A fluorescent probe for detecting cytochrome oxidase CYP2D6 and its application

Abstract

A fluorescent probe for detecting cytochrome oxidase CYP2D6 and its application, which belong to the field of biomedicine technology. The specific probe substrate can be used to determine the enzyme activity of CYP2D6 in biological systems. The process of CYP2D6 enzyme activity determination is as follows: the benzylmethoxy demethylation reaction in the meso-position pyridinium quaternary ammonium salt of Bodipy compounds is selected as the probe reaction, and the activity of CYP2D6 enzyme in various biological samples is determined by quantitatively detecting the amount of its metabolite generated per unit time. The probe is a selective probe for detecting CYP2D6 activity, which can achieve efficient discovery of CYP2D6 inhibitors (including mechanism-type inhibitors) and in-depth research on CYP2D6-related diseases. The probe can also be used for rapid in vitro screening of reversible and irreversible inhibitors, activators and inducers of CYP2D6, and for detecting drug-drug interactions of CYP2D6 at the in vivo level.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a fluorescent probe for detecting cytochrome oxidase CYP2D6 and an application thereof. Background Art

[0002] Cytochrome P450 is a superfamily of heme-thiolate proteins that play an important role in the metabolism of a variety of endogenous and exogenous substances, such as drugs, carcinogens, and environmental pollutants [Drug Discov Today. 2021, 26: 2456-64]. CYP2D6 is widely expressed in the liver, intestines, brain, and other parts of the body. According to statistics, CYP2D6 is an important subfamily of cytochrome P450, mediating the metabolism of approximately 25% (more than 150) of commonly used clinical drugs and is one of the important metabolic targets for studying human drug metabolism. Tricyclic antidepressants such as clomipramine, selective serotonin reuptake inhibitors such as fluoxetine, and opioids such as codeine are all substrates of CYP2D6 [Drug Metab Rev. 2009, 41: 573-643]. CYP2D6 is also involved in the metabolism of carcinogens and neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1,2,3,4-tetrahydroquinoline, and indolealkylamines, as well as endogenous substrates in the body such as ethyl serotonin and neurosteroids [J Biol Chem. 2013, 288: 4436-51; J Biol Chem. 2019, 294: 10336-48].

[0003] The CYP2D6 gene is located on chromosome 22q13.1 and consists of nine exons with an open reading frame of 1,491 base pairs encoding 497 amino acids [Br J Clin Pharmacol. 1987, 23:455-8]. Based on phenotype, the population can be divided into 2D6 ultra-rapid metabolizers, rapid metabolizers, intermediate metabolizers, and slow metabolizers, with the corresponding proportions in Caucasians being 3–5%, 70–80%, 10–17%, and 3–7%, respectively [Am J Hum Genet. 1997, 60:284-95]. Hu et al. reported that in the Han population, the variation in the two major metabolites of citalopram, desmethylcitalopram and N-oxycitalopram, was 38-129% and 13-138%, respectively [Drug Metab Pharmacokinet. 2016, 31: 133-8]. The relevant results also illustrate the significant variation in CYP2D6 metabolic levels among Chinese people.

[0004] To date, over 100 single nucleotide polymorphisms (SNPs) in CYP2D6 have been reported, including 33 non-synonymous SNPs with potential effects on CYP2D6 function. Furthermore, a growing number of clinical studies have demonstrated that CYP2D6 genetic polymorphisms can significantly influence drug clearance and efficacy [Drug Metab Rev. 2009, 41:573-643]. The variability in CYP2D6 function across populations further complicates the complexities of CYP2D6-mediated drug metabolic interactions. In particular, patients with inherently low metabolic function, combined with potent CYP2D6 inhibition induced by co-administered medications, are particularly susceptible to impaired drug metabolism, potentially exceeding the safety window and leading to adverse drug reactions. Furthermore, CYP2D6 regulates a wide range of drugs, including central nervous system drugs with narrow therapeutic indexes and potent side effects [Drug Metab Rev. 2009, 41:573-643]. Therefore, the efficient study of potent inhibitors of CYP2D6 and their inhibitory mechanisms has always been a hot topic in the field of drug metabolism.

[0005] Mechanism-based enzyme inactivation (MBI) converts substrates into highly unstable, highly active electrophilic metabolites, which covalently modify metabolic enzymes, leading to irreversible inactivation. This process is often highly correlated with adverse drug interactions and can easily induce serious adverse drug reactions [Chem Rev. 2018, 118:4037-70]. This indirect molecular inhibition of target enzyme activity is often more difficult to detect and predict, making related research challenging but highly significant.

[0006] Considering the safety issues of drugs that are preferentially metabolized by CYP2D6, and the fact that slow CYP2D6 metabolizers have low metabolic capacity and may be strongly inhibited by CYP2D6 inhibitors (including mechanism-based enzyme disinhibition), we need to develop fluorescent probes that can sensitively monitor CYP2D6 activity to help discover and evaluate MBI-related biological effects and drug safety. Summary of the Invention

[0007] To address the challenges of the prior art, the development of CYP2D6-selective probes is crucial for the efficient discovery of CYP2D6 inhibitors and in-depth research into related diseases. The development of highly selective CYP2D6 fluorescent probes and their associated high-throughput detection methods is of great practical value. The present invention provides a selective fluorescent probe for detecting cytochrome oxidase CYP2D6 and its applications. The fluorescent probe substrate is non-fluorescent, while its metabolites are fluorescent, enabling sensitive detection of CYP2D6 enzyme activity. This probe reaction can be used to quantitatively evaluate the distribution and function of CYP2D6 in a variety of biological systems.

[0008] The present invention provides a selective fluorescent probe for detecting cytochrome oxidase CYP2D6. The probe can be specifically catalyzed by CYP2D6 to generate a corresponding product, the general structural formula of which is shown in formula (1). The Bodipy derivative has the following general structural formula:

[0009]

[0010] Among them, R 1 is selected from hydrogen, methyl, ethyl, halogen, R 2 is selected from hydrogen, methyl, ethyl, halogen, R 3 Selected from methyl, p-methylphenyl, R 4 is selected from methyl, p-methylphenyl group; the probe substrate is selected from the following structures:

[0011]

[0012] The Bodipy compounds of the present invention have the characteristics of high selectivity of metabolic enzymes (mainly metabolized by CYP2D6), easy detection of metabolites, and high sensitivity.

[0013] The present invention also provides the selective fluorescent probe for detecting cytochrome oxidase CYP2D6, which uses the compound of formula (1) as a specific substrate of the CYP2D6 subtype to carry out a dealkylation reaction, and quantitatively measures the CYP2D6 activity in different biological systems (including recombinantly expressed CYP2D6 enzymes, human or animal tissue preparations, various tissue cells, and other biological systems) by quantitatively detecting the substrate elimination rate or the product generation rate per unit time. The specific determination method is:

[0014] Bodipy compounds are used as probe substrates in the system; the substrate concentration is selected from 1 / 10 to 10K m ; The optimal substrate concentration for single-point determination is K m .

[0015] In a buffer solution such as PBS, the reaction temperature is between 20°C and 60°C, preferably 37°C for the optimal reaction time; the pH of the incubation system is between 5.5 and 10.5, preferably pH 7.4 for the optimal reaction pH value;

[0016] The reaction time is 5 to 120 minutes, and the reaction is terminated when the corresponding hydroxylation products of the above substrates reach the quantitative limit and the substrate conversion rate does not exceed 20%;

[0017] The amount of substrate reduction or product generation per unit time is measured as an evaluation indicator of CYP2D6 activity.

[0018] The present invention provides a selective fluorescent probe for cytochrome oxidase CYP2D6 and its application. The probe substrate has no fluorescence, while its hydroxylation product has strong fluorescent properties. A fluorescence detector can be used to simultaneously achieve rapid and sensitive detection of the substrate and product. The fluorescence detection conditions for the hydroxylation product are: an excitation wavelength of 480-520 nm and a maximum emission wavelength of 540-600 nm.

[0019] This specific probe substrate is a fluorescent probe that is not susceptible to interference from the biological matrix and impurities during CYP2D6 activity detection. It can be used to quantitatively measure the activity of CYP2D6 and its isoenzymes in various recombinant CYP2D6, human and animal tissue preparations, and various tissue cells. It can also serve as a probe substrate for CYP2D6 in vivo and whole animals, assessing individual and species differences in the metabolic enzyme CYP2D6. The fluorescent detection method for this probe substrate and its hydroxylated metabolites can also be used for the rapid screening of CYP2D6 reversible and irreversible inhibitors, activators, and inducers, as well as for the quantitative evaluation of their activity-modulating and expression-inducing abilities.

[0020] Using recombinant cytochrome oxidase CYP2D6 and a liver microsome incubation system, the study demonstrated that Bodipy compounds can be specifically metabolized by cytochrome oxidase CYP2D6 to produce hydroxylated products, based on correlation analysis, reconstructed single-enzyme metabolic reactions, specific inhibition experiments, and enzyme reaction kinetics. Further investigations using metabolic evaluation systems such as freshly extracted hepatocytes, primary cultured hepatocytes, liver slices, and liver perfusion from various mammals revealed highly specific metabolic reactions.

[0021] As a selective fluorescent probe substrate for the highly specific cytochrome oxidase CYP2D6 enzyme, this compound can be used to detect the activity of CYP2D6. It is particularly suitable for the determination of CYP2D6 enzyme activity produced in bacteria, insect cells, mammalian cells, and yeast cloning expression systems, as well as the activity calibration of CYP2D6 in preparations such as microsomes and S9 derived from various mammalian tissues and organs.

[0022] This specific probe substrate can be used to measure CYP2D6 enzyme activity in biological systems. The CYP2D6 enzyme activity assay process is as follows: the benzylmethoxy demethylation reaction of the meso-pyridinium quaternary ammonium salt of a Bodipy compound is selected as the probe reaction, and the CYP2D6 enzyme activity in various biological samples is determined by quantitatively detecting the amount of metabolite produced per unit time. Using the two-photon fluorescent probe reaction for CYP2D6 enzyme described in this invention to detect CYP2D6 enzyme activity in vitro has the following significant advantages:

[0023] (1) High specificity: Bodipy compounds can be metabolized by cytochrome oxidase CYP2D6 enzyme with high specificity into a metabolite, namely the hydroxylation product.

[0024] (3) Cheap and easy to obtain: Bodipy compounds can be obtained by chemical synthesis, the synthesis process is simple and easy, and the detection cost of fluorescence method is low.

[0025] (4) High sensitivity: The hydroxylated products of Bodipy compounds have good fluorescence emission spectral characteristics, which can better reduce background fluorescence interference.

[0026] This type of probe exhibits excellent selectivity for recombinant human CYP2D6, while other CYP family enzymes rarely catalyze this probe reaction. The fluorescence response of probe 2D6-8 to CYP2D6 is 56 times higher than that of other CYP family enzymes. Therefore, this type of probe reaction can selectively detect CYP2D6 enzyme activity. Furthermore, this type of probe exhibits a low limit of detection, reaching 0.021 nM for probe 2D6-8. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the general structural formula of Bodipy compounds.

[0028] Figure 2 It's 2D6-8 1 H-NMR spectrum.

[0029] Figure 3 It's 2D6-8 13 C-NMR spectrum.

[0030] Figure 4 This is the high-resolution mass spectrum of 2D6-8.

[0031] Figure 5 This is the result of the 2D6-8 human CYP recombinant single enzyme screening test.

[0032] Figure 6 This is the result of inhibition by multiple CYP isoform inhibitors in human liver tissue.

[0033] Figure 7 This is a standard curve diagram for CYP2D6 protein concentration determination.

[0034] Figure 8 This is a laser confocal imaging image of HT-29 cells.

[0035] Figure 9 This is a laser confocal 3D imaging image of brain tissue.

[0036] Figure 10 This is the visualization high-throughput screening result of CYP2D6 inhibitors.

[0037] Figure 11 The metabolic pathway of 2D6-8 is mediated by CYP2D6.

[0038] Figure 12 is the reaction rate of CYP2D6 metabolizing 2D6-1 to 2D6-8. DETAILED DESCRIPTION

[0039] The present invention will be further described in the following examples, but are not intended to limit the present invention.

[0040]

[0041] Among them, R 1 、R 2 is hydrogen, R 3 and R 4 When it is methyl, the compound is named 2D6-1; R 1 、R 3 and R 4 is methyl, R 2 When R is hydrogen, the compound is named 2D6-2; 1 is hydrogen, R 2 is ethyl, R 3 and R 4 When it is methyl, the compound is named 2D6-3; R 1 is methyl, R 2 is ethyl, R 3 and R 4 When it is methyl, the compound is named 2D6-4; R 1 is methyl, R 2 is ethyl, R 3 is p-methylphenylvinyl, R 4 When it is methyl, the compound is named 2D6-5; R 1 、R 2 is hydrogen, R 3 and R 4 When it is p-methylphenyl group, the compound is named 2D6-6; R 1 is fluorine, R 2 is hydrogen, R 3 and R 4 When it is methyl, the compound is named 2D6-7; R 1 is fluorine, R 2 is chlorine, R 3 and R 4 When it is methyl, the compound is named 2D6-8.

[0042] Example 1. Synthesis of compounds 2D6-1 to 2D6-8

[0043] (1) Synthesis of Compound D-1

[0044]

[0045] 4-Pyridinecarboxaldehyde (428.4 mg, 4.0 mmol) and 2,4-dimethylpyrrole (951.4 mg, 10.0 mmol) were dissolved in 200 mL of anhydrous dichloromethane. Three drops of trifluoroacetic acid were added dropwise under nitrogen, and the reaction was stirred at room temperature overnight. Following completion of the reaction, chloranil (983.5 mg, 4.0 mmol) was added, and the mixture was stirred at room temperature for 2 h. The reaction solution was then washed with aqueous sodium bicarbonate solution, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was dissolved in 200 mL of anhydrous dichloromethane, and triethylamine (4.2 mL, 30 mmol) and boron trifluoride etherate (6.2 mL, 48.2 mmol) were added dropwise in an ice bath. After the additions were complete, the reaction system was stirred at room temperature for 8 h. The reaction was completed by TLC monitoring. The reaction solution was washed with aqueous sodium bicarbonate solution, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to remove the solvent. The residue was separated by silica gel column (developing solvent: dichloromethane) to obtain compound D-1 as an orange-red solid (426.6 mg, yield 32.8%). 1 H NMR (400MHz, CDCl3) δ8.83–8.75(m,2H),7.36–7.28(m,2H),6.01(s,2H),2.56(s,6H),1.41(s,6H). 13 C NMR(150MHz,DMSO-d6)δ156.06,151.04,142.97,142.62,138.87,130.20,123.66,122.27,14.57.HRMS(ESI-TOF):C 18 H 19 BF2N3 + ([M+H] + )Theoretical value is 326.1635, measured value is 326.1633.

[0046] (2) Synthesis of compound 2D6-1

[0047]

[0048] Compound D-1 (325.2 mg, 1.0 mmol) and 4-methoxybenzyl bromide (603.2 mg, 3.0 mmol) were dissolved in 20 mL of acetonitrile and stirred at room temperature for 15 h. Following completion of the reaction by TLC, the solvent was removed by distillation under reduced pressure, and the residue was used directly in the next reaction without purification. The residue was dissolved in acetone (20 mL), 5 mL of saturated aqueous ammonium hexafluorophosphate solution was added, and the mixture was stirred vigorously at room temperature for 2 h. The solvent was removed by distillation under reduced pressure, and the residue was separated by silica gel column chromatography (dichloromethane / methanol = 20 / 1, v / v) to obtain compound 2D6-1 as a dark red solid (316.9 mg, 53.6% yield). 1 H NMR(500MHz,DMSO-d6)δ9.39(d,J=6.5Hz,2H),8.46(d,J=6.5Hz,2H),7.53(d,J=8.6Hz,2H),7.0 2(d,J=8.6Hz,2H),6.27(s,2H),5.91(s,2H),3.77(s,3H),2.48(s,6H),1.33(d,J=20.3Hz,6H). 13 C NMR(125MHz,DMSO-d6)δ160.02,156.82,151.01,145.86,142.29,134.58,130.47,12 8.92,128.63,126.06,122.41,114.58,63.26,55.26,14.62,14.29.HRMS(ESI-TOF):C 26 H 27 BF2N3O + ([M-PF6] + )Theoretical value is 446.2210, and the measured value is 446.2197.

[0049] (3) Synthesis of Compound D-2

[0050]

[0051] Referring to the synthetic route of compound D-1, 3-methyl-4-pyridinecarboxaldehyde and 2,4-dimethylpyrrole were used as starting materials. After the reaction was completed, the residue was separated by silica gel column (developing solvent: dichloromethane) to obtain compound D-2 as an orange-red solid (390.7 mg, yield 28.8%). 1 H NMR (500MHz, CDCl3) δ8.60(d,J=5.5Hz,2H),7.18(d,J=4.9Hz,1H),6.01(s,2H),2.57(s,6H),2.23(s,3H),1.39(s,6H). 13C NMR (150MHz, DMSO-d6) δ156.17,151.95,148.79,142.55,142.06,138.00,130.88,129.59,123.08,122.21,15.96,14.75,13.95.HRMS(ESI-TOF):C 19 H 21 BF2N3 + ([M+H] + )Theoretical value is 340.1791, and the measured value is 340.1792.

[0052] (4) Synthesis of Compound 2D6-2

[0053]

[0054] Referring to the synthetic route of compound 2D6-1, compound D-2 and 4-methoxybenzyl bromide were used as starting materials. After completion of the reaction, the residue was separated on a silica gel column (developing solvent: dichloromethane / methanol = 20 / 1, V / V) to obtain compound 2D6-2 as a dark red solid (294.1 mg, yield 48.6%). 1 H NMR (500MHz, CDCl3) δ9.89 (s, 1H), 9.35 (d, J = 6.1Hz, 1H), 7.78 (d, J = 6.1Hz, 1H), 7.62 (d, J = 8.7Hz, 2H) ,6.92(d,J=8.7Hz,2H),6.33(s,2H),6.03(s,2H),3.81(s,3H),2.56(s,6H),2.50(s,3H),1.36(s,6H). 13 C NMR (125MHz, CDCl3) δ160.96,158.51,152.90,146.29,142.58,141.40,138.65,132.05,131.25 ,128.43,127.48,124.71,122.72,115.12,64.05,55.40,16.84,14.84,14.81.HRMS(ESI-TOF):C 27 H 29 BF2N3O + ([M-PF6] + )Theoretical value is 460.2366, and the measured value is 460.2361.

[0055] (5) Synthesis of Compound D-3

[0056]

[0057] Referring to the synthetic route of compound D-1, 4-pyridinecarboxaldehyde and 2,4-dimethyl-3-ethylpyrrole were used as starting materials. After the reaction was completed, the residue was separated by silica gel column (developing solvent: dichloromethane) to obtain compound D-3 as a red solid (416.3 mg, yield 27.3%). 1 H NMR (500MHz, CDCl3) δ8.77(d,J=5.5Hz,2H),7.31(d,J=5.5Hz,2H),2.55(d,J=8.9Hz,6H),2.30(q,J=7.5Hz,4H),1.31(s,6H),0.98(t,J=7.5Hz,6H). 13 CNMR(150MHz,DMSO-d6)δ154.41,150.97,143.37,138.25,137.47,133.50,129.49,123.96,16.84,14.95,12.75,11.94.HRMS(ESI-TOF):C 22 H 27 BF2N3 + ([M+H] + ) Theoretical value 382.2261, measured value 382.2267. (6) Synthesis of compound 2D6-3

[0058]

[0059] Referring to the synthetic route of compound 2D6-1, compound D-3 and 4-methoxybenzyl bromide were used as starting materials. After completion of the reaction, the residue was separated on a silica gel column (developing solvent: dichloromethane / methanol = 30 / 1, V / V) to obtain compound 2D6-3 as a dark red solid (296.4 mg, yield 45.8%). 1 H NMR (500MHz, CDCl3) δ9.72(d,J=5.3Hz,2H),7.93(d,J=5.5Hz,2H),7.63(d,J=8.4Hz,2H),6.93(d,J=8.4Hz,2 H),6.46(s,2H),3.81(s,3H),2.53(s,6H),2.27(dd,J=14.8,7.2Hz,4H),1.27(s,6H),0.95(t,J=7.5Hz,6H). 13C NMR (125MHz, CDCl3) δ161.04,156.93,154.42,145.54,136.85,134.69,131.29,130.94,12 8.70,128.44,124.49,115.20,64.48,55.42,17.04,14.47,13.01,12.78.HRMS(ESI-TOF):C 30 H 35 BF2N3O + ([M-PF6] + )Theoretical value is 502.2836, and the measured value is 502.2832.

[0060] (7) Synthesis of Compound D-4

[0061]

[0062] Referring to the synthetic route of compound D-1, 3-methyl-4-pyridinecarboxaldehyde and 2,4-dimethyl-3-ethylpyrrole were used as starting materials. After the reaction was completed, the residue was separated by silica gel column (developing solvent: dichloromethane) to obtain compound D-4 as a red solid (412.6 mg, yield 26.1%). 1 H NMR (500MHz, CDCl3) δ8.59(d,J=5.6Hz,2H),7.18(d,J=4.8Hz,1H),2.54(s,6H),2.31(q,J=7.5Hz,4H),1.29(s,6H),0.99(t,J=7.6Hz,6H). 13 C NMR (125MHz, CDCl3) δ154.70,151.53,148.25,143.77,137.41,135.68,133.24 ,131.54,129.09,123.14,17.07,16.22,14.61,12.61,11.25.HRMS(ESI-TOF):C 23 H 29 BF2N3 + ([M+H] + )Theoretical value is 396.2417, and the measured value is 396.2403.

[0063] (8) Synthesis of Compound 2D6-4

[0064]

[0065] Referring to the synthetic route of compound 2D6-1, compound D-4 and 4-methoxybenzyl bromide were used as starting materials. After completion of the reaction, the residue was separated on a silica gel column (developing solvent: dichloromethane / methanol = 30 / 1, V / V) to obtain compound 2D6-4 as a dark red solid (307.5 mg, yield 46.5%). 1 H NMR (500MHz, CDCl3) δ9.91(s,1H),9.45(s,1H),7.77(d,J=4.8Hz,1H),7.66(d,J=8.3Hz,2H),6.90(d,J=8.2Hz,2H ),6.37(s,2H),3.79(s,3H),2.53(s,6H),2.49(s,3H),2.27(q,J=7.5Hz,4H),1.24(s,6H),0.96(t,J=7.5Hz,6H). 13 C NMR (125MHz, CDCl3) δ160.86,156.82,153.83,146.05,142.57,138.85,136.41,134.46,131.21,130.5 5,127.78,127.73,124.98,115.04,63.91,55.38,17.04,16.91,14.50,12.79,12.19.HRMS(ESI-TOF):C 31 H 37 BF2N3O + ([M-PF6] + )Theoretical value is 516.2992, and the measured value is 516.2984.

[0066] (9) Synthesis of Compound D-5

[0067]

[0068] Compound D-4 (395.2 mg, 1.0 mmol) and p-methylbenzaldehyde (240.3 mg, 2.0 mmol) were dissolved in 60 mL of toluene. Glacial acetic acid (1.6 mL) and piperidine (1.92 mL) were added, respectively, and the reaction was refluxed under nitrogen for 24 h. After completion of the reaction, the reaction solution was diluted with ethyl acetate, and the organic phase was washed with water, saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and the residue was used directly in the next reaction without purification.

[0069] (10) Synthesis of Compound 2D6-5

[0070]

[0071] Referring to the synthetic route of compound 2D6-1, compound D-5 and 4-methoxybenzyl bromide were used as starting materials. After the reaction, the residue was separated on a silica gel column (developing solvent: dichloromethane / methanol = 40 / 1, v / v) to obtain compound 2D6-5 as a purple-red solid (88.5 mg, two-step reaction yield 11.6%). 1 H NMR (500MHz, CDCl3) δ9.84 (s, 1H), 9.39 (s, 1H), 7.79 (d, J = 5.6Hz, 1H), 7.73–7.57 (m, 3H),7.49(d,J=8.0Hz,2H),7.24(s,1H),7.20(d,J=8.0Hz,2H),6.92(d,J=8.3Hz,2H), 6.32(s,2H),3.80(s,3H),2.75–2.52(m,5H),2.50(s,3H),2.38(s,3H),2.29(dd,J=1 5.0,7.3Hz,2H),1.26(d,J=12.9Hz,6H),1.12(t,J=7.5Hz,3H),0.97(t,J=7.5Hz,3H). 13 C NMR (100MHz, CDCl3) δ160.90,157.81,154.02,151.96,146.14,142.51,139.4 4,139.14,137.34,136.66,136.34,135.15,134.60,134.19,131.20,129.58, 129.41,128.93,127.97,127.43,124.92,118.33,115.10,64.14,55.40,21.4 5,18.30,17.11,17.04,14.46,14.03,13.03,12.20,11.94.HRMS(ESI-TOF):C 39 H 43 BF2N3O + ([M-PF6] + )Theoretical value is 618.3462, and the measured value is 618.3469.

[0072] (11) Synthesis of Compound D-6

[0073]

[0074] Compound D-1 (365.2 mg, 1.0 mmol) and p-methylbenzaldehyde (480.6 mg, 4.0 mmol) were dissolved in 60 mL of toluene. Glacial acetic acid (1.6 mL) and piperidine (1.92 mL) were added, respectively, and the mixture was stirred and refluxed under nitrogen for 24 h. After completion of the reaction, the reaction solution was diluted with ethyl acetate. The organic phase was washed with water and saturated sodium chloride, then dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and the residue was separated on a silica gel column (developing solvent: dichloromethane / methanol = 200 / 1, v / v) to obtain compound D-6 as a purple solid (61.4 mg, 11.6% yield). 1 H NMR (500MHz, CDCl3) δ8.79(d,J=5.6Hz,2H),7.69(d,J=16.3Hz,2H),7.54(d,J=7.9Hz,4H),7 .35(d,J=5.6Hz,2H),7.27(s,1H),7.26–7.17(m,5H),6.65(s,2H),2.39(s,6H),1.47(s,6H). 13 C NMR (125MHz, CDCl3) δ153.44,150.60,143.87,141.32,139.48,137.03,134. 39,133.76,132.24,129.59,127.66,123.90,118.25,118.13,21.50,14.89.

[0075] (12) Synthesis of Compound 2D6-6

[0076]

[0077] Referring to the synthetic route of compound 2D6-1, compound D-6 and 4-methoxybenzyl bromide were used as starting materials. After completion of the reaction, the residue was separated on a silica gel column (developing solvent: dichloromethane / methanol = 40 / 1, V / V) to obtain compound 2D6-6 as a dark purple solid (303.8 mg, yield 38.2%). 1 H NMR (500MHz, CDCl3) δ9.61(s,2H),7.99(s,2H),7.61(dd,J=20.2,12.2Hz,4H),7.53(d,J=7.7Hz,4H),7.30(s,2H ),7.21(d,J=7.6Hz,4H),6.93(d,J=8.2Hz,2H),6.65(s,2H),6.38(s,2H),3.80(s,3H),2.39(s,6H),1.44(s,6H). 13C NMR (125MHz, CDCl3) δ161.01,154.59,153.60,145.44,140.35,140.05,138.64,133.46,131.33,131.21, 129.66,128.87,127.89,124.33,119.30,117.55,115.22,64.76,55.40,21.55,15.99.HRMS(ESI-TOF):C 42 H 39 BF2N3O + ([M-PF6] + )Theoretical value is 650.3149, and the measured value is 650.3156.

[0078] (13) Synthesis of Compound D-7

[0079]

[0080] Referring to the synthetic route of compound D-1, 3-fluoro-4-pyridinecarboxaldehyde and 2,4-dimethylpyrrole were used as starting materials. After the reaction was completed, the residue was separated by silica gel column (developing solvent: dichloromethane) to obtain compound D-7 as an orange-red solid (377.5 mg, yield 27.5%). 1 H NMR (500MHz, CDCl3) δ8.65 (s, 1H), 8.59 (d, J = 4.7Hz, 1H), 7.31 (t, J = 5.3Hz, 1H), 6.03 (s, 2H), 2.57 (s, 6H), 1.48 (s, 6H). 13 C NMR (125MHz, CDCl3) δ157.29,157.02,155.24,146.56,146.52,142.18,139.35,139. 16,130.94,130.82,130.72,130.33,124.86,121.99,14.68,14.01.HRMS(ESI-TOF):C 18 H 18 BF3N3Na + ([M+Na] + )Theoretical value is 366.1360, and the measured value is 366.1370.

[0081] (14) Synthesis of Compound 2D6-7

[0082]

[0083] Referring to the synthetic route of compound 2D6-1, compound D-7 and 4-methoxybenzyl bromide were used as starting materials. After completion of the reaction, the residue was separated on a silica gel column (developing solvent: dichloromethane / methanol = 40 / 1, v / v) to obtain compound 2D6-7 as a purple-red solid (275.3 mg, yield: 45.2%). 1 H NMR (400MHz, DMSO-d6) δ9.82(d,J=3.3Hz,1H),9.28(d,J=6.2Hz,1H),8.63(t,J=6.6Hz,1H),7.55(d,J =8.6Hz,2H),7.03(d,J=8.6Hz,2H),6.30(s,2H),5.89(s,2H),3.78(s,3H),2.49(s,6H),1.46(s,6H). 13 C NMR(100MHz,DMSO-d6)δ160.13,157.66,155.10,142.91,141.99,138.41,138.26,136.97,136.59,13 0.68,130.36,128.66,126.82,125.27,122.71,114.53,64.16,55.21,14.30,13.99.HRMS(ESI-TOF):C 26 H 26 BF3N3O + ([M-PF6] + )Theoretical value is 464.2116, and the measured value is 464.2093.

[0084] (15) Synthesis of Compound D-8

[0085]

[0086] Compound D-7 (343.1 mg, 1.0 mmol) and N-chlorosuccinimide (333.8 mg, 2.5 mmol) were dissolved in 30 mL of tetrahydrofuran and stirred at room temperature for 24 h. The reaction mixture was distilled under reduced pressure to remove the solvent, and the residue was dissolved in dichloromethane. The organic phase was washed with water, then with saturated sodium chloride, and dried over anhydrous sodium sulfate. The organic phase was filtered and the solvent was distilled under reduced pressure to remove the solvent. The residue was separated on a silica gel column (petroleum ether / dichloromethane = 1 / 1, v / v) to obtain compound D-8 as a red solid (195.7 mg, 47.6% yield). 1 H NMR (400MHz, CDCl3) δ8.69 (s, 1H), 8.64 (dd, J = 4.7, 1.1Hz, 1H), 7.29 (t, J = 5.3Hz, 1H), 2.60 (s, 6H), 1.48 (s, 6H). 13C NMR (125MHz, CDCl3) δ155.05,154.21,146.78,146.74,139.58,139.39,136. 94,131.39,130.04,128.59,124.52,123.54,12.58,11.56.HRMS(ESI-TOF):C 18 H 14 BCl2F3N3 - ([MH] - ) Theoretical value 410.0615, measured value 410.0617. (16) Synthesis of compound 2D6-8

[0087]

[0088] Referring to the synthetic route of compound 2D6-1, compound D-8 and 4-methoxybenzyl bromide were used as starting materials. After completion of the reaction, the residue was separated on a silica gel column (developing solvent: dichloromethane / methanol = 40 / 1, v / v) to obtain compound 2D6-8 as a purple-red solid (289.8 mg, yield: 42.8%). 1 H NMR (400MHz, DMSO-d6) δ9.85(d,J=3.5Hz,1H),9.31(d,J=6.2Hz,1H),8.64(t,J=6.5Hz,1H),7. 55(d,J=8.7Hz,2H),7.05(d,J=8.7Hz,2H),5.91(s,2H),3.79(s,3H),2.55(s,6H),1.48(s,6H). 13 CNMR(125MHz,DMSO-d6)δ160.72,157.80,155.77,155.19,143.52,137.74,137.57,131.33,130.7 7,130.13,128.50,127.72,125.42,123.54,115.15,64.92,55.76,12.96,12.26.HRMS(ESI-TOF):C 26 H 24 BCl2F3N3O + ([M-PF6] + )Theoretical value is 532.1336, and the measured value is 532.1329.

[0089] Note: Compound 2D6-8 1 H-NMR spectrum, 13 C-NMR spectra and high-resolution mass spectra such as Figure 2 、 3 , as shown in 4.

[0090] Example 2. In vitro determination of the catalytic selectivity of human recombinant CYP enzymes

[0091] Prepare 180 μL of CYP metabolic reaction system in advance, including pH 7.4 buffer (100 mM), glucose-6-phosphate (10 mM), glucose-6-phosphate dehydrogenase (1 unit / mL), MgCl2 (4 mM), recombinant human CYP single enzyme, and 2D6-8 with a final concentration of 10 μM. Pre-incubate at 37°C with shaking for 3 minutes; add 20 μL of 10 mM NADP to the reaction system. + Start the reaction; after 60 minutes, add 100 μL of glacial acetonitrile and shake vigorously to terminate the reaction; use a high-speed refrigerated centrifuge at 4°C, 20,000×g for 10 minutes, take the supernatant, and perform fluorescence detection (E x =520nm, E m =558nm).

[0092] from Figure 5 As can be seen from the figure, the probe has good selectivity for recombinant human CYP2D6 enzyme, while other CYP enzymes in the CYP family hardly catalyze this probe reaction. Therefore, this probe reaction can selectively detect the enzyme activity of CYP2D6.

[0093] Example 3. Inhibition test of various CYP subtypes in human liver tissue

[0094] A 180 μL CYP metabolic reaction system was prepared in advance, including pH 7.4 buffer (100 mM), glucose-6-phosphate (10 mM), glucose-6-phosphate dehydrogenase (1 unit / mL), MgCl2 (4 mM), commercial mixed human liver microsomes (0.3 mg / mL), and a final concentration of 2D6-8 of 10 μM. Chemical inhibitor samples of different CYP isoforms were set up separately, including α-naphthoflavone (0.5 μM), 8-methoxypsoralen (2.5 μM), triethylenethiophosphoramide (50 μM), montelukast (2 μM), sulfabenzazole (10 μM), quinidine (50 μM), clomethiazole (50 μM), ketoconazole (1 μM), and a control sample without inhibitors. All samples were pre-incubated at 37°C with shaking for 3 minutes, and 20 μL of 10 mM NADP was added to the reaction system. + Start the reaction; after 45 minutes, add 100 μL of glacial acetonitrile and shake vigorously to terminate the reaction; centrifuge at 4°C, 20,000×g for 10 minutes in a high-speed refrigerated centrifuge, take the supernatant, and perform fluorescence detection (E x =520nm, E m=558 nm). The percentage of residual activity of each inhibitor group sample at 558 nm was calculated by setting the control sample without inhibitor at 100%.

[0095] from Figure 6 As can be seen in the results, the CYP2D6 inhibitor quinidine showed a very significant inhibitory effect on the metabolism of 2D6-8 in human liver microsomes, while inhibitors of other CYP enzymes had little effect on the metabolic reaction of 2D6-8. These experimental results further confirmed the enzyme selectivity of 2D6-8.

[0096] Example 4. Determination of CYP2D6 protein concentration standard curve

[0097] The experiment was performed on a microplate reader using a 96-well plate, 2D6-8 (10 μM), glucose-6-phosphate (10 mM), glucose-6-phosphate dehydrogenase (1 unit / mL), MgCl2 (4 mM) NADP + (1 mM), CYP2D6 single enzyme (0-12.5 nM), pH 7.4 buffer 100 mM, a total volume of 200 μL, incubated at 37 ° C for 60 min, and the fluorescence intensity of the product and the protein concentration were used to make a standard curve. Figure 7 The left side shows the fluorescence intensity curve under different concentrations of CYP2D6 catalysis, and the right side shows the linear relationship between CYP2D6 protein concentration and fluorescence intensity. This result shows that the probe substrate has a wide linear range and can accurately quantify the content of CYP2D6.

[0098] Example 5. Detection limit determination of 2D6-8

[0099] The detection limit of the probe was calculated using the 3σ / k formula, where σ is the standard deviation and k is the slope. The calculated detection limit was 0.021 nM.

[0100] Example 6. Evaluation of CYP2D6 activity in HT-29 cells

[0101] The probe group and the probe plus CYP2D6 inhibitor group were set up separately. The cells were operated as follows: HT-29 cells were cultured at 1×10 5 Cells were seeded at a density of 100 cells (Φ = 20 mm) and incubated overnight at 37°C in a humidified incubator containing 5% CO2. Subsequently, the probe group cells were incubated with 10 μM 2D6-8 for 1 hour, the residual probes were washed with phosphate buffer solution, and the cells were photographed. The probe plus inhibitor group cells were added with CYP2D6 inhibitor 0.5 hours before the addition of 10 μM 2D6-8. The probe incubation time was also 1 hour. The residual probes were washed with phosphate buffer solution, and the cells were photographed (E x =514nm, E m =540-560nm). Figure 8As can be seen from the figure, the probe group exhibits strong fluorescence activity, while the fluorescence of the probe plus CYP2D6 inhibitor group is significantly weakened, indicating that the probe has high selectivity and detection sensitivity.

[0102] Example 7. 3D imaging study of brain tissue

[0103] Mouse brain frozen sections were thawed at room temperature and incubated in a 37°C constant temperature water bath for 60 minutes. During this period, 2D6-8 diluted with phosphate buffer solution was evenly added to the brain sections to maintain sufficient moisture of the tissue sections. Only PBS was added to the control group. DAPI reagent was added to each section sample for 15 minutes. The sections were then gently washed 3 times with phosphate buffer solution and placed in E x =514nm, E m = The fluorescence image of 2D6-8 was taken under the condition of 540-560 nm. Figure 9 This is the 3D imaging result of mouse brain slices.

[0104] Example 8. Visual screening of CYP2D6 inhibitors

[0105] The test group and the control group were set up separately. Briefly, 200 μL of CYP metabolic reaction system was prepared in advance, including pH 7.4 buffer (100 mM), test substance, 6-phosphate glucose (10 mM), glucose-6-phosphate dehydrogenase (1 unit / mL), MgCl2 (4 mM), enzyme, and pre-incubated at 37°C with shaking for 3 minutes. Then, NADP was added to the sample tube. + The reaction was started; after 30 minutes, 100 μL of glacial acetonitrile was added to the reaction system and the reaction was terminated after vigorous shaking; high-speed centrifugation was performed at 4°C and 20,000 × g for 10 minutes, and the supernatant was collected for fluorescence image analysis (E x :532nm,E m :570±15 nm; GE Amersham Typhoon). The percentage residual activity of the test compound group samples was calculated to evaluate the inhibitory ability of the test compound on CYP2D6. Figure 10 It is the visualization result of the inhibition of CYP2D6 by various Chinese herbal medicine extracts.

[0106] Example 9. Determination of Fluorescent Substrate Metabolic Rate

[0107] Prepare 180 μL of CYP metabolic reaction system in advance, including pH 7.4 buffer (100 mM), glucose-6-phosphate (10 mM), glucose-6-phosphate dehydrogenase (1 unit / mL), MgCl2 (4 mM), recombinant human CYP2D6 single enzyme, and 2D6-1-2D6-8 fluorescent substrate with a final concentration of 10 μM. Pre-incubate at 37°C with shaking for 3 minutes; add 20 μL of 10 mM NADP to the reaction system. + Start the reaction; after 60 minutes, add 100 μL of glacial acetonitrile and shake vigorously to terminate the reaction; use a high-speed refrigerated centrifuge at 4°C, 20,000×g for 10 minutes, collect the supernatant, and perform fluorescence detection; select the appropriate wavelength near the maximum absorption of the corresponding product for excitation, and calculate the metabolic rate of each sample by taking the fluorescence intensity corresponding to the maximum emission wavelength of each sample. The results are as follows: Figure 12 As shown in the figure, 2D6-5 and 2D6-6 cannot be metabolized by recombinant human CYP2D6 alone and therefore cannot be used as detection probes. Probes 2D6-1, 2D6-2, 2D6-3, 2D6-4, 2D6-7, and 2D6-8 can all be metabolized by recombinant human CYP2D6 alone, with 2D6-8 having the fastest metabolism rate.

Claims

1. A fluorescent probe for detecting cytochrome oxidase CYP2D6, characterized by: The probe substrate is a Bodipy compound derivative, and its general structural formula is as follows: Among them, R 1 is selected from hydrogen, methyl, ethyl, halogen, R 2 is selected from hydrogen, methyl, ethyl, halogen, R 3 Selected from methyl, p-methylphenyl, R 4 Selected from methyl, p-methylphenyl; The probe substrate is selected from the following structures: 。 2. The use of a fluorescent probe for detecting cytochrome oxidase CYP2D6 according to claim 1 for non-disease diagnosis and treatment purposes, characterized in that: The probe substrate is specifically catalyzed by CYP2D6 to generate corresponding products. The probe substrate is mixed with a biological sample containing CYP2D6 to undergo an enzymatic reaction. The activity of CYP2D6 in different biological systems is quantitatively determined by quantitatively detecting the substrate elimination rate or the generation rate of its hydroxylation product per unit time.

3. The use of a fluorescent probe for detecting cytochrome oxidase CYP2D6 for non-disease diagnosis and treatment purposes according to claim 2, characterized in that: The specific measurement methods and conditions are as follows: A. The concentration of the probe substrate in the system is 1 / 10~10 K m ; B. In PBS buffer, the reaction temperature is 20 o C to 60 o C; the pH of the incubation system is between 5.5 and 10.5; C. The reaction time is 5 to 120 minutes. The reaction is terminated when the corresponding hydroxylation product of the above probe substrate reaches the limit of quantification and the substrate conversion rate does not exceed 20%. D. Determine the amount of substrate reduction or product generation per unit time as an evaluation indicator of CYP2D6 activity; The fluorescence signals of the probe substrate and its hydroxylation product adopt an excitation wavelength of 480-520 nm and a maximum emission wavelength of 540-600 nm.

4. The use of a fluorescent probe for detecting cytochrome oxidase CYP2D6 for non-disease diagnosis and treatment purposes according to claim 2, characterized in that: The biological sample is any one of recombinantly expressed CYP2D6 enzyme, human or animal tissue preparation sample, various mammalian tissue cells and their preparations.

5. The use of a fluorescent probe for detecting cytochrome oxidase CYP2D6 for non-disease diagnosis and treatment purposes according to claim 2, characterized in that: The probe substrate is used for screening CYP2D6 reversible inhibitors, irreversible inhibitors and activators and for quantitative evaluation of regulatory capabilities.

6. The use of a fluorescent probe for detecting cytochrome oxidase CYP2D6 for non-disease diagnosis and treatment purposes according to claim 2, characterized in that: This probe substrate is used for screening CYP2D6 expression inducers and quantitatively evaluating the inducible expression ability, and evaluating individual and species differences in the metabolic enzyme CYP2D6.

7. The use of a fluorescent probe for detecting cytochrome oxidase CYP2D6 for non-disease diagnosis and treatment purposes according to claim 2, characterized in that: This probe substrate is used as a probe substrate for in vivo and overall CYP2D6 in experimental animals to evaluate individual and species differences in the metabolic enzyme CYP2D6.

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