The method for preparing the chemiluminescent probe LP according to claim 3, characterized in that: The synthetic route of LC is: ； 2-R4,-3-hydroxybenzaldehyde is converted to the chemiluminescent precursor LM through a six-step reaction, which includes the acetalization 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, and the phosphation reaction with trimethyl phosphite catalyzed by titanium tetrachloride. It is then further reacted with adamantanone in the presence of lithium diisopropylamide, and the silyl ether protection of the hydroxyl group is removed with hydrochloric acid. Finally, it is aldehyde-allylated with paraformaldehyde. The chemiluminescent precursor LM is then modified through the Wittig reaction, the Mohr reaction, and the nucleophilic addition of the aldehyde group to obtain chemiluminescent precursors LC modified with different chromophores.

5. Use of the chemiluminescent probe LP as claimed in claim 1 in determining CYP3A activity for non-disease diagnosis and treatment purposes.

6. The use according to claim 5, characterized in that The probe is mixed with a biological sample containing CYP3A and then an enzymatic reaction is carried out. The activity of CYP3A in different biological systems is quantitatively measured by quantitatively collecting and detecting the chemiluminescent signal generated per unit time or the generation rate of the corresponding product benzoate.

7. The use according to claim 6, characterized in that The specific steps of the test include the following: A. Set the reaction temperature between 20-60°C and the incubation system pH between 5.5-10.5; B. Add the prepared 0.1 M phosphate buffer to the reaction vessel, then add the probe at a concentration of 1.0-50 μM, and a biological sample containing CYP3A at a concentration of 0.02 mg / mL. Pre-incubate at 37°C for 3 minutes. C. Add NADPH to initiate the chemiluminescent reaction for 1 hour, ensuring that the above substrates reach the limit of quantification for the O-dealkylation product and the conversion of the substrate does not exceed 10%; D. Determine the maximum chemiluminescence signal within 3 hours as an evaluation indicator for measuring CYP3A activity in different complex biological systems.

8. The use according to claim 7, 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.

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