Fluorescent probe capable of responding to various cytochrome P450 enzymes as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe capable of responding to various cytochrome P450 enzymes and a preparation method and application thereof.According to the method, a traditional dye 4-bromo-1, 8-naphthalic anhydride serves as a parent nucleus, an aminoethyl side chain is introduced through a reaction with N-methylethylenediamine or ethanediamine, and then the fluorescent probe is obtained; and further performing coupling modification with 4-substituted phenylboronic acid to obtain a molecular structure with excellent fluorescence performance. In the prepared fluorescent probe, response groups such as substituted phenyl can be catalytically oxidized by CYPs, fluorescence on-off change is excited without coenzyme, and selective response to CYPs of various subtypes such as CYP 1A1, CYP 1A2, CYP 2B6, CYP 2D6 and the like is realized. Meanwhile, the prepared fluorescent probe has good water solubility, light stability and pH adaptability, shows high signal-to-noise ratio and low toxicity in a complex biological system, and has application potentials of later CYPs activity imaging, biomarker screening, disease diagnosis and the like.

Description

Technical Field

[0001] This invention belongs to the field of organic fluorescent probes and fluorescence detection technology of cytochrome P450 enzymes, specifically relating to a fluorescent probe that can respond to multiple cytochrome P450 enzymes, its preparation method, and its application. Background Technology

[0002] Cytochrome P450 enzymes (CYPs) are a class of monooxygenases containing a heme cofactor, widely distributed in eukaryotes and prokaryotes, and highly expressed, especially in the human liver. CYPs play crucial metabolic roles in the body, catalyzing the oxidation, reduction, and hydrolysis of various endogenous substances (such as lipids, cholesterol, and hormones) and exogenous compounds (such as drugs and environmental toxins). They are core enzymes in maintaining homeostasis, drug metabolism, toxic transformation, and the activation of carcinogens, among other important physiological and pathological processes. Currently, more than 50 human CYPs are known, with common ones including CYP1A1 and CYP3A4. They are characterized by complex enzyme profiles, broad substrate scope, and overlapping selectivity.

[0003] With the continuous development of precision medicine and drug metabolism research, higher demands are being placed on detection tools capable of efficiently and in real-time monitoring CYP activity. Fluorescent probes, due to their high sensitivity, high spatiotemporal resolution, and real-time imaging capabilities, have become an important tool in CYP research. However, most current CYP fluorescent probes are typically designed for single CYP subtypes, relying on specific substrate structures and responding only to a single CYP subtype, making it difficult to meet the needs of simultaneous detection of multiple subtypes. In the detection of multiple CYP subtypes, multiple sets of experiments need to be conducted in parallel, resulting in large sample consumption, cumbersome operations, and a tendency for excitation / emission spectrum overlap, causing signal crosstalk. Furthermore, multi-fluorescent probe mixtures are prone to competitive inhibition by metabolites, causing false positive or false negative signals, seriously affecting the reliability of detection results. Therefore, there is an urgent need to develop novel fluorescent probes with a universally applicable response site that can interact with multiple CYPs and produce significant fluorescence changes to meet the diverse application needs of high-throughput screening, in vivo imaging, and disease-related enzyme profiling studies. Summary of the Invention

[0004] The technical problem solved by this invention is to provide a fluorescent probe responsive to multiple cytochrome P450 enzymes and its preparation method. This method uses the traditional dye 4-bromo-1,8-naphthalenedicarboxylic anhydride as the core, introduces an aminoethyl side chain through reaction with N-methylethylenediamine or ethylenediamine, and further modifies it with 4-substituted phenylboronic acid to obtain a molecular structure with excellent fluorescence properties. The substituted phenyl and other responsive groups in the prepared fluorescent probe can be catalyzed and oxidized by CYPs, exciting a fluorescence "off-on" change without the need for coenzymes, achieving selective response to multiple CYP subtypes such as CYP 1A1, 1A2, 2B6, and 2D6. Simultaneously, the prepared fluorescent probe exhibits good water solubility, photostability, and pH adaptability, demonstrating high signal-to-noise ratio and low toxicity in complex biological systems, and possesses potential applications in CYP activity imaging, biomarker screening, and disease diagnosis.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a fluorescent probe that can respond to multiple cytochrome P450 enzymes, the structural formula of which is shown in Formula I:

[0006]

[0007] Where R1 is -CH2CH2NHR, and R is H or C. 1-6 Alkyl; R2 is a phenyl or substituted phenyl group, wherein the substituents on the substituted phenyl benzene ring are selected from C10. 1-6 Alkyl, C 1-3 Alkoxy or C 2-6 Alkylamide group.

[0008] Further specifying, R1 is -CH2CH2NH2 or -CH2CH2NHCH3; R2 is phenyl or 4-substituted phenyl, wherein the substituent on the 4-substituted phenyl benzene ring is selected from methyl, methoxy or acetamido.

[0009] The preparation method of the fluorescent probe responsive to multiple cytochrome P450 enzymes described in this invention comprises the following specific preparation steps:

[0010] Step S1: 4-Bromo-1,8-naphthalenedicarboxylic anhydride and an ethylenediamine compound are dissolved in solvent A and refluxed at 60–85 °C. After the reaction is complete, the mixture is purified by filtration, washing with cold methanol, and column chromatography to obtain the compound shown in Formula II. The ethylenediamine compound is N-methylethylenediamine or ethylenediamine. The general structural formula of the compound shown in Formula II is:

[0011]

[0012] Where R1 is -CH2CH2NH2 or -CH2CH2NHCH3;

[0013] In step S2, the compound shown in Formula II, the 4-substituted phenylboronic acid compound, tetrakis(triphenylphosphine)palladium and potassium carbonate are added to solvent B and refluxed at 110-120°C. After the reaction is completed, the target product, the compound shown in Formula I, is obtained by filtration, extraction and column chromatography separation and purification. This compound can be used as a fluorescent probe for various cytochrome P450 enzymes. The 4-substituted phenylboronic acid compound is 4-methylphenylboronic acid, 4-methoxyphenylboronic acid or 4-acetamidophenylboronic acid.

[0014] Further specified, the molar ratio of 4-bromo-1,8-naphthoic anhydride to ethylenediamine compounds in step S1 is 1:1 to 1.5; the molar ratio of the compound shown in Formula II, 4-substituted phenylboronic acid compounds, tetrakis(triphenylphosphine)palladium to potassium carbonate in step S2 is 1:4:0.1 to 0.5:10.

[0015] Further specifying, solvent A in step S1 is anhydrous ethanol or anhydrous ethanol / N,N-dimethylformamide mixed solvent; solvent B in step S2 is toluene / ethanol / water mixed solvent, wherein the volume ratio of toluene / ethanol / water is 7-9:1-2:1.

[0016] Further specifying, in step S1, the column chromatography separation and purification process uses a silica gel column, and the eluent is a dichloromethane / methanol mixed solution with a volume ratio of 300:1; in step S2, the column chromatography separation and purification process uses a silica gel column, and the eluent is a dichloromethane / methanol mixed solution with a volume ratio of 150:2 or 200:3; and in step S2, the extraction solvent used in the extraction process is dichloromethane.

[0017] The fluorescent probe described in this invention is used to detect various cytochrome P450 enzymes. The fluorescent probe is water-soluble, photostable, and pH-adaptable, and has low toxicity and high signal-to-noise ratio when applied to complex biological systems.

[0018] The application of the fluorescent probe described in this invention in the preparation of detection tools for selectively responding to multiple cytochrome P450 enzymes in interfering environments, wherein the interfering environment is one or more of ions, amino acids, and macromolecules, wherein the ions are SnCl2, CaCl2·2H2O, NaHSO4, CuSO4, FeCl2·7H2O, MnSO4, KCl, Al(SO4)3, MgSO4, Zn(NO3)2, CdCl2, KH2PO4, AgNO3, Li2CO3, CoCl2, HgCl2, NiCl2·6H2O, Na2I, and N. One or more of aHSO3; amino acids including glycyl-L-phenylalanine, lysine, serine, glutamine, methionine, glutamic acid, glycyl-L-tyrosine, D-methionine, glycine, dithiothreitol, L-aspartic acid, DL-leucine, L-glutamic acid, DL-threonine, arginine, cystine, homocysteine, valine, hypoxanthine, L-cysteine, and reduced glutathione; macromolecules including bovine lipase, β-amylase, proteinase K, trypsin, fetal bovine serum albumin, pepsin, hemoglobin, and bovine serum albumin.

[0019] The fluorescent probe described in this invention is used in the preparation of tools for detecting metabolic enzyme activity or screening biomarkers. The fluorescent probe has good fluorescence response sensitivity and dynamic range to liver microsomes in animal or human metabolic systems and can be used to evaluate the metabolic activity of various cytochrome P450 enzymes in liver microsomes in vitro.

[0020] The fluorescent probe described in this invention is used as an imaging reagent for various cytochrome P450 enzyme activities, wherein the fluorescent probe has low cytotoxicity and good biocompatibility.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] (1) This invention proposes a novel CYP-responsive fluorescent probe. This probe uses 4-bromo-1,8-naphthoic anhydride and N-methylethylenediamine or ethylenediamine as raw materials to prepare the intermediate product 6-bromo-2-(2-(R-amino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione, where R = CH3 or H. The intermediate product is then further reacted with 4-substituted phenylboronic acid to prepare the final product. This fluorescent probe enables the fluorescence detection of various CYPs, providing a reference for the design of other similar molecules and the detection of target substances.

[0023] (2) The fluorescent probe prepared in this invention incorporates a nitrogen-methyl group and a substituted phenyl structure that are sensitive to CYPs. Even without the need for an external coenzyme system (such as NADPH), it can still specifically respond to CYPs, triggering a significant fluorescence enhancement effect and a blue shift in emission wavelength. It exhibits high sensitivity and specificity, enabling direct detection of CYP activity in complex biological environments. This fluorescent probe is suitable for the fluorescence detection of various CYPs such as CYP2B6, CYP1A2, CYP1A1, and CYP2D6. Furthermore, the fluorescent probe possesses good water solubility, photostability, and pH adaptability, demonstrating high signal-to-noise ratio and low toxicity in complex biological systems, and exhibiting excellent universality and imaging potential. Attached Figure Description

[0024] Figure 1 The images show the 1H and 1C NMR spectra of the Probe A intermediate in Example 1.

[0025] Figure 2 The images show the 1H and 1C NMR spectra of the Probe B intermediate in Example 1.

[0026] Figure 3 The images show the 1H and 1C NMR spectra of the fluorescent probe Probe A in Example 1.

[0027] Figure 4 The images show the 1H and 1C NMR spectra of the fluorescent probe Probe B in Example 1.

[0028] Figure 5 The images show the high-resolution mass spectra of fluorescent probes Probe A and Probe B from Example 1.

[0029] Figure 6 The UV-Vis absorption and fluorescence emission spectra of fluorescent probes Probe A and Probe B are shown, where: (a) is the absorption spectrum of the fluorescent probe (40 μM) in Tris-HCl buffer (25 mM, pH 7.4); and (b) is the fluorescence emission spectrum under the same conditions.

[0030] Figure 7 The fluorescence spectra of the response of fluorescent probes Probe A and Probe B to different concentrations of mouse liver microsomes (RLM) are shown.

[0031] Figure 8 The fluorescence spectra of the response of fluorescent probes Probe A and Probe B to different concentrations of human liver microsomes (HLM) are shown.

[0032] Figure 9The following are the concentration response spectra of fluorescent probes Probe A and Probe B to different subtypes of P450 enzymes: (a) CYP 2B6; (b) CYP 1A2; (c) CYP 2D6; (d) CYP 1A1.

[0033] Figure 10 The results show the solubility of fluorescent probes A and B in the aqueous phase.

[0034] Figure 11 The results show the photostability of fluorescent probes A and B.

[0035] Figure 12 The results show the fluorescence stability of fluorescent probes A and B under different pH conditions.

[0036] Figure 13 The results show the selectivity of fluorescent probes A and B for common macromolecules.

[0037] Figure 14 The results show the ion and amino acid selectivity assays for fluorescent probes A and B.

[0038] Figure 15 The effect of fluorescent probes Probe A and Probe B at different concentrations on cell viability (cytotoxicity assessment). Detailed Implementation

[0039] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0040] Materials used: 4-Bromo-1,8-naphthalenedicarboxylic anhydride, 98%, Anhui Zesheng Technology Co., Ltd.; N-methylethylenediamine, 98%, Anhui Zesheng Technology Co., Ltd.; ethylenediamine, Anhui Zesheng Technology Co., Ltd.; 4-methoxyphenylboronic acid, 99%, Anhui Zesheng Technology Co., Ltd.; tetrakis(triphenylphosphine)palladium, 99%, Anhui Zesheng Technology Co., Ltd.; anhydrous potassium carbonate, Tianjin Guangfu Technology Development Co., Ltd.; anhydrous ethanol, analytical grade, Tianjin De'en Chemical Reagent Co., Ltd.; toluene, analytical grade, Chengdu Kelong Chemical Co., Ltd.; dichloromethane, analytical grade, Tianjin De'en Chemical Reagent Co., Ltd.; methanol, analytical grade, Tianjin De'en Chemical Reagent Co., Ltd.; mixed male SD rat liver microsomes, Wuhan Plete Biomedical Technology Co., Ltd.; mixed male human liver microsomes, Wuhan Plete Biomedical Technology Co., Ltd.; cytochrome P450 enzymes (CYP2B6, CYP1A1, CYP1A2, CYP2D6), Corning Incorporated.

[0041] The synthetic routes for fluorescent probes ProbeA (R=CH3) and ProbeA (R=H) are as follows:

[0042]

[0043] Example 1

[0044] The preparation steps of the fluorescent probes ProbeA (R=CH3) and ProbeB (R=H) that can respond to multiple CYPs in this embodiment are as follows:

[0045] (1) Synthesis of ProbeA intermediate and ProbeB intermediate

[0046] 4-Bromo-1,8-naphthalenedicarboxylic anhydride (10 mmol) was dissolved in anhydrous ethanol (50 mL), and after thorough mixing, N-methylethylenediamine (10 mmol) and ethylenediamine (10 mmol) were added separately. The mixture was refluxed at 80 °C for 5 h. The product was filtered, washed with cold methanol, and purified by silica gel column chromatography (elution: V(dichloromethane):V(methanol) = 300:1) to obtain 6-bromo-2-(2-(methylamino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (Probe A intermediate, yield 78%) and 6-bromo-2-(2-(amino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (Probe B intermediate, yield 63%). NMR characterization data are as follows: Probe A intermediate, 1HNMR (600MHz, CDCl3) δ8.65(d,J=7.7Hz,1H),8.56(d,J=8.3Hz,1H),8.41(d,J=7.8Hz,1H),8.03 (d,J=7.8Hz,1H),7.86–7.81(m,1H),4.34(t,J=6.5Hz,2H),2.97(t,J=6.5Hz,2H),2.48(s,3H). 13 CNMR (151MHz, CDCl3) δ 163.90, 133.33, 132.14, 131.32, 131.10, 130.64, 130.32, 129.10, 128.08, 123.07, 122.21, 49.90, 39.93, 36.37; ProbeB intermediate, 1 HNMR (600MHz, DMSO-d6) δ8.58(s,2H),8.32(s,1H),8.22(s,1H),7.99(s,1H),4.08(t,J=6.7Hz,2H),2.82(s,2H). 13 CNMR (151MHz, DMSO-d6) δ 163.70, 163.65, 133.00, 132.00, 131.84, 131.40, 130.31, 129.44, 129.31, 128.95, 123.52, 122.76, 43.09, 40.54. The corresponding proton and carbon NMR spectra are as follows: Figure 1 and Figure 2 As shown.

[0047] (2) Synthesis of fluorescent probes Probe A (R=CH3) and Probe B (R=H)

[0048] Under nitrogen protection, the intermediate corresponding to the fluorescent probe (5 mmol), 4-methoxyphenylboronic acid (20 mmol), tetrakis(triphenylphosphine)palladium (0.5 mmol), and anhydrous potassium carbonate (50 mmol) were added to a two-necked flask containing 50 mL of degassed mixed solvent (toluene:ethanol:water = 8:1:1, v / v / v), and refluxed at 115 °C for 12 h. After cooling, the mixture was filtered, the filtrate was poured into water, extracted three times with dichloromethane, the organic layers were combined, washed with hydrochloric acid, dried over magnesium sulfate, and filtered. Purification by silica gel column chromatography (elution: V(dichloromethane):V(methanol) = 200:3) yielded pale yellow solids, namely fluorescent probe A (yield 68%) and fluorescent probe B (yield 65%). NMR characterization data are as follows: Fluorescent probe A, 1H NMR(600MHz, CDCl3)δ8.62(d,J=7.6Hz,2H),8.30(dd,J=8.5,1.0Hz,1H),7.71–7.65(m,2H),7.46–7.41(m,2 H),7.09–7.06(m,2H),4.40(t,J=6.4Hz,2H),3.91(s,3H),3.03(t,J=6.3Hz,2H),2.52(s,3H),2.17(s,1H). 13 C NMR (151MHz, CDCl3) δ 163.70, 163.55, 158.94, 145.88, 131.91, 130.28, 130.13, 130.06, 130.04, 129.18, 127.86, 126.76, 125.68, 121.73, 120.20, 113.16, 54.44, 48.77, 38.48, 35.01, 34.93, 29.91, 28.68, 21.68, 13.10. Fluorescent probe Probe B, 1 H NMR (600MHz, CDCl3) δ8.63(d,J=7.3Hz,2H),8.31(d,J=8.5Hz,1H),7.69(dd,J=13.4,7.1Hz,2H) ,7.45(d,J=7.6Hz,2H),7.08(d,J=7.5Hz,2H),4.32(t,J=5.8Hz,2H),3.92(s,3H),3.12(s,2H). 13 C10 NMR (151 MHz, DMSO-d6) δ 163.87, 163.65, 159.64, 145.91, 132.28, 131.20, 130.73, 130.47, 130.46, 129.45, 128.23, 127.91, 127.38, 122.70, 121.11, 114.38, 55.38, 54.94, 48.65, 42.13, 40.06, 29.07. The corresponding 1H and 1C NMR spectra of the products are shown below. Figure 3 and Figure 4 As shown.

[0049] Example 2

[0050] The specific preparation steps of the fluorescent probes Probe A (R=CH3) and Probe B (R=H) that can respond to multiple CYPs in this embodiment are as follows:

[0051] (1) Synthesis of Probe A intermediate and Probe B intermediate

[0052] 4-Bromo-1,8-naphthalenedicarboxylic anhydride (10 mmol) was dissolved in anhydrous ethanol (60 mL), stirred and mixed thoroughly, and then N-methylethylenediamine (15 mmol) and ethylenediamine (15 mmol) were added separately. The mixture was refluxed at 70 °C for 3 h. The post-processing was the same as in Example 1, and Probe A intermediate (yield 81%) and Probe B intermediate (yield 68%) were obtained.

[0053] (2) Synthesis of fluorescent probes Probe A (R=CH3) and Probe B (R=H)

[0054] Under nitrogen protection, the intermediate corresponding to the fluorescent probe (5 mmol), 4-methoxyphenylboronic acid (25 mmol), tetrakis(triphenylphosphine)palladium (0.25 mmol), and anhydrous potassium carbonate (40 mmol) were added to 60 mL of degassed mixed solvent (toluene:ethanol:water = 7:2:1, v / v / v) and refluxed at 110 °C for 10 h. The post-processing was the same as in Example 1, finally yielding fluorescent probe A (70% yield) and fluorescent probe B (67% yield).

[0055] Example 3

[0056] The specific preparation steps of the fluorescent probes Probe A (R=CH3) and Probe B (R=H) that can respond to multiple CYPs in this embodiment are as follows:

[0057] (1) Synthesis of Probe A intermediate and Probe B intermediate

[0058] 4-Bromo-1,8-naphthalenedicarboxylic anhydride (8 mmol) was dissolved in a mixed solvent of anhydrous ethanol (40 mL) and a small amount of DMF (5 mL). After stirring and mixing thoroughly, N-methylethylenediamine (12 mmol) and ethylenediamine (12 mmol) were added separately, and the mixture was refluxed at 85 °C for 2.5 h. The post-treatment process was the same as in Example 1, yielding Probe A intermediate (79% yield) and Probe B intermediate (64% yield).

[0059] (2) Synthesis of fluorescent probes Probe A (R=CH3) and Probe B (R=H)

[0060] Under nitrogen protection, the intermediate corresponding to the fluorescent probe (4 mmol), 4-methoxyphenylboronic acid (16 mmol), tetrakis(triphenylphosphine)palladium (0.2 mmol), and anhydrous potassium carbonate (40 mmol) were added to 40 mL of degassed mixed solvent (toluene:ethanol:water = 9:1:1, v / v / v) and refluxed at 118 °C for 11 h. After cooling, extraction was performed in the same manner as in Example 1. The eluent used in the column chromatography separation and purification process was a dichloromethane / methanol mixture with a volume ratio of 150:2, ultimately yielding fluorescent probes Probe A (66% yield) and Probe B (62% yield).

[0061] Example 4

[0062] Absorption and emission spectra of fluorescent probes A and B:

[0063] The fluorescent probes Probe A and Probe B prepared in Example 1 were each prepared into 6 mM DMSO stock solutions. 20 μL of each stock solution was added to 3 mL of pH 7.40 Tris-HCl buffer to prepare test solutions. The absorption spectra were measured using a UV-Vis spectrophotometer, and the absorption wavelength corresponding to the maximum absorbance was recorded. Using this absorption peak as the excitation wavelength, fluorescence emission spectra in the range of 400–700 nm were collected, and the emission wavelength corresponding to the maximum fluorescence intensity was recorded (see results in…). Figure 6 Using quinine sulfate as a standard reference, the molar extinction coefficients and relative fluorescence quantum yields of fluorescent probes Probe A and Probe B were calculated. The results showed that both Probe A and Probe B possessed high molar extinction coefficients and relatively low fluorescence quantum yields, making them suitable for designing as "off-on" type CYPs fluorescent probes.

[0064] Example 5

[0065] Response spectra of fluorescent probes Probe A and Probe B to different concentrations of mouse liver microsomes (RLM):

[0066] DMSO stock solutions of fluorescent probes Probe A and Probe B were diluted to a final concentration of 40 μM in Tris-HCl buffer at pH 7.40. Different concentrations of RLM solution were added according to a pre-defined gradient, and the solutions were thoroughly mixed at room temperature. The emission spectra in the wavelength range of 400–700 nm were recorded using a fluorescence spectrophotometer, with the excitation wavelength being the maximum absorption peak. The results showed that with increasing RLM concentration, the fluorescence intensity of both Probe A and Probe B increased in a concentration-dependent manner, and the wavelengths also exhibited a certain degree of blue shift. Figure 7 As shown.

[0067] Example 6

[0068] Response spectra of fluorescent probes Probe A and Probe B to different concentrations of human liver microsomes (HLM):

[0069] The method was the same as in Example 5, except that RLM was replaced with an equal amount of HLM. HLM solutions were added sequentially to Tris-HCl buffer containing either fluorescent probe Probe A or Probe B according to a predetermined concentration gradient. The solutions were mixed thoroughly at room temperature, and then the fluorescence emission spectra were measured. The results showed that the response trends of fluorescent probes Probe A and Probe B to HLM were consistent with those of RLM; the fluorescence signal significantly increased with increasing HLM concentration, and the wavelength underwent a certain degree of blue shift. The results are as follows: Figure 8 As shown.

[0070] Example 7

[0071] Response spectra of fluorescent probes Probe A and Probe B to different CYP isotypes:

[0072] DMSO stock solutions of fluorescent probes Probe A and Probe B were diluted to a final concentration of 40 μM in Tris-HCl buffer at pH 7.40. Following a set concentration gradient, CYP 2B6, CYP 1A2, CYP 1A1, and CYP 2D6 (final concentration range: 0–0.024 nmol / mL) were added and thoroughly mixed at room temperature. The fluorescence intensity was monitored as a function of enzyme concentration using a fluorescence spectrophotometer. Experimental results showed that both fluorescent probes Probe A and Probe B exhibited significant concentration-dependent fluorescence enhancement responses to the different CYP isotypes, with a certain degree of blue shift in wavelength. The results are shown below. Figure 9 As shown.

[0073] Example 8

[0074] Detection of water solubility of fluorescent probes Probe A and Probe B

[0075] Take 6 mM DMSO stock solutions of fluorescent probes Probe A and Probe B, and add 1 μL each time to a cuvette containing 3 mL of pH 7.40 Tris-HCl buffer, stirring thoroughly after each addition to ensure complete dissolution and that the final DMSO volume fraction is less than 0.1%. Measure the absorbance at each concentration using a UV spectrophotometer. Stop the test when the absorbance shows uneven changes. Then process the data using Origin software. The results are shown below. Figure 10As shown in the figure. All experiments were performed in five parallel determinations. The experimental results show that both fluorescent probes Probe A and Probe B exhibit good water solubility, which meets the basic requirements for their subsequent application in complex biological systems.

[0076] Example 9

[0077] Photostability testing of fluorescent probes ProbeA and Probe B:

[0078] Fluorescent probes A and B were prepared in Tris-HCl buffer solution at pH 7.40, with a final concentration of 40 μM and a DMSO volume fraction controlled below 0.5%. During the test, a cold trap containing saturated sodium nitrite solution was placed between the cuvette and the light source (500W iodine-tungsten lamp), and the distance between the light source and the test sample was controlled at 30 cm. The test samples were continuously irradiated at set time intervals, and the fluorescence intensity changes at the maximum emission wavelength were recorded using a fluorescence spectrophotometer. All experiments were performed in triplicate, and the results are shown below. Figure 11 As shown in the figure, both fluorescent probes ProbeA and ProbeB exhibit good photostability.

[0079] Example 10

[0080] Stability tests of fluorescent probes ProbeA and ProbeB under different pH conditions:

[0081] The solution was prepared using high-purity water, and the pH was gradually adjusted to 2–12 using hydrochloric acid (HCl) and sodium hydroxide (NaOH) solutions, with pH intervals of approximately 0.4–0.5. 3.0 mL of each pH solution was taken, and fluorescent probes Probe A and Probe B were added respectively to achieve a final concentration of 40 μM. The fluorescence emission spectra of the two fluorescent probes in solutions at different pH values ​​were measured using a fluorescence spectrophotometer. Throughout the testing process, experimental conditions were kept consistent, including excitation wavelength, slit width, and sample concentration. All data were obtained from five parallel experiments. The results are as follows: Figure 12 As shown in the figure, the fluorescence intensity of fluorescent probes ProbeA and ProbeB remains almost unchanged in the pH range of 2 to 12.

[0082] Example 11

[0083] Ion, amino acid, and macromolecular selectivity assays for fluorescent probes ProbeA and ProbeB:

[0084] (1) Macromolecular interference test:

[0085] To 40 μM of fluorescent probes ProbeA and ProbeB in Tris-HCl buffer (pH 7.40), macromolecular solutions of lipase, β-amylase, proteinase K, trypsin, 10 wt% fetal bovine serum, pepsin, hemoglobin, and bovine serum albumin (10 mg / mL) were added, respectively, in 20-fold equivalents. The fluorescence intensity of each sample at the maximum emission wavelength was measured using a fluorescence spectrophotometer. The experiment was repeated 5 times. The results are as follows: Figure 13 As shown in the figure, fluorescent probes ProbeA and ProbeB also exhibit good tolerance to interference from these macromolecules, with no significant change in probe fluorescence intensity.

[0086] (2) Ion interference test:

[0087] Solutions of fluorescent probes Probe A and Probe B with a final concentration of 40 μM were prepared in Tris-HCl buffer (pH 7.40). Ionic solutions (30 mM) of the following ions were added to each sample: SnCl2, CaCl2·2H2O, NaHSO4, CuSO4, FeCl2·7H2O, MnSO4, KCl, Al(SO4)3, MgSO4, Zn(NO3)2, CdCl2, KH2PO4, AgNO3, Li2CO3, CoCl2, HgCl2, NiCl2·6H2O, Na2I, and NaHSO3, respectively. Each ion was added in 20 equivalents, and the fluorescence intensity at the maximum emission wavelength was recorded using a fluorescence spectrophotometer. Each experiment was performed in duplicate five times. The results are attached. Figure 14 As shown in Figure (a), the fluorescence intensity of fluorescent probes ProbeA and ProbeB is almost unaffected under various ion conditions, indicating that they have high resistance to ion interference. This characteristic is beneficial for their subsequent application in complex biological systems.

[0088] (3) Amino acid interference test:

[0089] Using the same method as the ion interference test, solutions of glycyl-L-phenylalanine, lysine, serine, glutamine, methionine, glutamic acid, glycyl-L-tyrosine, D-methionine, glycine, dithiothreitol, L-aspartic acid, DL-leucine, L-glutamic acid, DL-threonine, arginine, cystine, homocysteine, valine, hypoxanthine, L-cysteine, and reduced glutathione (10 mM) were added to 40 μM of the fluorescent probes ProbeA and ProbeB in Tris-HCl buffer (pH 7.40), respectively, in cumulative amounts of 20 equivalents. The fluorescence intensity of the samples at the maximum emission wavelength was measured, and the experiment was repeated 5 times. Results are as follows: Figure 14As shown in Figure (b), it can be seen that the above amino acids have little interference with the fluorescent probes ProbeA and ProbeB, and the fluorescence intensity remains basically stable.

[0090] Example 12

[0091] Cytotoxicity assays of fluorescent probes ProbeA and ProbeB:

[0092] (1) Cell culture:

[0093] Both HepG2 and BRL3A cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences. The cell culture medium was DMEM complete medium (WelGene) containing 10 wt% fetal bovine serum (FBS) and 1 wt% penicillin / streptomycin. Cells were cultured routinely at 37°C in a 5% CO2 incubator. One day prior to cytotoxicity, cells were sputtered at 5 × 10⁻⁶ cells / day. 4 The cells were seeded at a density of 100 μL / well in 96-well plates and incubated for 24 hours to allow them to adhere and grow.

[0094] (2) Cytotoxicity test:

[0095] Fluorescent probes ProbeA and ProbeB were prepared into working solutions at different concentrations (5 μM, 10 μM, 15 μM, and 20 μM). After discarding the culture medium, culture medium containing different concentrations of fluorescent probes was added to the corresponding wells in a 96-well plate, and incubation was continued for 24 hours. A blank control group was included in the experiment, and all experiments were performed in parallel wells (5 wells each). After incubation, 20 μL of 5.0 mg / mL MTT solution was added to each well, and the plate was incubated at 37°C for 4 hours. Subsequently, the culture medium was removed, and 150 μL of LDMSO was added. The absorbance at 490 nm was measured using a microplate reader, and the experiment was repeated three times. Cell viability was calculated, and the average of the five experiments was taken. The results are as follows: Figure 15 As shown in the figure, fluorescent probes ProbeA and Probe B still exhibited low cytotoxicity at 20 μM, demonstrating good biocompatibility and suitability for subsequent in vivo CYPs activity imaging studies.

[0096] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A fluorescent probe responsive to multiple cytochrome P450 enzymes, characterized in that... The structural formula of the fluorescent probe is shown in Formula I: Where R1 is -CH2CH2NHR, and R is H or C. 1-6 Alkyl; R2 is a phenyl or substituted phenyl group, wherein the substituents on the substituted phenyl benzene ring are selected from C10. 1-6 Alkyl, C 1-3 Alkoxy or C 2-6 Alkylamide group.

2. The fluorescent probe responsive to multiple cytochrome P450 enzymes according to claim 1, characterized in that: R1 is -CH2CH2NH2 or -CH2CH2NHCH3; R2 is phenyl or 4-substituted phenyl, wherein the substituent on the 4-substituted phenyl benzene ring is selected from methyl, methoxy or acetamido.

3. A method for preparing a fluorescent probe responsive to multiple cytochrome P450 enzymes as described in claim 1 or 2, characterized in that... The specific preparation steps are as follows: Step S1: 4-Bromo-1,8-naphthalenedicarboxylic anhydride and an ethylenediamine compound are dissolved in solvent A and refluxed at 60–85 °C. After the reaction is complete, the mixture is purified by filtration, washing with cold methanol, and column chromatography to obtain the compound shown in Formula II. The ethylenediamine compound is N-methylethylenediamine or ethylenediamine. The general structural formula of the compound shown in Formula II is: Where R1 is -CH2CH2NH2 or -CH2CH2NHCH3; In step S2, the compound shown in Formula II, the 4-substituted phenylboronic acid compound, tetrakis(triphenylphosphine)palladium and potassium carbonate are added to solvent B and refluxed at 110-120°C. After the reaction is completed, the target product, the compound shown in Formula I, is obtained by filtration, extraction and column chromatography separation and purification. This compound can be used as a fluorescent probe for various cytochrome P450 enzymes. The 4-substituted phenylboronic acid compound is 4-methylphenylboronic acid, 4-methoxyphenylboronic acid or 4-acetamidophenylboronic acid.

4. The method for preparing a fluorescent probe responsive to multiple cytochrome P450 enzymes according to claim 3, characterized in that: In step S1, the molar ratio of 4-bromo-1,8-naphthoic anhydride to ethylenediamine compounds is 1:1 to 1.5; in step S2, the molar ratio of the compound shown in Formula II, 4-substituted phenylboronic acid compounds, tetrakis(triphenylphosphine)palladium to potassium carbonate is 1:4:0.1 to 0.5:

10.

5. The method for preparing a fluorescent probe responsive to multiple cytochrome P450 enzymes according to claim 3, characterized in that: In step S1, solvent A is anhydrous ethanol or anhydrous ethanol / N,N-dimethylformamide mixed solvent; in step S2, solvent B is toluene / ethanol / water mixed solvent, wherein the volume ratio of toluene / ethanol / water is 7-9:1-2:

1.

6. The method for preparing a fluorescent probe responsive to multiple cytochrome P450 enzymes according to claim 3, characterized in that: In step S1, the column chromatography separation and purification process uses a silica gel column, and the eluent is a dichloromethane / methanol mixed solution with a volume ratio of 300:1; in step S2, the column chromatography separation and purification process uses a silica gel column, and the eluent is a dichloromethane / methanol mixed solution with a volume ratio of 150:2 or 200:3; in step S2, the extraction solvent used in the extraction process is dichloromethane.

7. The application of the fluorescent probe according to claim 1 or 2 in the detection of various cytochrome P450 enzymes, wherein the fluorescent probe is water-soluble, photostable, and pH-adaptable, and has low toxicity and high signal-to-noise ratio when applied to complex biological systems.

8. The application of the fluorescent probe according to claim 1 or 2 in the preparation of a detection tool for selectively responding to multiple cytochrome P450 enzymes in an interfering environment, wherein the interfering environment is one or more of ions, amino acids, and macromolecules, wherein the ions are SnCl2, CaCl2·2H2O, NaHSO4, CuSO4, FeCl2·7H2O, MnSO4, KCl, Al(SO4)3, MgSO4, Zn(NO3)2, CdCl2, KH2PO4, AgNO3, Li2CO3, CoCl2, HgCl2, NiCl2·6H2O, Na One or more of 2I and NaHSO3; amino acids are one or more of glycyl-L-phenylalanine, lysine, serine, glutamine, methionine, glutamic acid, glycyl-L-tyrosine, D-methionine, glycine, dithiothreitol, L-aspartic acid, DL-leucine, L-glutamic acid, DL-threonine, arginine, cystine, homocysteine, valine, hypoxanthine, L-cysteine, and reduced glutathione; macromolecules are one or more of bovine lipase, β-amylase, proteinase K, trypsin, fetal bovine serum, pepsin, hemoglobin, and bovine serum albumin.

9. The application of the fluorescent probe according to claim 1 or 2 in the preparation of metabolic enzyme activity detection tools or biomarker screening tools, wherein the fluorescent probe has good fluorescence response sensitivity and response dynamic range to liver microsomes in animal or human metabolic systems, and can be used to evaluate the metabolic activity of various cytochrome P450 enzymes in liver microsomes in vitro.

10. The application of the fluorescent probe according to claim 1 or 2 as an imaging reagent for various cytochrome P450 enzyme activities, wherein the fluorescent probe has low cytotoxicity and good biocompatibility.

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