A near-infrared fluorescent molecular probe capable of specifically identifying cytochrome oxidase CYP3A4 and a preparation method thereof

Abstract

A near-infrared fluorescent molecular probe that can specifically identify cytochrome oxidase CYP3A4 and its preparation and application. The fluorescent molecular probe has a structural formula shown in Formula I: the probe itself has no fluorescence, and the hydroxylation product produced after reacting with cytochrome oxidase CYP3A4 has fluorescence, and its maximum emission wavelength is in the near-infrared region, reaching 721nm, which can reduce background fluorescence interference, has less light damage, and has better tissue penetration. It can be used for various recombinant CYP3A4, the determination of CYP3A4 enzyme activity in various tissue cells, as well as the rapid screening of CYP3A4 inhibitors and the quantitative evaluation of inhibition ability.

Description

Technical Field

[0001] The invention relates to an organic small molecule fluorescent probe, in particular to a near-infrared fluorescent molecular probe capable of specifically identifying cytochrome oxidase CYP3A4 and a preparation method thereof, belonging to the technical field of biosensor. Background Art

[0002] Cytochrome P450 (CYP) is a large family of self-oxidizable heme proteins, belonging to the class of monooxygenases, named for its specific absorption peak at 450 nanometers. It is involved in the metabolism of endogenous substances and exogenous substances including drugs and environmental compounds. According to the degree of homology of the amino acid sequence, its members are divided into three levels: family, subfamily and enzyme individual. Among them, CYP3A4 has a high abundance in the liver and a wide substrate spectrum. It is considered to be the most important CYP isoform in humans and contributes to the metabolism of more than 50% of clinical drugs. Unfortunately, CYP3A4 activity can be affected by many clinical drugs, leading to the occurrence of adverse drug interactions and even life-threatening. Therefore, there is an urgent need to develop technologies that can monitor CYP3A4 activity in real time to accurately characterize the activity of CYP3A4 in biological systems.

[0003] At present, traditional detection methods such as mass spectrometry or high-performance liquid chromatography have been developed, but due to the defects of complex operation steps, high cost, and the need for pretreatment such as cell or tissue lysis, their further application in living cells or in vivo is limited. In comparison, fluorescence analysis has attracted widespread attention due to its excellent sensitivity and specificity, especially the ability to achieve real-time non-invasive cell / tissue imaging. Among them, compared with light in the ultraviolet / visible light (<700nm) region, near-infrared light (700-900nm) causes less photodamage to biological samples, has better tissue penetration, and is less susceptible to interference from the autofluorescence of complex life systems, making it more suitable for biological imaging.

[0004] For example, although the invention patents with application number CN202111426695.6 and title “Application of a broad-spectrum fluorescent probe for detecting cytochrome oxidase CYP3A” and application number CN201910158458.2 and title “Application of a two-photon fluorescent probe for detecting cytochrome oxidase CYP3A4” can both detect cytochrome oxidase CYP3A4, their emission wavelengths are relatively short and their anti-interference capabilities are relatively weak. Therefore, it is of great significance to develop near-infrared fluorescent molecular probes that can specifically identify cytochrome oxidase CYP3A4. Summary of the invention

[0005] In view of the problem that the current fluorescent molecular probes for detecting cytochrome oxidase CYP3A4 have weak anti-interference ability, the present invention proposes a near-infrared fluorescent molecular probe that can specifically identify cytochrome oxidase CYP3A4 and a preparation method thereof. The probe has a maximum emission wavelength in the near-infrared region of 721 nm, can reduce background fluorescence interference, and has less light damage and better tissue penetration.

[0006] The technical means adopted by the present invention to solve the above problems are: a near-infrared fluorescent molecular probe that can specifically identify cytochrome oxidase CYP3A4, having a structural formula shown in Formula I:

[0007]

[0008] A method for preparing a near-infrared fluorescent molecular probe that can specifically identify cytochrome oxidase CYP3A4 is as follows:

[0009] (1) Add chromone-3-formaldehyde and bromoacetaldehyde glycol triphenylphosphine salt to dichloromethane, stir at room temperature for 30 minutes, add NaOH solution, continue stirring for 3 hours, add concentrated hydrochloric acid, continue stirring for 30 minutes, pour the reaction system into water, extract with dichloromethane, combine and dry the organic layers, concentrate under reduced pressure, and purify by column chromatography to obtain compound I; wherein the molar ratio of chromone-3-formaldehyde to bromoacetaldehyde glycol triphenylphosphine salt is 1:1.1 to 1:1.5, the amount ratio of chromone-3-formaldehyde to dichloromethane is 1:3 to 1:10 mmol / mL, the molar ratio of chromone-3-formaldehyde to NaOH is 1:1 to 1:2, the amount ratio of chromone-3-formaldehyde to concentrated hydrochloric acid is 1:2 to 1:2.5 mmol / mL, and the concentration of NaOH solution is 1 mmol / mL;

[0010] (2) Compound I, 2-methylpyridinium salt and piperidine are dissolved in an alcoholic solvent such as methanol or ethanol, heated to reflux at 90°C until completion, the solvent is removed by rotary evaporation, and purified by column chromatography to obtain a fluorescent molecular probe represented by formula I; wherein the molar ratio of compound I to 2-methylpyridinium salt is 1:1.1 to 1:1.5, the molar ratio of compound I to piperidine is 5:1 to 8:1, and the amount ratio of compound I to solvent is 1:7 to 1:10 mmol / mL:

[0011]

[0012] The present invention provides a novel near-infrared fluorescent molecular probe that can specifically identify cytochrome oxidase CYP3A4 and its application. The probe has no fluorescence, and its hydroxylation product has fluorescence, and its maximum emission wavelength is in the near-infrared region (maximum emission wavelength is 721nm). A fluorescence detector can be used for specific detection of cytochrome oxidase CYP3A4. The detection principle is as follows:

[0013]

[0014] This specific probe is not easily interfered by biological system matrix and impurities during the CYP3A4 activity detection process, and can be used for the determination of various recombinant CYP3A4 and CYP3A4 enzyme activities in various tissue cells, as well as the rapid screening of CYP3A4 inhibitors and the quantitative evaluation of inhibition ability.

[0015] The beneficial effects of the present invention are:

[0016] 1. The fluorescent molecular probe provided by the present invention can specifically interact with CYP3A4 to produce changes in the fluorescence spectrum, while interacting with other substances cannot lead to obvious changes in the fluorescence spectrum, thereby achieving quantitative and specific identification of CYP3A4.

[0017] 2. The fluorescent molecular probe provided by the present invention has simple synthesis steps, readily available raw materials, simple detection methods and low costs.

[0018] 3. The maximum emission wavelength of the fluorescent molecular probe provided by the present invention is in the near-infrared region, and has the advantages of low light absorption by biological tissues or low self-light intensity, strong penetration into biological tissues, high imaging signal-to-noise ratio and resolution, and good water solubility, which is more conducive to the determination of CYP3A4 enzyme activity in various tissue cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 1. An emission spectrum showing the fluorescence intensity of the fluorescent molecular probe as a function of CYP3A4 concentration in the implementation of the present invention;

[0020] 2. The linear relationship between the fluorescence intensity of the fluorescent molecular probe and the concentration of CYP3A4 in the implementation of the present invention;

[0021] 3. Selectivity diagram of the fluorescent molecular probe for CYP3A4 in the implementation of the present invention;

[0022] 4. Fluorescence confocal imaging of the fluorescent molecular probe in HepG2 cells in the implementation of the present invention, wherein a) is the cells treated with 10 μM ketoconazole for 30 min and then with 50 μM probe; b) is the cells treated with 10 μM rifampicin for 30 min and then with 50 μM probe. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings.

[0024] Embodiment 1

[0025] Synthesis of Compound Ⅰ

[0026] Chromone-3-carboxaldehyde (870.78 mg, 5 mmol) and bromoacetaldehyde glycol triphenylphosphine salt (2361.10 mg, 5.5 mmol) were placed in a 100 mL round-bottom flask, 25 mL CH2Cl2 was added to completely dissolve, and stirred at room temperature for 30 min. Then 5 mL NaOH solution (1.5 g, 5 mmol) was slowly added to the reaction system. After stirring for 3 h, 10 mL concentrated hydrochloric acid was added, and stirring was continued for 30 min. The reaction system was poured into water and extracted with CH2Cl2. The organic layers were combined, dried over anhydrous sodium sulfate, and then concentrated in vacuo. Finally, column chromatography was used for separation and purification to obtain 757.73 mg of compound with a yield of 75.7%. 1 H NMR (400MHz, CDCl3) δ (ppm): 9.68 (s, 1H), 8.08 (d, J = 8.0Hz, 1H), 7.74 (t, J = 7.9Hz, 1H), 7.58 (d, J = 8.2Hz, 1H), 7, 47-7.44 (m, 2H), 6.72 (s, 1H), 6.29 (d, J = 6.8Hz, 1H).

[0027] Synthesis of the compound represented by formula I

[0028] Compound I (600.58 mg, 3 mmol) and 25 mL of ethanol were added to a 50 mL round-bottom flask. After sufficient dissolution, 2-methylpyridinium salt (846.25 mg, 3.6 mmol) and 0.5 mmol of piperidine were added and refluxed at 90° C. The reaction was monitored by TLC until completion. After cooling, the solvent was removed by rotary evaporation and purified by column chromatography to obtain 657.16 mg of the product with a yield of 52.5%. 1 H NMR (400MHz, CDCl3): δ8.86(d,J=8.1Hz,1H),8.10-8.08(m,2H),7.74(t,J=8.0Hz,1H),7.66(t,J=8.1Hz,1H),7.58(d,J= 8.2Hz,1H),7.47(t,J=8.0Hz,1H),7.29(d,J=8.2Hz,1H),6.72-6.68(m,3H),6.53-6.51(m,2H),4.36(s,3H).HRMS-ESI(C 19 H 16 INO2)m / z:calc.for[MI] + :290.1176; found,290.9782.

[0029] Embodiment 2

[0030] Fluorescence response experiment of fluorescent molecular probe to human recombinant CYP3A4

[0031] 1.8 mL of CYP metabolic reaction system (including PBS buffer solution (0.01 M, pH = 7.4), different concentrations of recombinant human CYP3A4, 10 μM fluorescent molecular probe of formula I) was pre-incubated at 37°C for 3 minutes under shaking conditions, and 0.2 mL of 10 mM NADPH generation solution (1 mM NADP + , 10 mM glucose-6-phosphate, 1 unit / mL glucose-6-phosphate dehydrogenase and 4 mM MgCl2) to start the reaction. After 40 minutes, 50 μL of glacial acetonitrile was added. After vigorous shaking, the reaction was terminated. Then, high-speed centrifugation was performed at 4°C and 20,000×g for 20 minutes. The supernatant was taken for fluorescence detection. The results are as follows Figure 1 The results showed that as the concentration of CYP3A4 increased (0-0.1 mg / mL), the fluorescence at 721 nm gradually increased, and within the concentration range of 0-0.02 mg / mL, the fluorescence intensity at 721 nm had a good linear relationship (R 2 =0.98945)(e.g. Figure 2 shown).

[0032] Embodiment 3

[0033] Selective experiment of fluorescent molecular probe for human recombinant CYP3A4

[0034] 1.8 mL of CYP metabolic reaction system (including PBS buffer solution (0.01 M, pH = 7.4), different concentrations of recombinant human CYP enzymes, and 10 μM of the fluorescent molecular probe shown in Formula I) was pre-incubated at 37°C for 3 minutes under shaking conditions, and 0.2 mL of 10 mM NADPH generation solution (1 mM NADP + , 10 mM glucose-6-phosphate, 1 unit / mL glucose-6-phosphate dehydrogenase and 4 mM MgCl2) to start the reaction. After 40 minutes, 50 μL of glacial acetonitrile was added. After vigorous shaking, the reaction was terminated. Then, high-speed centrifugation was performed at 4°C and 20,000×g for 20 minutes. The supernatant was taken for fluorescence detection. The results are as follows Figure 3 The results showed that the probe had excellent specificity for CYP3A4 and other CYP enzymes did not interfere with its response.

[0035] Embodiment 4

[0036] Imaging of CYP3A4 in living cells using fluorescent molecular probes

[0037] We selected HepG2 cells that can express various CYP isozymes as model cells. HepG2 cells that can be plated and cryopreserved were inoculated in MEM / EBSS medium (containing 10% FBS) and incubated overnight at 37°C in a humidified incubator containing 5% CO2. The adherent cells were washed twice with medium without FBS and divided into two groups. One group was treated with 10 μM ketoconazole (an inhibitor of CYP3A4) and incubated in a 5% CO2 incubator at 37°C for 30 minutes, and the other group was treated with 10 μM rifampicin (an inducer of CYP3A4) and incubated in a 5% CO2 incubator at 37°C for 30 minutes. Then, 50 μM of the fluorescent molecular probe shown in Formula I was added. Finally, the cells were incubated at 37°C for another 60 minutes, and then rinsed three times with PBS (0.01 M, pH = 7.4) to remove the extracellular probe and imaged. The results are shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that the cells with ketoconazole have almost no fluorescence ( Figure 4 In the cells treated with rifampicin, obvious red fluorescence was observed ( Figure 4 The probe can be used to image endogenous CYP3A4 in living cells.

[0038] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Technicians in the relevant technical field may make various modifications and changes without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions should also fall within the protection scope of the present invention, and the protection scope of the present invention should be defined by the claims.

Claims

1. A near-infrared fluorescent molecular probe that can specifically identify cytochrome oxidase CYP3A4, characterized in that: It has the structural formula shown in Formula I:

2. A method for preparing a near-infrared fluorescent molecular probe capable of specifically identifying cytochrome oxidase CYP3A4 according to claim 1, characterized in that: Here’s how: (1) Add chromone-3-carboxaldehyde and bromoacetaldehyde glycol triphenylphosphine salt to dichloromethane, stir, then add NaOH solution, continue stirring, then add concentrated hydrochloric acid, continue stirring, then pour the reaction system into water, extract, dry, and concentrate and purify to obtain compound I, wherein the structural formula of compound I is: (2) Compound I, 2-methylpyridinium salt and piperidine are dissolved in an alcoholic solvent, heated under reflux until completion, and the solvent is removed by rotary evaporation and purified to obtain a fluorescent molecular probe represented by formula I, wherein the alcoholic solvent is methanol or ethanol.

3. The method for preparing a near-infrared fluorescent molecular probe capable of specifically identifying cytochrome oxidase CYP3A4 according to claim 2, characterized in that: In the step (1), the stirring time after adding dichloromethane to chromone-3-carboxaldehyde and bromoacetaldehyde glycol triphenylphosphine salt is 30 minutes, the stirring time after adding NaOH solution is 3 hours, and the stirring time after adding concentrated hydrochloric acid is 30 minutes.

4. The method for preparing a near-infrared fluorescent molecular probe capable of specifically identifying cytochrome oxidase CYP3A4 according to claim 2, characterized in that: In the step (1), the molar ratio of chromone-3-formaldehyde to bromoacetaldehyde glycol triphenylphosphine salt is 1:1.1-1:1.5, the dosage ratio of chromone-3-formaldehyde to dichloromethane is 1:3-1:10 mmol / mL, the molar ratio of chromone-3-formaldehyde to NaOH is 1:1-1:2, and the dosage ratio of chromone-3-formaldehyde to concentrated hydrochloric acid is 1:2-1:2.5 mmol / mL.

5. The method for preparing a near-infrared fluorescent molecular probe capable of specifically identifying cytochrome oxidase CYP3A4 according to claim 2, characterized in that: The concentration of the NaOH solution in step (1) is 1 mmol / mL.

6. The method for preparing a near-infrared fluorescent molecular probe capable of specifically identifying cytochrome oxidase CYP3A4 according to claim 2, characterized in that: The extractant in step (1) is dichloromethane.

7. The method for preparing a near-infrared fluorescent molecular probe capable of specifically identifying cytochrome oxidase CYP3A4 according to claim 2, characterized in that: In the step (2), the molar ratio of compound I to 2-methylpyridinium salt is 1:1.1 to 1:1.5, the molar ratio of compound I to piperidine is 5:1 to 8:1, and the amount ratio of compound I to solvent is 1:7 to 1:10 mmol / mL.

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