Near-infrared fluorescent probe targeting CYP1B1 enzyme and its preparation and use

Abstract

The present invention provides a near-infrared fluorescent probe targeting CYP1B1 enzyme and its preparation and use. The near-infrared fluorescent probe has a structural formula as shown in formula (I): wherein X=O or X=NH, located at the 4' position or the 5' position; Y is a connecting chain, comprising multiple ethylene glycol fragments or multiple alkyl groups. The present invention also relates to the use of the aforementioned probe, wherein the probe targets the specifically expressed tumor marker CYP1B1 enzyme and images the tumor site by specifically binding to the CYP1B1 enzyme. The probe will promote the application of molecular imaging probes in tumor imaging and has good application prospects and clinical transformation value in early diagnosis of tumors.

Description

Technical Field

[0001] The present invention belongs to the field of molecular imaging probes, and in particular, relates to a near-infrared fluorescent probe targeting CYP1B1 enzyme and a preparation method and application thereof. Background Art

[0002] Cytochrome P450 1B1 (CYP1B1) is a member of the cytochrome P4501 family and has been shown to be closely related to the occurrence and development of various cancers, especially hormone-induced cancers, including prostate cancer, breast cancer, lung cancer, bladder cancer, etc. (Go, RE, KA Hwang, and KC Choi. Cytochrome P450 1 family and cancers. Journal of Steroid Biochemistry and Molecular Biology, 2015, 147: 24-30.). As an important intracellular oxidase, CYP1B1 not only participates in the metabolism of exogenous procarcinogens (such as benzanthracene compounds, benzopyrene, 1-ethynylpyrene, etc.), but also metabolizes the endogenous substance 17-β-estrogen into 4-hydroxy 17-β estradiol with genotoxicity (Nebert, DWand TP Dalton. The role of cytochrome P450 enzymes in endogenous signaling pathways and environmental carcinogenesis. Nat Rev Cancer, 2006, 6: 947-960.). Studies have confirmed that CYP1B1 is highly expressed in tumor tissues, but almost not expressed in normal tissues, and can also be induced to express by some procarcinogens. The expression of CYP1B1 may be regulated by estrogen receptors (ER) and aryl hydrocarbon receptors (AhR), which induce CYP1B1 expression after binding to the corresponding ligands. Therefore, CYP1B1 is considered to be one of the important markers of tumors and is expected to play a role as a target in the diagnosis and treatment of tumors.

[0003] Early diagnosis and efficacy evaluation of tumors through bioimaging is a challenging and practical topic. Molecular probes can reflect changes in molecular levels in vivo in a non-invasive or minimally invasive way, making disease parameters specific and accurate, and reflecting small changes in the early stages of tumor development, which has great potential in early tumor diagnosis (Weissleder, R. and MJPittet. Imaging in the era of molecular oncology. Nature, 2008, 452: 580-589.). Near infrared (NIR) imaging technology is a type of optical molecular imaging. Compared with PET, MRI and other imaging methods, NIR imaging has no radiation and low cost. It also has the advantages of non-destructive, high specificity and high sensitivity. It can realize in situ, real-time and quantitative monitoring of specific tissues, and has good application prospects in tumor diagnosis, treatment and prognosis (Zeng, ZL, S Liew, X. Wei, and K. Y. Pu. Hemicyanine-Based Near-Infrared Activatable Probes for Imaging and Diagnosis of Diseases. Angewandte Chemie-International Edition, 2021, 60: 26454-26475.). However, since the target may also have a certain distribution in normal tissues, the current near-infrared fluorescent molecular probes still have the problems of high background signal and insufficient imaging specificity. The emergence and application of the new target CYP1B1 is expected to provide a certain reference for solving this problem.

[0004] At present, CYP1B1 enzyme is not widely used as a molecular imaging target in molecular imaging research in tumor diagnosis. In the research before the present invention, based on the binding mode of α-naphthoflavone (ANF, an effective inhibitor of CYP1B1) and CYP1B1 enzyme, a variety of probes and probe precursors derived from ANF were successfully synthesized (ZL201810373475.3, ZL201910731604.6, ZL202111057949.1). These probes or probe precursors show the ability to indicate the tumor site in vivo by binding to CYP1B1. However, in the process of introducing the connecting chain and the fluorescent group, the inhibitory activity of α-naphthoflavone derivatives on the CYP1B1 enzyme has been greatly reduced. It may be that the orientation of the introduced group is not the entrance direction of the inhibitor to the active pocket of the CYP1B1 protein, resulting in a certain influence on the binding of the probe to the active pocket of CYP1B1. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a near-infrared fluorescent small molecule probe targeting CYP1B1 enzyme and its preparation and application in view of the deficiencies in the prior art. The present invention, through the study of the single crystal structure of ANF and ancient mammal CYP1B1 enzyme (PDB ID: 6OYU), discovered that the active pocket remains in an open state, as well as the possible opening direction of the pocket when the CYP1B1 enzyme binds to a substrate or inhibitor. Based on this idea, the present invention designs a novel near-infrared fluorescent small molecule probe targeting CYP1B1 enzyme, which provides a method for early diagnosis of tumors by performing near-infrared in vitro imaging of intracellular CYP1B1 enzymes and displaying their expression sites and levels in animals, thereby solving the problems of low specificity of existing molecular probes and difficulty in diagnosis in current tumor treatment.

[0006] The principle of the present invention is that the CYP1B1 enzyme is found to be specifically highly expressed in tumor cells and induced to be expressed in early cancer cells. It is considered to be one of the tumor markers and has the potential to be used as a target for early diagnosis of tumors. In previous studies, based on the compound 3'-fluoro-6,7,10-trimethyloxy-α-naphthoflavone, which has the strongest inhibitory activity on CYP1B1, a substituent was introduced at its 3-position to synthesize a class of near-infrared fluorescent probes and successfully applied to the imaging of tumor sites, but the probe also has the problem of insufficient binding ability with the CYP1B1 enzyme. In order to further explore probes with stronger binding ability, the present invention is based on the study of the single crystal structure of the complex of α-naphthoflavone and the ancient mammal CYP1B1 enzyme, and found that the active pocket of the CYP1B1 enzyme remains in an open state. The reason may be that the difference in the amino acid sequence in F Helix makes a common break in the CYP1 family enzymes not exist in this ancient mammal CYP1B1 enzyme, resulting in F Helix being more rigid and easier to remain in an open state. Under the premise of retaining the F atom, the introduction of a substituent at the meta position (5' position) or para position (4' position) of the ANF benzene ring can point to the direction of the pocket entrance. Therefore, the present invention intends to use the α-naphthoflavone derivatives (CYP1B1 IC 50 =0.043nM) is introduced into the 4' or 5' position through easily derivatized hydroxyl, amino and other functional groups, so as to further connect the signal group to obtain a new near-infrared fluorescent probe targeting CYP1B1 enzyme. The present invention selects 2-4 ethylene glycol fragments (n=1,2,3) or full alkyl chains as connecting chains, and the near-infrared fluorescent molecule Cy5.5 with good tissue permeability as the signal group, and introduces the two into α-naphthoflavone derivatives to prepare the molecular probe of the present invention. The present invention further explores and obtains a molecular probe targeting CYP1B1 enzyme with stronger binding ability, promoting the application of CYP1B1 enzyme in tumor diagnosis and personalized treatment.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] In the first aspect, the present invention provides a near-infrared fluorescent probe targeting the CYP1B1 enzyme, characterized in that it includes an affinity ligand, a signal group and a connecting chain for connecting the affinity ligand and the signal group; the connecting chain contains multiple ethylene glycol fragments or multiple alkyl groups; the affinity ligand includes an α-naphthoflavone derivative, and the signal group includes a near-infrared fluorescent molecule (preferably Cy5.5).

[0009] Preferably, the structural formula of the fluorescent probe is as shown in Formula I:

[0010] Wherein, X=O or X=NH, located at the 4' or 5' position, Y is a connecting chain, and the near-infrared fluorescent molecule is Cy5.5; when X=O, Y is n=1, 2, 3; when X=NH, Y is n=1, 2, 3 or Y is n=2,3,4.

[0011] More preferably, the structural formula of the fluorescent probe is as shown in Formula I:

[0012] Wherein, X=O or X=NH, located at the 5' position, Y is the connecting chain, and the near-infrared fluorescent molecule is Cy5.5; when X=O, Y is n=3; when X=NH, Y is n=3 or Y is n=4.

[0013] More preferably, the fluorescent probe comprises at least one of the following structures:

[0014]

[0015] In a second aspect, the present invention provides a method for preparing a near-infrared fluorescent probe targeting CYP1B1 enzyme, comprising the following steps:

[0016] A. The X group is protected to obtain compound V

[0017] B. and Reaction to generate compound II

[0018] C. Compound II undergoes rearrangement, cyclization, and removal of the protecting group under acidic conditions to generate derivative III

[0019] D. Introduce a connecting chain into derivative III to generate derivative IV

[0020] E. The derivative IV reacts with water-soluble Cy5.5-NHS to obtain the near-infrared fluorescent probe.

[0021] Preferably, in step A, P in the compound V is a protecting group; wherein, when X=O, the reaction is carried out in the presence of MOMCl, and P=MOM; when X=NH, the reaction is carried out in the presence of (Boc)2O, and P=Boc.

[0022] Preferably, in step A, when X=O, The molar ratio of MOMCl is 1:1 to 1:10; when X = NH, The molar ratio of (Boc)2O is 1:2 to 1:6.

[0023] Preferably, the reaction of step B is carried out in the presence of DMAP and EDC·HCl.

[0024] Preferably, in step B, The molar ratio of compound V, DMAP, and EDC·HCl is 1:1:0.1:1 to 1:5:1:10.

[0025] Preferably, the reaction of step C is carried out in the presence of t-BuOK and concentrated sulfuric acid.

[0026] Preferably, in step C, the molar ratio of compound II, t-BuOK and concentrated sulfuric acid is 1:1:20 to 1:10:50, and the concentration of the concentrated sulfuric acid is 18.4 mol / L.

[0027] Preferably, when X=O, the reaction of step D is carried out in the presence of Cs2CO3 and hydrochloric acid; when X=NH, the reaction of step D is carried out in the presence of HATU and DIPEA and hydrochloric acid.

[0028] Preferably, in step D, when X=O, the molar ratio of derivative III, Cs2CO3, and hydrochloric acid is 1:2:20 to 1:10:80; when X=NH, the molar ratio of derivative III, HATU, DIPEA, and hydrochloric acid is 1:1:1:20 to 1:5:10:80, and the concentration of hydrochloric acid is 12 mol / L.

[0029] Preferably, in step D, when X=O, the connecting chain is a chain comprising multiple ethylene glycol segments. n=1, 2, 3, forming an ether bond with derivative III, and removing the Boc protecting group to obtain derivative IV; when X=NH, the connecting chain is composed of multiple ethylene glycol fragments n=1,2,3 or contains multiple alkyl groups n=2, 3, 4, form an amide bond with derivative III, and remove the Boc protecting group to obtain derivative IV.

[0030] In a third aspect, the present invention also provides a use of a near-infrared fluorescent probe targeting CYP1B1 enzyme in the preparation of a tumor diagnostic reagent.

[0031] Compared with the probe prepared by introducing substituents at other sites (such as site 3) of α-naphthoflavone, the present invention designs a near-infrared probe that can bind to the CYP1B1 enzyme, significantly improving the affinity of the probe targeting the CYP1B1 enzyme, obtaining a better near-infrared tumor imaging effect, and further applying it to photoacoustic imaging of tumors.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1) The near-infrared fluorescent molecular probe described in the present invention is designed based on the study of the binding mode of α-naphthoflavone with the open CYP1B1 enzyme. The study found that a CYP1B1 enzyme maintained in an open state indicates the possible opening direction in the dynamic process of the substrate binding to the active pocket of the CYP1B1 enzyme. The present invention introduces a connecting chain in the opening direction to effectively avoid the introduction of a signal group to significantly reduce the inhibitory activity of the α-naphthoflavone derivative ligand on the CYP1B1 enzyme.

[0034] 2) Compared with the probes obtained in the previous laboratory research, the molecular probe provided by the present invention has a stronger ability to specifically bind to the CYP1B1 enzyme (Kd value can reach below 100nM), and can better enrich in tumor cells and indicate the tumor site. Moreover, in both near-infrared fluorescence and photoacoustic imaging modalities, the probes described in the present invention can specifically bind to the CYP1B1 enzyme to achieve real-time tumor imaging in vivo.

[0035] 3) The target CYP1B1 enzyme selected by the present invention is a specific marker for tumors. In the previous studies using it as a biological target for molecular probe in vivo imaging, there was still a problem of insufficient ability of the probe to bind to the CYP1B1 enzyme. The probe described in the present invention has a more novel structure and a stronger ability to target the CYP1B1 enzyme, which will effectively promote the CYP1B1 enzyme to play a target role in tumor in vivo imaging, and will have good application prospects and clinical transformation value in early diagnosis of tumors.

[0036] 4) The present invention further studies the application of probes targeting CYP1B1 enzyme in tumor photoacoustic imaging. The molecular probe provided by the present invention can specifically target tumors and has good photoacoustic imaging effects, and has the potential to be used in tumor photoacoustic imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0038] Figure 1 The preparation route map of the near-infrared fluorescent probe of structural formula I prepared in the present invention;

[0039] Figure 2 is the spectrum measurement result of the probe; where: Figure 2 a UV absorption spectrum; Figure 2 b is the fluorescence emission spectrum; Figure 2 c-2e is the fluorescence stability measurement result of the probe;

[0040] Figure 3 To detect the expression of CYP1B1 enzyme in different cells; Figure 3 a is the result of Western Blot experiment; Figure 3 b is the quantification result of CYP1B1 enzyme expression relative to the internal reference;

[0041] Figure 4 is the saturation curve of the near-infrared fluorescent probe of the present invention in colon cancer cell HCT-15; wherein: Figure 4 a is the saturation curve of I-1 in colon cancer cell HCT-15; Figure 4 b is the saturation curve of I-2 in colon cancer cells HCT-15; Figure 4 c is the saturation curve of I-3 in colon cancer cells HCT-15;

[0042] Figure 5 It is the confocal microscopic imaging result of the near-infrared fluorescent probe of the present invention in cells; wherein: Figure 5 a is the confocal microscopic imaging result of probe I-1 in colon cancer cell HCT-15; Figure 5 b is the confocal microscopic imaging result of probe I-1 in skin fibroblast HFF-1; Figure 5 c is the confocal microscopic imaging result of probe I-2 in colon cancer cell HCT-15; Figure 5 d is the confocal microscopic imaging result of probe I-2 in skin fibroblast HFF-1; Figure 5 e is the confocal microscopic imaging result of probe I-3 in colon cancer cell HCT-15; Figure 5 f is the confocal microscopic imaging result of probe I-3 in skin fibroblast HFF-1; DAPI indicates cell nucleus staining, Cy5.5 indicates Cy5.5 fluorescence, and Merge indicates the image of the superposition of the two channel signals;

[0043] Figure 6The results are the near-infrared fluorescence in vivo imaging results and the quantitative results of the fluorescence intensity in the tumor site of HCT-15 colon cancer cell-bearing mice at different time points after injection of I-1 only (Imaging) and co-injection of I-1 and blocker ANF (Blocking); among which: Figure 6 a is the result of near-infrared fluorescence in vivo imaging; Figure 6 b is the quantification result of fluorescence intensity at the tumor site;

[0044] Figure 7 The biodistribution and quantitative results of I-1 in the main in vitro tissues of mice with colon cancer cell HCT-15 transplanted tumors after injection of I-1 (Imaging) and co-injection of I-1 and blocker ANF (Blocking); Among them: Figure 7 a is the biological distribution; Figure 7 b is the quantitative result of biodistribution;

[0045] Figure 8 The results of in vivo photoacoustic imaging and quantification of photoacoustic intensity at the tumor site in mice with colon cancer cell HCT-15 transplanted tumors at different time points after injection of I-1 (Imaging) and PBS (PBS); among them: Figure 8 a is the result of in vivo photoacoustic imaging; Figure 8 b is the quantification result of photoacoustic intensity at the tumor site. DETAILED DESCRIPTION

[0046] The present invention is described in detail below in conjunction with specific embodiments and accompanying drawings. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0047] Example 1

[0048] This embodiment relates to a method for preparing a near-infrared fluorescent probe I-1 derived from 6,7,10-trimethoxy-3'-fluoro-α-naphthoflavone having a structural formula I, such as Figure 1 As shown, the following steps are included:

[0049] Step 1: After 3-fluoro-5-hydroxybenzoic acid (6.4mmol) is dissolved in dichloromethane (30mL), DIPEA (32.0mmol) is slowly added dropwise to mix well, and MOMCl (32.0mmol) is slowly added under nitrogen protection in an ice-water bath. The ice-water bath is removed 10 minutes after the addition is complete, and the reaction solution is stirred at room temperature overnight. After the reaction is completed, quench with saturated NH4Cl solution, extract with dichloromethane, combine the organic phases, wash with saturated NaCl solution in turn, and dry with anhydrous Na2SO4. Use a rotary evaporator to concentrate under reduced pressure to obtain a white solid. The white solid is dissolved in methanol (50ml), and NaOH aqueous solution (2.4g dissolved in 50ml water) is added, and heated at 70°C for 3 hours. After the reaction was completed, the mixture was cooled to room temperature and the pH of the reaction solution was adjusted to about 5 with dilute hydrochloric acid. A large amount of white precipitate was observed. After standing, the mixture was filtered to obtain a white powdery solid 3-fluoro-5-(methoxymethoxy)benzoic acid V-1 with a yield of 60.1%. 1 HNMR (400MHz, DMSO-d6): δ7.40 (dd, J=2.5, 1.3Hz, 1H), 7.30 (m, 1H), 7.16 (m 1H), 5.26 (s, 2H), 3.39 (s, 3H). 13 C NMR (101MHz, DMSO-d6): δ165.92,163.71,158.14,133.71,112.89,109.13,108.31,94.17,55.86.

[0050] Step 2: Dissolve 1-(1-hydroxy-4,5,8-trimethoxynaphthalen-2-yl)ethane-1-one (1.8 mmol, for the synthesis method, see the published literature Journal of Chemical Research, 2012: 264-265.), compound V (2.7 mmol), and EDC·HCl (3.6 mmol) in dichloromethane (Superdry, 20 ml), add DMAP (0.45 mmol), protect with nitrogen, and stir overnight at room temperature. After the reaction is completed, wash the reaction solution with an equal volume of water, extract with dichloromethane, combine the organic phases, wash with saturated NaCl solution in turn, dry with anhydrous Na2SO4, and dry under reduced pressure to obtain a light yellow mixed solid. Column chromatography was used for further purification, and a mixture of petroleum ether and ethyl acetate (5:1) was used for elution. The eluates containing the target product were combined and dried to obtain a light yellow solid 2-acetyl-4,5,8-trimethoxynaphthalen-1-yl-3-fluoro-5-(methoxymethoxy)benzoate II-1. Yield: 90.4%. 1H NMR (400MHz, Chloroform-d): δ7.72(d,J=2.5Hz,1H),7.63-7.58(m,1H),7.23(s,1H),7.09(s,1H),6.96-6.92(d,J= 8.7Hz,1H),6.81(d,J=8.7Hz,1H),5.24(d,J=1.4Hz,2H),4.01(s,3H),3.90(s,3H),3.51-3.53(m,6H),2.57(s,3H). 13 C NMR (101 MHz, Chloroform-d):δ

[0051] 198.13,164.56,164.33,162.10,158.64,155.06,151.05,140.35,132.59,129.13,122.37,121.39,114. 03,111.48,110.41,109.19,108.26,105.37,94.73,58.09,56.89,56.37,56.28,31.43.ESI-HRMS:calcd for C 24 H 24 FO8 + 459.1450; found 459.1457[M+H] + .

[0052] Step 3: Dissolve compound Ⅱ-1 (1.1mmol) in THF (Superdry, 30ml), and add t-BuOK (2.2mmol) in batches in an ice-water bath. After the addition is complete, remove the ice-water bath, stir at room temperature for 2 hours, and the color of the reaction solution gradually deepens. After the reaction is completed, add an equal volume of ice water to quench, and adjust the pH to 4-5 with glacial acetic acid, and a red precipitate can be observed. Extract with dichloromethane several times, combine the organic phases, wash with saturated NaCl solution in turn, dry with anhydrous Na2SO4, and spin dry to obtain an orange-red solid. Dissolve the solid in CH3COOH, slowly add concentrated H2SO4 (1.5mL) while stirring, and heat to reflux at 75°C for 1 hour. After the reaction is completed, cool to room temperature, neutralize the excess acid with saturated NaHCO3 solution, and a yellow precipitate can be seen. Let stand, filter, wash with a small amount of water, and dry to obtain a yellow solid 3'-fluoro-5'-hydroxy-6,7,10-trimethoxy-α-naphthoflavone Ⅲ-1. Yield: 85.4%. 1HNMR (400MHz, DMSO-d6): δ10.70(s,1H),7.54-7.50(m,2H),7.30(s,3H),7.14 (s,1H),6.94(dt,J=10.5,2.3Hz,1H),4.08(s,3H),3.94(s,3H),3.84(s,3H). 13 C NMR (176MHz, DMSO-d6): δ176.11,163.82,162.45,160.92,159.79,154.34,150.74,148.09,134.09,121.23,1 20.87,117.39,113.35,110.05,109.78,107.01,105.61,103.59,97.92,57.47,56.55,56.20.ESI-HRMS:calcd for C 22 H 18 FO6 + 397.1082;found 397.1081[M+H] + .

[0053] Step 4: Dissolve compound III-1 (0.13mmol) in DMF (Superdry, 5mL), then drop tert-butyloxy 2-(2-(2-(2-iodoethoxy)ethoxy)ethoxy)ethylamine (0.15mmol, see CN201910731604.6 for the synthesis method) in DMF solution (5mL), add cesium carbonate (0.63mmol) in batches under ice-water bath conditions, and stir overnight at 65°C under nitrogen protection. After the reaction is completed, wash the reaction solution with 2 volumes of water, extract with ethyl acetate, combine the organic phases, wash with saturated NaCl solution several times, dry with anhydrous Na2SO4, and spin dry to obtain a yellow crude product, which is purified by column chromatography, and the elution solvent is dichloromethane-methanol (50:1). After concentrated by reduced pressure evaporation, a yellow oily product is obtained. The product is dissolved in ethyl acetate (6mL), and 1mL of ethyl acetate-hydrochloric acid (5.14mmol) mixed solution is added under nitrogen protection, and stirred at room temperature for 5 hours. After the reaction, add saturated NaHCO3 solution to adjust the pH to about 7, extract with dichloromethane-methanol (3:1) mixed solution, combine the organic phases, dry with anhydrous Na2SO4, and concentrate under reduced pressure to obtain a yellow solid. Purify by column chromatography, the elution solvent is dichloromethane-methanol (5:1), combine the elution solvent groups with the same Rf value, and concentrate under reduced pressure to obtain a yellow powdery solid. 2-(3'-(2-(2-aminoethoxy)ethoxy)-5'-fluorophenyl)-6,7,10-trimethoxy-α-naphthoflavone IV-1. Yield: 77.4%. 1H NMR(400MHz,DMSO-d6):δ7.57(d,J=6.5Hz,2H),7.23-7.15(m,4H),7.04(dd,J=10.6,2.4Hz,1H),4.20(d,J=4.9Hz,2H),4.00(s,3H),3.90(s,3H),3.80(m,5H),3.63(dt,J=5.3,2.7Hz,2H),3.58–3.52(m,4H),3.51–3.47(m,2H),3.37-3.33(m,2H),2.64(t,J=6.0Hz,2H). 13 C NMR(101MHz,DMSO-d6):δ175.99,163.16(d,J=242.5Hz),160.31,160.14,154.16,150.68,150.51,147.83,134.06(d,J=10.9Hz),121.10,120.71,117.17,113.04,109.66,109.11,107.09,104.99(d,J=24.9Hz),104.51(d,J=25.2Hz),97.76,72.67,69.97,69.84,69.79,69.57,68.79,68.08,57.29,56.18,56.01,41.15.ESI-HRMS:calcdfor C 30 H 35 FNO9 + 572.2290;found572.2291[M+H] + .

[0054] Step 5: Compound IV-1 (6.3 μmol) and Cy5.5-NHS (3.0 μmol) were dissolved in dry DMSO (0.6 mL), and 40 μL TEA was added. The mixture was stirred overnight at room temperature, and the reaction was monitored by high performance liquid chromatography. After confirming that the reaction was fully reacted, two equivalents of TFA were added to the reaction solution to neutralize TEA, and the reaction solution was purified by semi-preparative HPLC to separate the product. A C18 packing column (250 mm × 10 mm) was used, and 200 μL was injected at a time. The mobile phase was gradient eluted from 95% water (25 mM ammonium acetate) / 5% acetonitrile to 5% water (containing 25 mM ammonium acetate) / 95% acetonitrile within 10 min. From 10 min to 30 min, 5% water (25 mM ammonium acetate) / 95% acetonitrile was isocratic elution, the flow rate was 4 mL / min, the UV monitoring wavelength was 254 nm, and Rt = 18 min. The collected product solutions were combined, and the acetonitrile was removed by decompression using a rotary evaporator. The remaining liquid was placed in a -80°C refrigerator and frozen overnight. After freezing, the product was freeze-dried for 48 hours to obtain a blue solid, namely probe I-1. Yield: 68.9%. 1 H NMR (400MHz, Methanol-d4): δ9.08(dd,J=9.1,2.9Hz,2H),8.75(s,2H),8.62(d,J=1.6Hz,2H),8.33-8.26(m,2H),7.99(s,1H),7.70( dd,J=11.6,9.3Hz,2H),7.55(d,J=6.7Hz,2H),7.32(s,1H),7.14(d,J=2.6Hz,2H),7.05(s,1H),6.92(d,J=10.4Hz,1H),6.47(s,1H),6 .20-6.12(m,2H),4.24-4.16(m,4H),4.10(s,2H),4.04(s,3H),3.93(s,3H),3.83(d,J=4.4Hz,2H),3.79(s,3H),3.71-3.52(m,10H), 3.47(t,J=5.2Hz,2H),2.17(t,J=7.2Hz,2H),2.02-1.91(m,12H),1.79(d,J=8.9Hz,2H),1.65(t,J=7.6Hz,2H),1.42(t,J=7.1Hz,3H). 13C NMR (176MHz, Methanol-d4): δ179.47,175.81,175.66,175.58,164.60,16 3.27,159.32,156.37,154.84,154.60,152.78,152.49,150.51,144.94,14 4.88,144.06,141.82,141.38,136.31,136.04,135.45(d,J=10.7Hz),130 .61,129.69,129.64,128.90,127.45,125.19,123.45,123.23,122.14,118 .87,115.44,113.62,113.38,111.03,110.82,107.91,106.71,106.56,10 5.74(d,J=25.0Hz),104.23,104.13,98.88,71.76,71.65,71.61,71.20,70 .67,70.49,69.61,64.42,58.46,57.60,57.48,57.10,56.84,52.77,52.66 ,44.92,40.31,36.59,28.28,27.92,27.78,27.23,26.42.ESI-HRMS:calcd for C 71 H 74 F3O 22 S4 2- 734.6899; found 734.6658[M] 2- .

[0055] Example 2

[0056] This embodiment relates to a method for preparing a near-infrared fluorescent probe I-2 derived from 6,7,10-trimethoxy-3'-fluoro-α-naphthoflavone having a structural formula I, such as Figure 1 As shown, the following steps are included:

[0057] Step 1: Dissolve 3-fluoro-5-aminobenzoic acid (12.9mmol) and DMAP (3.9mmol) in dichloromethane (30mL), slowly add TEA (64.5mmol) and mix well. Add (Boc)2O (38.7mmol) in dichloromethane (10mL) under nitrogen protection and ice-water bath. After the addition is complete, remove the ice-water bath and stir the reaction solution at room temperature overnight. After the reaction is completed, wash with an equal volume of water and extract with dichloromethane. The organic phase is washed with saturated NaCl solution in turn and dried over anhydrous Na2SO4. Use a rotary evaporator to concentrate under reduced pressure to obtain a yellow solid. Dissolve the yellow solid in a mixed solution of methanol-tetrahydrofuran (3:2) (30ml), add NaOH aqueous solution (1M, 30ml), and heat at 70°C for 6 hours. After the reaction is completed, the mixture is cooled to room temperature, the organic solvent is dried by rotary evaporation, and the reaction solution is acidified with dilute hydrochloric acid to a pH of about 5. A large amount of precipitation is observed. The mixture is allowed to stand and filtered to obtain a slightly yellow solid 3-fluoro-5-((tert-butoxycarbonyl)amino)benzoic acid V-2. Yield: 85.6%. 1 H NMR (400MHz, DMSO-d6): δ9.80 (s, 1H), 7.92 (s, 1H), 7.55 (d, J = 11.2Hz, 1H), 7.24 (m, 1H), 1.48 (s, 9H). 13 C NMR (101MHz, DMSO-d6): δ166.20, 162.03 (d, J = 241.7Hz), 152.62, 141.65 (d, J = 11 .3Hz),133.30(d,J=8.9Hz),114.86,108.94(d,J=23.3Hz),108.53,79.89,28.03.

[0058] Step 2: Same as step 2 of Example 1, except that 3-fluoro-5-((tert-butoxycarbonyl)amino)benzoic acid is used instead of 3-fluoro-5-(methoxymethoxy)benzoic acid to obtain 2-acetyl-4,5,8-trimethoxynaphthalen-1-yl-3-((tert-butoxycarbonyl)amino)-5-fluorobenzoate II-2, yield: 83.9%. 1 H NMR(400MHz,Chloroform-d)δ8.09(d,J=11.0Hz,1H),7.86(s,1H),7.47-7.40(m,2H),7.27(s,1H),6.98( d,J=8.7Hz,1H),6.63(d,J=8.8Hz,1H),4.00(s,3H),3.93(s,3H),3.33(s,3H),2.33(s,3H),1.60(s,9H). 13C NMR(101MHz,Chloroform-d)δ197.56,164.26,163.38(d,J=244.1Hz),154.49,152.69,151.19,150.71,141.19(d,J=11.0Hz),131.81(d,J=8.9Hz),128 .38,122.76,121.04,115.25,113.08,110.94,110.68(d,J=4.8Hz),110.37, 107.76,105.78,81.45,58.84,56.81,55.81,31.63,28.55.ESI-HRMS:calcd for C 27 H 29 FNO8 + 514.1872;found 514.1879[M+H] + .

[0059] Step 3: The same as step 3 of Example 1, except that II-2 is used instead of II-1 to obtain 3'-fluoro-5'-amino-6,7,10-trimethoxy-α-naphthoflavone III-1, with a yield of 76.1%. 1 H NMR (400MHz, DMSO-d6): δ7.24-7.14(m,5H),6.93(s,1H),6.57(d,J=11.2Hz,1H),5.74(s,2H),4.01(s,3H),3.90(s,3H),3.81(s,3H). 13 CNMR (101MHz, DMSO-d6): δ175.97,163.54(d,J=239.2Hz),161.63,154.18,151.26(d,J=10.7Hz),150.70,148.02,133.80(d,J=10.4Hz),12 1.16,120.76,117.34,113.05,109.79,107.34,106.51,102.81(d,J=24.0Hz),100.15,99.90,97.88,57.38,56.55,56.10.ESI-HRMS:calcd for C 22 H 19 FNO5 + 396.1292;found 396.1296[M+H] + .

[0060] Step 4: Dissolve compound III-1 (0.13mmol), 9-((tert-butoxycarbonyl)amino)nonanoic acid (0.20mmol), and HATU (0.20mmol) in DMF (Superdry, 8mL), then add DIPEA (0.26mmol) dropwise, and stir overnight at room temperature under nitrogen protection. After the reaction is completed, wash the reaction solution with 2 volumes of water, extract with ethyl acetate, wash the organic phase with saturated NaCl solution several times, dry with anhydrous Na2SO4, and spin dry to obtain a yellow solid. Dissolve the solid in dichloromethane (6mL), add 1mL of dichloromethane-hydrochloric acid (5.14mmol) mixed solution under nitrogen protection, the solution turns dark red, and stir at room temperature for 5h. After the reaction is completed, add saturated NaHCO3 solution to adjust the pH to alkaline, the solution turns yellow, extract with a mixed solution of dichloromethane-methanol (3:1), combine the organic phases, dry with anhydrous Na2SO4, and concentrate under reduced pressure to obtain a yellow solid. Column chromatography was used for purification, and the elution solvent was dichloromethane-methanol (5:1). After concentration under reduced pressure, a yellow powder solid 9-amino-N-(3-fluoro-5-(6,7,10-trimethoxy-α-naphthoflavone))nonanamide IV-2 was obtained with a yield of 61.8%. 1 H NMR (400MHz, DMSO-d6): δ10.38(s,1H),8.13(s,1H),7.72(dd,J=14.1,10.4Hz,3H),7.30(s,2H),7.02(s,1H),4.04(s,3H),3.94(s,3H),3.85 (s,3H),2.65(t,J=7.4Hz,2H),2.38(t,J=7.4Hz,2H),2.13(t,J=7.5Hz,1H),1.99(d,J=9.3Hz,1H),1.61(q,J=7.2Hz,4H),1.55-1.37(m,6H). 13 CNMR (101MHz, DMSO-d6): δ175.91, 172.00, 162.43 (d, J = 241.3Hz), 160.75, 154.41, 15 0.78,148.14,141.45,133.88(d,J=10.1Hz),124.24,121.25,120.87,117.37,113.39, 112.77,110.06,108.83(d,J=26.0Hz),107.58(d,J=25.4Hz),107.18,97.86,57.45,5 6.64,56.19,40.91,36.41,31.16,29.85,29.02,28.61,26.26,24.98.ESI-HRMS:calcd for C 31 H 36FN2O6 + 551.2552;found 551.2553[M+H] + .

[0061] Step 5: Same as step 5 of Example 1, except that IV-2 replaces IV-1 to obtain probe I-2 with a yield of 75.0%. 1 H NMR (400MHz, DMSO-d6): δ10.40(s,1H),9.00(d,J=9.2Hz,2H),8.43(d,J=12.6Hz,4H),8.19(d,J=4.2Hz,2 H),7.74(q,J=9.0,8.3Hz,5H),7.34-7.27(m,3H),7.09(s,1H),6.62(t,J=12.2Hz,2H),6.35(d,J=13.6Hz, 2H),4.23(s,4H),4.04(s,3H),3.94(s,3H),3.84(s,3H),2.96(d,J=6.6Hz,2H),2.32(d,J=7.4Hz,2H),2. 02(d,J=7.8Hz,2H),1.95(s,8H),1.87(s,12H),1.75(s,2H),1.56(d,J=8.1Hz,4H),1.33(t,J=7.0Hz,7H). 13 C NMR (176MHz, DMSO-d6): δ175.94,173.18,172.64,172.03,171.61,161.74,160.87,154.43,153.21,153.12,150.82,150.77,148.19,145.69,1 45.56,139.72,139.31,133.97(d,J=10.1Hz),133.53,132.56,132.02, 129.73,127.59,127.50,126.83,125.46,123.44,121.63,121.22,120. 91,118.91,117.42,113.41,111.14,110.14,109.15,107.70,107.57,1 07.33,105.10,104.92,102.81,102.40,97.87,57.44,56.72,56.21,50 .72,45.68,43.46,38.33,36.38,35.09,29.09,29.02,28.74,28.56,27.05,26.95,26.35,25.70,24.96,24.90,21.51,12.42.ESI-HRMS:calcd for C72 H 75 FN4O 19 S4 2- 724.3030; found 723.3754[M] 2- .

[0062] Example 3

[0063] This embodiment relates to a method for preparing a near-infrared fluorescent probe I-3 derived from 6,7,10-trimethoxy-3'-fluoro-α-naphthoflavone having a structural formula I, such as Figure 1 As shown, the following steps are included:

[0064] Step 1: Same as step 1 in Example 2

[0065] Step 2: Same as step 2 in Example 2

[0066] Step 3: Same as step 3 in Example 2

[0067] Step 4: The same as step 4 of Example 2, except that 9-((tert-butoxycarbonyl)amino)nonanoic acid was replaced with 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-oic acid to obtain compound 2-(2-(2-aminoethoxy)ethoxy-ethoxy)-N-(3-fluoro-5-(6,7,10-trimethoxy-α-naphthoflavone))acetamide IV-3, yield: 65.9%. 1 H NMR (400MHz, DMSO-d6): δ8.19(s,1H),7.80(t,J=8.3,2H),7.28(s,3H),7.06(s,1H),4.17(s,2H),4.04(s,3H),3.93(s ,3H),3.84(s,3H),3.72(dd,J=5.7,3.2Hz,2H),3.65(dd,J=5.9,3.2Hz,2H),3.60-3.51(m,6H),2.63(t,J=5.8Hz,2H). 13CNMR (176MHz, DMSO-d6): δ175.92, 169.11, 162.37 (d, J = 241.7Hz), 160.53, 154.40, 150. 76,150.71,148.08,140.50(d,J=11.4Hz),133.86(d,J=10.0Hz),121.19,120.86,117.3 1,113.48,113.26,109.92,109.49(d,J=25.8Hz),108.16(d,J=24.7Hz),107.18,97.79, 72.64,70.47,70.23,69.82,69.66,69.50,57.39,56.56,56.15,41.13.ESI-HRMS:calcd for C 30 H 34 FN2O9 + 585.2243; found 585.2248 [M+H] + .

[0068] Step 5: The same as step 5 of Example 1, except that IV-3 replaces IV-1 to obtain probe I-3, with a yield of 65.0%. 1 HNMR (400MHz, Methanol-d4): δ9.07(t,J=8.9Hz,2H),8.74(d,J=7.6Hz,2H),8.62(t,J=1.6Hz,1H),8.26(q,J=12.2Hz,3H),8.13(s,1H), 7.78(d,J=9.5Hz,2H),7.70(t,J=8.8Hz,2H),7.64(d,J=9.4Hz,1H),7.32(s,1H),7.12(s,2H),7.01(s,1H),6.45(s,1H),6.18(d,J=13.7H z,1H),6.09(d,J=13.7Hz,1H),4.19-4.24(m,2H),4.16(s,2H),4.02-4.07(m,5H),3.93(s,3H),3.82–3.63(m,9H),3.61(t,J=4.2Hz,2H), 3.50(d,J=5.4Hz,2H),3.13(s,2H),2.16(t,J=6.9Hz,2H),2.07–1.84(m,12H),1.76(s,2H),1.63(t,J=7.8Hz,2H),1.40(t,J=7.2Hz,5H). 13C NMR (176MHz, Methanol-d4): δ179.22,177.73,175.85,175.58,171.33,164.11,162.91,156.35,154.86,154.54,152.60,152.40,150.40,148.53,1 44.93,144.05,141.80,141.37,136.34,136.00,133.70(d,J=15.0Hz),13 0.61,129.67,128.82,127.46,123.31,123.15,122.12,118.69,117.27,1 16.76,115.20,114.36,113.58,113.42,111.30,111.11,110.84,107.92, 104.84,104.13,101.43,101.11,98.78,71.92,71.52,71.34,70.95,70.6 0,64.42,58.32,57.60,57.48,57.36,57.05,56.79,52.74,52.60,40.27, 36.57,28.18,27.90,27.79,27.23,26.31,22.35,12.83.ESI-HRMS:calcd for C 71 H 75 N4O 22 FS4 1482.3740; found1482.3707[M].

[0069] Example 4

[0070] The inhibitory activities of α-naphthoflavone derivatives III and IV obtained in Examples 1, 2 and 3 on CYP1A1, CYP1A2 and CYP1B1 enzymes were determined.

[0071] This experiment used the classic EROD test to determine its inhibitory activity and selectivity against CYP1A1, CYP1A2, and CYP1B1 enzymes. The total volume of the reaction system was 200 μL, which contained recombinant CYP1 enzyme (60 fmol CYP1A1, or 100 fmol CYP1A2, or 20 fmol CYP1B1), 7-ethoxy-3H-phenoxazine-3-one (150 nM), NADPH (0.54 mM), MgCl2 (3.3 mM), Tris-HCl buffer (50 mM, pH = 7.4, containing 1% BSA) and various concentrations of α-naphthoflavone derivatives (0.1, 1, 10, 50, 250, 500, 1000, 2000 nM). α-naphthoflavone was used as a positive control, and 3 replicate wells were set up for each experimental group or control group as parallel experiments. First, 50 μL of α-naphthoflavone derivatives and 75 μL of a mixture containing CYP enzymes and 7-ethoxy-3H-phenoxazine-3-one were added to a blank 96-well plate. DMSO was used instead of α-naphthoflavone derivatives in the control group. After incubation at 37°C for 5 minutes, 75 μL of NADPH regeneration solution was added to start the reaction. The reaction system containing CYP1A1 enzyme was incubated at 37°C for 15 minutes, the system containing CYP1A2 enzyme was incubated for 50 minutes, and the system containing CYP1B1 enzyme was incubated for 35 minutes. After the incubation, 100 μL of pre-cooled CH3OH was added to terminate the reaction. The fluorescence value was detected by a multifunctional microplate reader within 10 minutes, and the excitation and emission wavelengths were 545 nm and 590 nm, respectively. IC was calculated by nonlinear regression analysis using GraphPad Prism software. 50 The final IC 50 The results were the average of three repeated experiments.

[0072] The experimental results are shown in Table 1:

[0073] Table 1. Inhibitory activity and selectivity of III and IV against CYP1A1, CYP1A2 and CYP1B1 enzymes

[0074]

[0075] From the results in Table 1, it can be seen that compared with ANF, the inhibitory activity of the hydroxyl-containing derivative III-1 on the CYP1B1 enzyme has decreased, but it still remains at the nanomolar level. The compound IV-1 obtained by introducing the connecting chain has greatly improved the inhibitory activity and selectivity, especially the selectivity between CYP1A2 and 1B1, which has increased by more than 9000 times. The amino-containing derivative III-2 and the compounds IV-2 and IV-3 obtained by introducing the connecting chain have better activity in inhibiting the CYP1B1 enzyme than ANF, and also have high selectivity between CYP1A2 and 1B1. This also indicates that the probe synthesized based on compound IV has the ability to specifically target the CYP1B1 enzyme.

[0076] Example 5

[0077] This example will measure the UV and fluorescence spectra of the probe and the fluorescence stability of the probe. The steps are as follows:

[0078] The probe was diluted to a concentration of 1 μM with PBS and then transferred to a quartz cuvette. The maximum absorption spectrum of the probe was first measured using a UV-visible spectrophotometer, with a scanning wavelength range of 500-800 nm. The quartz cuvette was then transferred to a fluorescence spectrophotometer to measure the fluorescence spectrum, and the excitation wavelength was set according to the measured maximum UV absorption wavelength, with a scanning wavelength range of 600-800 nm. The experimental results are shown in the attached figure. Figure 2 As shown in a and 2b, the maximum fluorescence emission wavelengths of probes I-1, I-2, and I-3 are 698.4 nm, 696.6 nm, and 698.0 nm, respectively.

[0079] When measuring the fluorescence stability of the probe, the probe was diluted to a concentration of 1 μM with DMEM medium (containing 10% FBS) and incubated in a 37°C incubator. After incubation for 0 h, 2 h, 4 h, 6 h, 12 h and 24 h, the fluorescence spectrum was measured with a fluorescence spectrophotometer, with a scanning wavelength range of 685-800 nm. The excitation wavelength is the maximum ultraviolet absorption wavelength of the probe. The experimental results are shown in the attached Figure 2 As shown in c-2e, the probe has good fluorescence stability.

[0080] Example 6

[0081] In this example, the expression of CYP1B1 enzyme in different cells was determined by Western blot. The selected cell lines were normal human skin fibroblast HFF-1, tumor human colon cancer cell HCT-15, human breast cancer cell MDA-MB-231, and human lung cancer cell NCI-H1299. The steps are as follows:

[0082] The protein in the cells was extracted with RIPA lysis buffer (containing 1% PMSF protease inhibitor), and the protein was quantified using a BCA protein concentration assay kit. Then, 1 / 4 volume of SDS-PAGE protein loading buffer (5X) was added to the protein solution, and the protein was fully denatured by boiling in a 96°C water bath for 15 minutes, and then stored at low temperature after natural cooling.

[0083] Prepare 12% SDS-PAGE gel according to the instructions of the gel preparation kit, with a protein loading of 50 μg per well, and recombinant CYP1B1 protein as a control. Electrophoresis was performed at 80V for 30 minutes, and then at 120V for 75 minutes. After the electrophoresis, the gel was removed and the protein blot was transferred to a nitrocellulose membrane (NC membrane) using a wet transfer method. The transfer current was 300 mA for 120 minutes. After the transfer, the NC membrane was removed, washed with TBST washing solution, and then blocked with skimmed milk powder blocking solution at room temperature for 1 hour. According to the marker band, the NC membrane was cut open, and the membrane strips containing the internal reference protein were incubated with rabbit anti-GAPDH polyclonal antibodies (4000 times diluted with TBST), and the membrane strips containing the target protein were incubated with mouse anti-CYP1B1 monoclonal antibodies (1000 times diluted with TBST). During incubation, ensure that the membrane strips are completely immersed in the antibody working solution and incubate overnight at 4°C. After the primary antibody incubation, the membrane was washed three times with TBST washing solution, and the corresponding secondary antibody was added for incubation at room temperature for 70 minutes. The membrane strips containing the internal reference protein were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) (TBST diluted 5000 times), and the membrane strips containing the target protein were incubated with horseradish peroxidase-labeled mouse IgG1 binding protein (m-IgG1 BP) (TBST diluted 1000 times). After the secondary antibody incubation, the membrane was washed twice with TBST, then washed twice with TBS, and the ECL luminescent reagent was prepared and developed in an imager.

[0084] The experimental results are as attached Figure 3 As shown in the figure, CYP1B1 was expressed in tumor cells HCT-15, MDA-MB-231, and NCI-H1299, while CYP1B1 was not detected in normal cells HFF-1, further indicating that CYP1B1 enzyme is specifically highly expressed in tumor cells. Using GAPDH as an internal reference, the expression of CYP1B1 in HCT-15 cells is relatively high.

[0085] Example 7

[0086] This example measured the equilibrium dissociation constant Kd of the near-infrared fluorescent probes I-1, I-2, and I-3 with the CYP1B1 enzyme at the cellular level.

[0087] The colon cancer cells HCT-15 with high expression of CYP1B1 enzyme determined in Example 6 were plated at 2×10 5The cells were inoculated at a density of 1000 μg / cm2 into a six-well plate and cultured overnight to adhere to the wall. Experimental group, blocking control group and blank control group were set up. Different concentrations of probe dilution were added to the experimental group, and the probe and 5-fold concentration of blocker (Ⅳ) were added to the blocking control group at the same time. No probe was added to the blank control group. After incubation at 37°C for 1 hour, the culture medium was removed and the cells were washed three times with PBS. After trypsin digestion for 90 seconds, the cells were transferred to a centrifuge tube, and then the cells were collected by centrifugation (1000 r / min, 3 min), washed twice with PBS, and then resuspended in 800 μL PBS. The fluorescence intensity of the cells after incubation with different concentrations of probes was measured by flow cytometry, and the fluorescence values ​​of nonspecific binding at each concentration were deducted (blocking control group data). The probe concentration was used as the horizontal axis and the fluorescence intensity was used as the vertical axis. GraphPad Prism was used to draw the saturation binding curve and calculate the Kd value. The experimental results are shown in the attached figure. Figure 4 As shown, the Kd values ​​of probes I-1, I-2, and I-3 are 93.66 nM, 212.93 nM, and 168.00 nM, respectively, all at the nanomolar level, indicating that the probes synthesized by the present invention can bind to the CYP1B1 enzyme with high affinity and have the potential to be applied to in vivo tumor imaging.

[0088] Example 8

[0089] In this example, confocal microscopic imaging studies of near-infrared fluorescent probes I-1, I-2, and I-3 on colon cancer cells HCT-15 that highly express CYP1B1 enzyme and skin fibroblasts HFF-1 that do not express CYP1B1 enzyme were performed.

[0090] First, place the cell slides into the bottom of a 24-well plate, and then place the cells at 5×10 4 The density of each well was inoculated into a 24-well plate, and incubated overnight at 37°C to allow the cells to evenly adhere to the cell slide. An imaging group and a blocking group were set up. The cells in the imaging group were incubated with 0.5μM or 1.0μM probes, and the cells in the blocking group were incubated with 1.0μM probes and 5.0μM blocker (IV) at the same time. After incubation at 37°C for 1 hour, the culture medium was removed, the cells were washed 3 times with PBS, and 200μL 4% paraformaldehyde was added to fix the cells. After 10 minutes, wash twice with PBS, and add 100μL DAPI staining solution to stain the cell nuclei. After 5-10 minutes, wash with PBS to remove excess staining solution, take out the cell slide, and cover it upside down on a slide with anti-fluorescence quencher. Each group of cells was then imaged using a confocal microscope, and the imaging results are shown in the attached figure. Figure 5 In HCT-15 cells, the fluorescence intensity of the blocking group was significantly lower than that of the imaging group, while in HFF-1 cells, no fluorescence signal of the probe could be observed in both the imaging group and the blocking group, further indicating that probe I was enriched in the cell by specifically binding to the CYPIB1 enzyme.

[0091] Example 9

[0092] This example is an experiment to evaluate the near-infrared imaging ability and biodistribution of probe I-1 in living tumor-bearing mice (colon cancer cell HCT-15).

[0093] 5×10 6 HCT-15 cells were injected into the axilla of the right forelimb of BALB / C nude mice. When the tumor diameter reached 0.5-1 cm, the tumor-bearing mice were randomly divided into an imaging group and a blocking group (5 mice in each group). The mice in the imaging group were injected with probe I-1 (12 nmol / mouse, 100 μL / mouse) dissolved in PBS through the tail vein, and the mice in the blocking group were injected with a mixed solution (100 μL / mouse) of probe I-1 (12 nmol / mouse) and inhibitor α-naphthoflavone (20 mg / kg) intravenously. Near-infrared imaging of the two groups of mice was performed using IVIS small animal in vivo imaging equipment 2 hours, 4 hours, 6 hours, and 24 hours after injection. The experimental results are shown in the attached figure. Figure 6 As shown in the figure, the probe was enriched in the tumor site 2 hours after injection, and the tumor site was still clearly visible 24 hours later. The fluorescence intensity of the tumor site in the blocking group was lower than that in the imaging group, and the difference between the two groups was most significant at the 6th hour (P<0.001). This confirmed in vivo that the probe I-1 can indicate the tumor site by specifically binding to the CYP1B1 enzyme.

[0094] After the 24-hour imaging was completed, major organs and tissues (heart, liver, spleen, lung, kidney, intestine, tumor, and muscle) were immediately collected for ex vivo near-infrared imaging. Figure 7 As shown, the probe was mainly distributed in the tumor tissue, and the fluorescence intensity of the tumor tissue in the imaging group was significantly stronger than that in the blocking group (p<0.01), further confirming that probe I-1 can enrich in the tumor tissue by specifically binding to the CYP1B1 enzyme, thereby enabling tumor imaging.

[0095] Example 10

[0096] This example is an experiment to evaluate the in vivo photoacoustic imaging ability of probe I-1 in tumor-bearing mice (colon cancer cells HCT-15).

[0097] In order to further expand the application scope of the probe, the photoacoustic imaging ability of the probe was evaluated. The construction of the tumor-bearing mouse model was the same as in Example 9. The mice were divided into an imaging group and a control group. The imaging group was injected with probe I-1 dissolved in PBS (12nmol / mouse, 100μL / mouse), and the control group was injected with the same dose of PBS. Photoacoustic imaging was performed on the two groups of mice using a photoacoustic imaging system 2 hours, 4 hours, 6 hours, and 24 hours after injection. The results are shown in the attached figure. Figure 8As shown, in the imaging group, the photoacoustic signal was widely distributed in the tumor epidermis and inside, while in the PBS control group, only a small amount of photoacoustic signal was found in the tumor epidermis. The probe was enriched in the tumor site 2 hours after injection, and then the probe gradually diffused into the tumor. Photoacoustic signals could still be observed after 24 hours. At all time points, the photoacoustic signals in the imaging group were significantly different from those in the control group (p<0.01), indicating that probe I-1 can specifically target tumors and has good photoacoustic imaging effects, and has the potential to be used for tumor photoacoustic imaging.

[0098] In summary, this patent designs and synthesizes a new near-infrared fluorescent probe I that specifically targets the CYP1B1 enzyme based on the newly discovered co-crystal structure of ANF and CYP1B1. The evaluation of imaging capabilities at the cellular level showed that the probe can bind to CYP1B1 with high affinity and be enriched in tumor cells. In the subsequent in vivo imaging capability evaluation, probe I-1 was significantly enriched in the tumor site under both near-infrared imaging and photoacoustic imaging modalities, showing good tumor site imaging capabilities. The research results show that the new probe I designed by the present invention has the ability to specifically target the CYP1B1 enzyme and image tumors, which provides a reference for promoting the application of molecular probes in clinical tumor diagnosis.

[0099] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A near-infrared fluorescent probe targeting CYP1B1 enzyme, characterized in that: The structural formula of the fluorescent probe is shown in Formula I: Formula I; wherein, X = O or X = NH, located at the 4' position or the 5' position, Y is a connecting chain, and the near-infrared fluorescent molecule is Cy5.5; when X = O, Y is n=1, 2, 3; when X = NH, Y is n=1, 2, 3 or Y is n=2, 3, 4.

2. The near-infrared fluorescent probe targeting CYP1B1 enzyme according to claim 1, characterized in that: The structural formula of the fluorescent probe is shown in Formula I: Formula I; wherein, X = O or X = NH, located at the 5' position, Y is a connecting chain, and the near-infrared fluorescent molecule is Cy5.5; when X = O, Y is n = 3; when X = NH, Y is n=3 or Y is n=4.

3. A method for preparing a near-infrared fluorescent probe targeting CYP1B1 enzyme according to claim 1 or 2, characterized in that: The following steps are involved: A. The X group is protected to obtain compound V ; In step A, P in the compound V is a protecting group; wherein, when X = O, the reaction is carried out in the presence of MOMCl, P = MOM; when X = NH, the reaction is carried out in the presence of (Boc)2O, P = Boc; B. and Reaction to generate compound II ; The reaction of step B is carried out in the presence of DMAP and EDC·HCl; C. Rearrange and cyclize compound II and generate derivative III under acidic conditions ; The reaction of step C is carried out in the presence of t-BuOK and concentrated sulfuric acid; D. Introduce a connecting chain into derivative III to generate derivative IV ; When X = O, the reaction of step D is carried out in the presence of Cs2CO3 and hydrochloric acid; when X = NH, the reaction of step D is carried out in the presence of HATU and DIPEA and hydrochloric acid; In step D, when X = O, the connecting chain is a chain containing multiple ethylene glycol fragments. n = 1, 2, 3, forming an ether bond with derivative III, and removing the Boc protecting group to obtain derivative IV; when X = NH, the connecting chain contains multiple ethylene glycol fragments n = 1, 2, 3 or contains multiple alkyl groups n=2, 3, 4, forming an amide bond with derivative III, and removing the Boc protecting group to obtain derivative IV; E. The derivative IV reacts with water-soluble Cy5.5-NHS to obtain the near-infrared fluorescent probe.

4. Use of the near-infrared fluorescent probe targeting CYP1B1 enzyme according to claim 1 in the preparation of tumor diagnostic reagents.

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