A probe prodrug and probe for detecting OX-LDL and its application in detecting vascular atherosclerosis

Abstract

The present invention discloses a probe prodrug for detecting OX-LDL and a method for preparing the probe and its application to early detection of vascular atherosclerosis. The prepared organic fluorescent material is applied to a macrophage model of atherosclerosis. Since the positive charge of the probe prodrug can be combined with the negative charge in the inner membrane of the mitochondria, the aggregation of MIT-FP in the inner membrane of the mitochondria can be achieved. Due to the special mitochondrial peroxidation environment of the model, the positively charged MIT-FP will be hydrolyzed by HOCl in the mitochondria, releasing the probe FP with a hydrophobic structure. The FP molecule is very easy to combine with OX-LDL in the cytoplasmic matrix, emitting green fluorescence, achieving the purpose of atherosclerosis model detection, and providing a clever method for realizing early detection of atherosclerosis and oxidation evaluation of lipoproteins in the future. The design of the prodrug improves the recognition effect of the probe FP molecule, reduces the interference of the background signal, and also provides a "dual targeting" strategy at the same time.

Description

Technical Field

[0001] The present invention relates to the field of early diagnosis of atherosclerosis, and is a material for a probe prodrug which utilizes the mitochondrial peroxidation environment to activate and specifically target OX-LDL, an early marker of atherosclerosis, and a preparation method thereof. Background Art

[0002] Cardiovascular disease is the leading cause of death worldwide, most commonly due to atherosclerosis leading to adverse clinical events such as myocardial infarction and stroke. Atherosclerosis is a complex and progressive disease characterized by abnormal lipid deposition and abnormal inflammatory response. Numerous studies have shown that atherosclerosis is caused by abnormal disposal of cholesterol-laden low-density lipoprotein (LDL) in the arterial wall, which is the result of endothelial dysfunction or even endothelial dysfunction in the presence of hyperlipidemia and hypertension [see: J. BMC Med., 2013, 11, 13; JB Su World J. Cardiol., 2015, 7, 719-741.]. LDL in the arterial endothelium is easily oxidized into oxidized low-density lipoprotein (OX-LDL), accompanied by mild inflammation, which can activate innate and adoptive immune responses. Therefore, smooth muscle cells and endothelial cells in the vascular wall secrete chemical attractants and adhesion factors (such as vascular cell adhesion molecule-1 [VCAM-1], P and E-selectin) to promote the recruitment of leukocytes to endothelial cells, such as monocytes and T lymphocytes [see: C. Cochain and A. Zernecke, Pflugers Arch., 2017, 469, 485-499.]. Macrophage colony stimulating factor (M-CSF) drives monocytes to differentiate into macrophages that contain overexpressed scavenger receptors (SR), such as SR-A, SR-B1, CD36 and LDLs receptor-1 (LOX-1) [see: OL Francone, L. Royer, G. Boucher, M. Haghpassand, A. Freeman, D. Brees and R. J. Aiello, Arterioscler., Thromb., Vasc. Biol., 2005, 25, 1198-1205.]. Macrophages internalize and oxidize LDL to form foam cells containing a large number of lipid droplets, produce inflammatory factors, and further enhance the retention of LDL and the differentiation of T lymphocytes and B lymphocytes [see: F. Abdolmaleki, SM Gheibi Hayat, V. Bianconi, TP Johnston and A. Sahebkar, Trends Cardiovasc. Med., 2019, 29, 363-371.]. These immune cells, together with lipids, form atherosclerotic plaques, in which lipids and other tissue factors derived from necrotic and apoptotic foam cells constitute a vulnerable necrotic core rich in hydrophobic lipids. The growth factors and reactive oxygen species (ROS) released by these cells stimulate smooth muscle cell migration and collagen deposition, leading to the development of atherosclerotic plaques.

[0003] Macrophages are a large heterogeneous subpopulation that can be differentiated into different functional subpopulations according to their exposure to the local microenvironment [see: S. Gordon and A. P. M. Estrada, Immunol. Rev., 2014, 262, 207.]. In general, macrophages can be subdivided into pro-inflammatory M1 and anti-inflammatory M2 phenotypes. M1 macrophages gather in the shoulder, the most vulnerable area of ​​the plaque, and the necrotic core, while macrophages in stable plaques mainly express M2 markers [see: S. Yang, H. Q. Yuan, Y. M. Hao, Z. Ren, S. L. Qu, L. S. Liu, D. H. Wei, Z. H. Tang, J. F. Zhang and Z. S. Jiang, Clin. Chim. Acta, 2020, 501, 142-146.]. M1 and M2 macrophages coexist in the fibrous cap, where M2 macrophages promote fibrosis and damage repair to counteract inflammatory M1 macrophages, thereby improving plaque stability. M1 macrophages are dominant in unstable plaques, while the M2 phenotype is more abundant in stable plaques due to its inherent anti-inflammatory properties [see: H. Mangge and G. Almer, Molecules, 2019, 24, 1-13.]. Therefore, distinguishing between M1 and M2 cells also helps to distinguish between severe atherosclerotic cells and mild or anti-inflammatory atherosclerotic cells.

[0004] In addition, mitochondria produce superoxide anions at physiological levels, and may become pathological due to excessive ROS (such as H2O2 and HClO, etc.) due to mitochondrial dysfunction or due to the failure of antioxidant mechanisms. Accelerated atherosclerosis and elevated mitochondrial ROS can be seen in experiments involving the loss of the antioxidant system in ApoE-KO mice, indicating that mitochondrial ROS plays an important role in the formation of atherosclerosis [see: Madamanchi NR, Runge MS. Mitochondrial dysfunction in atherosclerosis. Circ Res. 2007; 100 (4): 460-473.].

[0005] Therefore, it is of great scientific and practical significance to design materials for early detection of atherosclerosis based on the unique ROS and highly hydrophobic atherosclerotic plaque environment of atherosclerosis. Summary of the invention

[0006] The present invention is to design a method and application for early detection of atherosclerosis, and provide a material and a preparation method thereof that can realize early detection of OX-LDL, an early marker of atherosclerosis.

[0007] The present invention cleverly designs an organic fluorescent molecular rotor MIT-FP, which has almost no fluorescence in the blood circulation system at pH 7.4. The borate functional group gives the probe prodrug MIT-FP the property of being easily oxidized in the peroxidation environment of atherosclerosis, thereby releasing the probe molecule FP that responds to OX-LDL. In the cells of the atherosclerosis model, the compound can target the mitochondrial environment of the cell and undergo a series of oxidation reactions in the mitochondria, and finally generate a hydrophobic probe molecule FP that is free to the cytoplasmic matrix. The probe molecule FP then combines with the highly hydrophobic OX-LDL in the cytoplasmic matrix, releasing strong green fluorescence, thereby selectively identifying the early atherosclerosis model, greatly improving the recognition effect of the probe molecule FP, and reducing the interference of background signals.

[0008] In order to solve the technical problem of the present invention, the proposed technical solution is: a probe prodrug having the following structure:

[0009]

[0010] The reaction route is as follows:

[0011] Glycine tert-butyl ester hydrochloride is dispersed in tert-butyl alcohol, and N-ethylcarbazole-3-carboxaldehyde is added to continue the reaction. Then the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester is prepared and added, and stirring is continued. After the reaction is completed, the mixture is extracted with water and CH2Cl2. The organic layer is dried over anhydrous sodium sulfate, and the solvent is removed by a rotary evaporator. The residue is purified by column chromatography to obtain a yellow solid.

[0012] The intermediate tert-butyl 2-(1-isopropoxymethyleneamino)acetate needs to be prepared and used immediately, and is synthesized by the following method: dissolving glycine tert-butyl hydrochloride, ethyl acetimidate hydrochloride, and sodium carbonate in a mixed solution of water / ether, followed by high-speed stirring. After the reaction is completed, the ether phase is collected, dried over anhydrous sodium sulfate, and the solvent is removed to obtain a white oily liquid.

[0013] Then, the yellow solid was mixed with pyridine-4-carboxaldehyde and refluxed for 10-15 hours in the presence of a catalyst. The obtained red solution was extracted with water and CH2Cl2, dried over anhydrous magnesium sulfate, and purified by silica gel chromatography to obtain a red solid.

[0014] Finally, the red solid was mixed with 4-bromomethylphenylboronic acid pinacol ester, and acetonitrile was used as a solvent at room temperature. The reaction was carried out overnight to obtain a purple solution, which was filtered and washed with acetonitrile, petroleum ether and cyclohexane to obtain a dark purple solid.

[0015] The synthetic route is as follows:

[0016]

[0017] In order to solve the technical problem of the present invention, another technical solution is proposed: a probe molecule, characterized in having the following structure:

[0018]

[0019] The method comprises the following steps: dispersing glycine tert-butyl ester hydrochloride in tert-butyl alcohol, adding 4-formyl-pyridine to continue the reaction. Then preparing and adding the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester, and continuing stirring. After the reaction is completed, the mixture is extracted with water and CH2Cl2. The organic layer is dried over anhydrous sodium sulfate, and then the solvent is removed by a rotary evaporator. The residue is purified by column chromatography to obtain a yellow solid.

[0020] The intermediate tert-butyl 2-(1-isopropoxymethyleneamino)acetate needs to be prepared and used immediately, and is synthesized by the following method: dissolving glycine tert-butyl hydrochloride, ethyl acetimidate hydrochloride, and sodium carbonate in a mixed solution of water / ether, followed by high-speed stirring. After the reaction is completed, the ether phase is collected, dried over anhydrous sodium sulfate, and the solvent is removed to obtain a white oily liquid.

[0021] Then, the yellow solid was mixed with pyridine-4-carboxaldehyde and refluxed for 10-15 hours in the presence of a catalyst. The obtained red solution was extracted with water and CH2Cl2, dried over anhydrous magnesium sulfate, and purified by silica gel chromatography to obtain a red solid.

[0022] The reaction route is as follows:

[0023]

[0024] In order to solve the technical problem of the present invention, another technical solution is proposed: a pharmaceutical composition comprising the probe prodrug MIT-FP or probe FP and a pharmaceutically acceptable carrier thereof.

[0025] In order to solve the technical problem of the present invention, another technical solution is proposed: a pharmaceutical composition, which is composed of the active ingredient of the probe prodrug MIT-FP or probe FP and pharmaceutically acceptable pharmaceutical excipients to form a clinically applicable spray, lyophilized powder injection, and kit.

[0026] In order to solve the technical problem of the present invention, another technical solution is proposed: using tert-butyl alcohol as a reaction solvent and the synthesis application of reaction conditions such as the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester.

[0027] The example is as follows: Synthesis of carbazole-based FP: Glycine tert-butyl ester hydrochloride is dispersed in tert-butyl alcohol, and N-ethylcarbazole-3-carboxaldehyde is added to continue the reaction. Then, the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester is prepared and added, and stirring is continued. After the reaction is completed, the mixture is extracted with water and CH2Cl2. The organic layer is dried over anhydrous sodium sulfate, and the solvent is removed by a rotary evaporator. The residue is purified by column chromatography to obtain a yellow solid.

[0028] The intermediate tert-butyl 2-(1-isopropoxymethyleneamino)acetate needs to be prepared and used immediately, and is synthesized by the following method: dissolving glycine tert-butyl hydrochloride, ethyl acetimidate hydrochloride, and sodium carbonate in a mixed solution of water / ether, followed by high-speed stirring. After the reaction is completed, the ether phase is collected, dried over anhydrous sodium sulfate, and the solvent is removed to obtain a white oily liquid.

[0029] The invention discloses an application of the probe prodrug MIT-FP or the probe molecule FP in the preparation of HDL (high-density lipoprotein), LDL (low-density lipoprotein), OX-LDL (oxidized low-density lipoprotein), and relates to early diagnostic reagents and therapeutic drugs for atherosclerosis.

[0030] A use of the probe prodrug MIT-FP or the probe molecule FP in the preparation of lipoprotein (HDL, LDL, OX-LDL) oxidation evaluation.

[0031] Beneficial effects of the present invention

[0032] The invention discloses a preparation method and application of a probe for early detection of atherosclerosis. A probe prodrug MIT-FP and a probe FP are prepared by organic synthesis, and the method relates to the field of early detection of atherosclerosis. The prepared organic fluorescent material is applied to the macrophage RAW264.7 cell model of atherosclerosis. Since the positive charge of the probe prodrug can be combined with the negative charge in the mitochondrial inner membrane, the aggregation of MIT-FP in the mitochondrial inner membrane can be achieved. Due to the special mitochondrial peroxidation environment of the model, the positively charged MIT-FP will be hydrolyzed by HOCl in the mitochondria to release the probe FP with a hydrophobic structure. The FP molecule is very easy to combine with OX-LDL (oxidized low-density lipoprotein) in the cytoplasmic matrix, emitting green fluorescence, achieving the purpose of atherosclerosis model detection, and providing a clever method for realizing early detection of atherosclerosis and oxidation evaluation of lipoproteins in the future. The design of the prodrug improves the recognition effect of the probe FP molecule, reduces the interference of the background signal, and also provides a "dual targeting" strategy.

[0033] Compared with the prior art, the present invention has the following significant advantages: a new "dual-targeted" probe prodrug that can be activated by an oxidative environment and responds to viscosity is invented. After oxidative cleavage, it can bind to highly hydrophobic OX-LDL and release strong green fluorescence, thereby selectively identifying early atherosclerosis models, greatly improving the recognition effect of the probe FP molecule and reducing the interference of background signals. It is an excellent probe prodrug material.

[0034] Activatable photosensitizer prodrugs can utilize the special oxidative environment of atherosclerosis and the characteristics of specific highly hydrophobic OX-LDL to selectively activate fluorescence at the lesion. This strategy can not only achieve the targeting of OX-LDL at atherosclerosis, but also reduce the interference of background fluorescence during imaging. For example, the upregulation of traditional reactive oxygen species can lead to a series of biological pathological processes, such as cancer, inflammation and other diseases. In addition, for the increase of reactive oxygen species caused by inflammation in the organism, traditional single reactive oxygen probes can lead to false positives and high background fluorescence results. The formation of atherosclerosis originates from the increase of intracellular reactive oxygen species in the early stage, and the peroxidative environment further promotes the generation of OX-LDL (see: Kattoor, AJ, Pothineni, NVK, Palagiri, D. et al. Oxidative Stress in Atherosclerosis. Curr Atheroscler Rep 19, 42 (2017).). Therefore, the "dual response" recognition mechanism of the probe prodrug MIT-FP to the above-mentioned markers of reactive oxygen species and OX-LDL will avoid the above process.

[0035] In the present invention, the characteristics of oxidized cells and high OX-LDL in atherosclerosis can be used to distinguish pathological tissues from normal tissues. The borate ester part can specifically recognize HOCl in the cell to produce a probe molecule FP, which is further transferred to the cytoplasm and combined with OX-LDL to produce green fluorescence. The recognition process of the probe prodrug MIT-FP makes good use of the pathological characteristics of high oxidized and highly hydrophobic OX-LDL in atherosclerosis, thereby achieving a better recognition effect. In addition, the probe is insensitive to other related factors of the cell such as pH, anions, cations, thiols, etc., and has good selectivity and specificity.

[0036] In summary, the probe prodrug MIT-FP in the present invention can achieve the "dual response" function of HOCl and OX-LDL for coronary artery plaques at the cellular and in vivo levels, and the material has almost no toxicity in the blood circulation system, further reducing the damage to normal tissues and reducing the side effects on the body. Therefore, the probe prodrug MIT-FP can achieve the effect of specifically identifying cells in the atherosclerosis model. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The probe FP of the present invention 1 H NMR (500 MHz, DMSO-d6) spectrum;

[0038] Figure 2 The probe FP of the present invention 13 C NMR (500 MHz, DMSO-d 6 ) spectrum;

[0039] Figure 3 is a high-resolution mass spectrum of the probe FP of the present invention;

[0040] Figure 4 The probe prodrug MIT-FP of the present invention 1 H NMR spectrum;

[0041] Figure 5 The probe prodrug MIT-FP of the present invention 13 C NMR (500 MHz, DMSO-d 6 ) spectrum;

[0042] Figure 6 This is a high-resolution mass spectrum of the probe prodrug MIT-FP of the present invention;

[0043] Figure 7 This is a mechanism description of the probe prodrug MIT-FP of the present invention;

[0044] Figure 8 The probe prodrug MIT-FP of the present invention is HOCl / ClO - and UV absorption spectra of OX-LDL response;

[0045] Fig. 9 The probe prodrug MIT-FP of the present invention is added with 1.2 equivalents of HOCl / ClO - Later OX-LDL titration fluorescence spectra;

[0046] Fig.10 The probe prodrug MIT-FP of the present invention is added with 1.2 equivalents of HOCl / ClO - The fluorescence quantum yield of subsequent OX-LDL titrations;

[0047] Fig.11 The fluorescence spectra of the probe FP of the present invention in equal amounts of HDL, LDL, OX-LDL and cholesterol solutions of different concentrations;

[0048] Fig.12 This is a selectivity experiment of the probe FP of the present invention in different proteins;

[0049] Fig.13 The probe prodrug MIT-FP of the present invention reacts with HOCl / ClO in water and glycerol respectively. - Fluorescence spectra before and after response;

[0050] Fig.14 The probe prodrug MIT-FP of the present invention is added with 1.2 equivalents of HOCl / ClO - Later visco titration fluorescence spectroscopy;

[0051] Fig.15 The fluorescence spectra of the probe prodrug MIT-FP of the present invention vary with pH in different viscosity systems;

[0052] Fig.16 The probe prodrug MIT-FP of the present invention is added with 1.2 equivalents of HOCl / ClO - Fluorescence lifetime spectra of subsequent viscometric titrations;

[0053] Fig.17 The probe prodrug MIT-FP of the present invention is added with HOCl / ClO in pure water containing 90% glycerol by volume. - Later titration fluorescence lifetime spectra;

[0054] Fig.18 The probe prodrug MIT-FP of the present invention is added with 1.2 equivalents of HOCl / ClO in pure water containing 90% glycerol by volume. - The fluorescence lifetime spectrum that changes with pH later;

[0055] Fig.19 The time-varying fluorescence spectrum of the probe prodrug MIT-FP of the present invention in an aqueous solution containing 50% ethanol;

[0056] Fig. 20 This is a test of the response of the probe prodrug MIT-FP of the present invention to solutions of different polarities;

[0057] Fig.21 The mass spectrometry mechanism of the probe prodrug MIT-FP of the present invention;

[0058] Fig. 22 The recognition effect of the probe prodrug MIT-FP of the present invention in cells;

[0059] Fig.23 The probe prodrug MIT-FP of the present invention has a distinguishing effect on M0, M1 and M2 cells;

[0060] Fig.24 In vivo fluorescence imaging of arterial plaques in an early atherosclerosis mouse model using the probe prodrug MIT-FP of the present invention;

[0061] Fig.25 Confocal fluorescence imaging of the probe prodrug MIT-FP of the present invention at the arterial plaque tissue of an early atherosclerosis mouse model;

[0062] Fig.26 This is the bright field imaging of the probe prodrug MIT-FP of the present invention at the arterial plaque tissue of the early atherosclerosis mouse model. DETAILED DESCRIPTION

[0063] Example 1: A method and application for early detection of atherosclerosis, and a material capable of achieving early detection of atherosclerosis and a preparation method thereof. The synthesis scheme is as follows:

[0064] Glycine tert-butyl ester hydrochloride (1.0 eq.) and sodium hydroxide (1.1 eq.) were added to tert-butyl alcohol and stirred at room temperature until the sodium hydroxide was completely dissolved. Then N-ethylcarbazole-3-carboxaldehyde (1.0 eq.) was added and stirred at room temperature overnight. Then the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester (1.3 eq.) was prepared and added, and the mixture was stirred at room temperature overnight. The mixture was then extracted with water and CH2Cl2. The organic layer was dried over anhydrous sodium sulfate and the solvent was removed by a rotary evaporator. The residue was purified by column chromatography using petroleum ether: ethyl acetate (3:1) as eluent to obtain a yellow solid. Then, the yellow solid (1.0 eq.) was mixed with pyridine-3-carboxaldehyde (1.0 eq.), and 1,4-dioxane was used as solvent. Under the catalysis of acetic acid and piperidine, the reaction was refluxed at 100°C using a pressure tube, and a silica gel chromatography plate was used to monitor the reaction. After the reaction, it was extracted with water and CH2Cl2, dried over anhydrous magnesium sulfate, and purified by a silica gel chromatography column using petroleum ether: ethyl acetate (3:1) as solvent. After purification, recrystallization was performed to obtain a red solid. Probe FP 1 H NMR spectrum ( Figure 1 ): 1H NMR (400MHz, CDCl3) δ8.92(s,1H),8.69(d,J=4.6Hz,2H),8.52(d,J=8.7Hz,1H),8.16(d,J=7.7Hz,1H),7.98(dd,J=15.8,2.8Hz,1H),7 .60–7.37(m,6H),6.87(dd,J=15.8,1.9Hz,1H),4.48(d,J=1.7Hz,2H),4.39(dd,J=7.2,3.0Hz,2H),1.48(s,9H),1.45(d,J=1.6Hz,3H). Probe FP 13 CNMR spectrum( Figure 2 ): 13 C NMR (126MHz, CD3CN)δ170.09,166.93,156.11,150.76,142.60,141.65,140.62,136.79,136.38,131.83,131.08,126.48,126.39,125.81,123.75,123.29,121.60,120.91,120.08,117.91,109.12,83.37,42.40,37.95,28.11,13.99. High resolution mass spectrometry ( Figure 3 ): calculate m / z[M+H] + is 507.2391, measured 507.2384.

[0065] Finally, the red solid (1.0 eq.) was mixed with 4-bromomethylphenylboronic acid pinacol ester (1.1 eq.), and acetonitrile was used as solvent at room temperature to react overnight to obtain a purple solution, which was filtered and washed with a mixture of acetonitrile, petroleum ether, and cyclohexane to obtain a dark purple solid probe prodrug MIT-FP. 1 H NMR spectrum ( Figure 3 ): 1 H NMR (400MHz, DMSO-d6) δ9.25 (d, J = 6.4 Hz, 2H), 9.16 (s, 1H), 8.58 (t, J = 8.8 Hz, 3H), 8.38–8.20 (m, 1H), 7.91 (d, J = 15.8 Hz, 1H), 7.80–7.67 (m, 4H), 7.57 (d, J = 7.5 Hz, 3H), 7.47 (s, 1H), 7.32 (t, J = 7.5 Hz, 1H), 5.87 (s, 2H), 4.71 (s, 2H), 4.52 (d, J = 6.9 Hz, 2H), 1.43 (s, 9H), 1.37 (t, J = 7.0 Hz, 3H), 1.29 (s, 12H).13 C NMR spectrum ( Figure 5 ): 13 C NMR (126MHz, DMSO) δ 169.17, 167.16, 156.22, 151.02, 144.90, 141.25, 140.18, 137.43, 136.13, 135.16, 132.69, 131.20, 129.52, 128.15, 126.60, 126.26, 125.95, 125.19, 122.91, 122.28, 120.91, 119.99, 109.81, 83.88, 82.13, 62.59, 42.07, 37.31, 27.65, 24.62, 13.77. High resolution mass spectrometry ( Figure 6 ): calculate m / z[M+H] + is 724.3785, measured 724.3705.

[0066] Example 2: The above-mentioned probe prodrug MIT-FP capable of targeting atherosclerosis model cells is first activated in mitochondria and then embedded in OX-LDL ( Figure 7 ).

[0067] The prepared organic fluorescent material was applied to the RAW264.7 cell model of atherosclerosis. Since the positive charge of the probe prodrug MIT-FP can combine with the negative charge in the mitochondrial inner membrane, the probe prodrug MIT-FP can be aggregated in the mitochondrial inner membrane. Due to the special mitochondrial peroxidation environment of the model, the positively charged probe prodrug MIT-FP will be hydrolyzed by HOCl in the mitochondria to obtain electrons, activate and release the viscosity-sensitive molecular rotor, and become a neutral probe FP, which is then transferred to the cytoplasmic matrix. Since the hydrolyzed molecule has an obvious more hydrophobic structure, the molecule is very easy to bind to the overexpressed and hydrophobic OX-LDL in the cytoplasmic matrix, and the internal rotation of the recognition molecule embedded therein is hindered, resulting in the restriction of the non-radiative transition of the molecule, thereby increasing the radiative transition of the molecule, and then releasing green fluorescence, thereby achieving the purpose of atherosclerosis model detection, providing an ingenious method for the future early detection of atherosclerosis.

[0068] Example 3: HOCl / ClO of the above-mentioned probe prodrug MIT-FP capable of targeting atherosclerosis model cells and activating in mitochondria - UV absorption spectrum ( Figure 8 ).

[0069] First, an aqueous solution containing 1.2 equivalents of HOCl probe prodrug MIT-FP was prepared, and then different concentrations of OX-LDL were added to it to prepare a 10 μmol solution, and the changes in UV absorption after the addition of OX-LDL were recorded. - Afterwards, the pyridinium salt in the probe prodrug MIT-FP molecule breaks, followed by a rearrangement of the charge within the molecule, resulting in a significant blue shift in the spectrum (~60 nm).

[0070] Example 4: Fluorescence spectrum of the above-mentioned probe prodrug MIT-FP that can target the characteristic marker of atherosclerosis OX-LDL ( Fig. 9 ).

[0071] The probe prodrug MIT-FP was tested in a pure water system after adding HOCl / ClO - The recognition effect of OX-LDL can be seen by adding HOCl / ClO into the pure water system. - After that, the fluorescence intensity of the system hardly changed. However, after adding OX-LDL, the fluorescence intensity of the probe prodrug MIT-FP at 565nm increased significantly. The fluorescence intensity before and after the reaction changed by about 700 times, and the absolute fluorescence quantum yield changed by about 500 times ( Fig.10 ). This indicates that the probe prodrug MIT-FP can be - After activation, it has a significant recognition effect on OX-LDL.

[0072] Example 5: Fluorescence spectra of the probe FP of the present invention in equal amounts of HDL, LDL, OX-LDL and cholesterol solutions of different concentrations ( Fig.11 ).

[0073] Considering that the probe will be used to evaluate different oxidation degrees of lipoproteins, the probe was tested for fluorescence spectroscopy in solutions containing equal amounts of HDL, LDL, and OX-LDL. The results showed that as the degree of lipoprotein oxidation gradually increased, its fluorescence signal also gradually increased. In addition, it was found that even high concentrations of cholesterol (10 mmol) could not cause significant changes in the fluorescence signal of FP, which also indicated that the selective effect of probe FP on lipoproteins was not affected by cholesterol in environmental cells.

[0074] Example 6: Protein selectivity experiment of the above-mentioned probe prodrug MIT-FP ( Fig.12 ).

[0075] The recognition effect of the probe prodrug MIT-FP in OX-LDL and other dozen protein solutions was tested in a pure water system. It can be seen that after adding OX-LDL to the pure water system, the fluorescence intensity of the system hardly changed. However, after adding OX-LDL, the fluorescence intensity of the probe prodrug MIT-FP at 565nm increased significantly. The fluorescence intensity before and after the reaction changed by about 700 times, and the absolute fluorescence quantum yield changed by about 500 times. This shows that the probe prodrug MIT-FP can be used in the presence of HOCl / ClO - After activation, it has a significant recognition effect on OX-LDL.

[0076] Example 7: Fluorescence spectra of the above-mentioned probe prodrug MIT-FP before and after recognition in glycerol and pure water ( Fig.13 ).

[0077] The probe prodrug MIT-FP was tested for HOCl / ClO in a pure water system and a pure water system containing 90% glycerol. - The recognition effect can be seen by adding HOCl / ClO into the pure water system. - After that, the fluorescence intensity of the system hardly changed. However, in the pure water system containing 90% glycerol, the fluorescence intensity of the probe prodrug MIT-FP was only slightly enhanced at around 650nm. However, after adding HOCl / ClO - Afterwards, the fluorescence intensity of the probe prodrug MIT-FP at 565nm increased significantly, and the fluorescence intensity before and after the reaction changed by about 124 times, and the absolute fluorescence quantum yield changed by about 148 times. This shows that the probe prodrug MIT-FP can be - After activation, it has an obvious recognition effect on the viscosity change of the system.

[0078] Example 8: The above-mentioned probe prodrug MIT-FP is added with HOCl / ClO - Visco titration fluorescence spectrum after reaction ( Fig.14 ).

[0079] Similarly, in order to explore the sensitivity of the probe prodrug MIT-FP to viscosity, the probe prodrug MIT-FP was tested as follows: the same concentration of HOCl / ClO was added to glycerol / water solutions with different water contents. - The fluorescence spectra were tested respectively. The results showed that as the proportion of glycerol in the system gradually increased, the fluorescence signal of MIT-FP gradually increased, indicating that the probe prodrug MIT-FP could be oxidized by HOCl / ClO - It has good viscosity response after activation.

[0080] Example 9: Fluorescence spectra of the above-mentioned probe prodrug MIT-FP changing with pH in different viscosity systems ( Fig.15 ).

[0081] In this experiment, the pH-dependent fluorescence response of MIT-FP was measured. After excitation at 470nm, the emission spectrum of MIT-FP was recorded to show a maximum value near 590nm. The results show that MIT-FP has a good recognition effect only when the viscosity of the system increases and the pH is 7-10. As the pH decreases from 6 to 4, the fluorescence intensity of the system after the reaction decreases, which may be attributed to the reaction of HOCl with ClO in a more acidic system. - The HOCl content in the solution gradually increased, and the oxidized form of HOCl was - The pH value of the probe prodrug is low, which results in a relative decrease in fluorescence emission at a lower pH. The normal physiological pH environment of cells is about 6-9, and the probe prodrug can work normally within this range, indicating that the probe prodrug has good stability.

[0082] Example 10: The above-mentioned probe prodrug MIT-FP is added with HOCl / ClO - The fluorescence lifetime spectrum of the subsequent viscosity titration ( Fig.16 ).

[0083] For the above-mentioned probe prodrug MIT-FP, 1.2 equivalents of HOCl / ClO were first used. - After pretreatment, the fluorescence lifetime change under different viscosities was tested. The results showed that the fluorescence lifetime of MIT-FP increased significantly in high viscosity solutions. The time-resolved fluorescence decay curve was fitted with a double exponential decay function, and the average lifetime increased from 0.84ns to 2.46ns. This helps to use the fluorescence lifetime to calibrate the viscosity value of the system.

[0084] Example 11: Fluorescence lifetime spectrum of the above-mentioned probe prodrug MIT-FP in pure water containing 90% glycerol by volume ( Fig.17 ).

[0085] Due to the HOCl / ClO - The level of HOCl / ClO may cause changes in fluorescence intensity. Therefore, in order to eliminate this effect, a fluorescence lifetime control experiment is required. Here, a 90% HOCl / ClO - The titration experiment showed that as HOCl / ClO - The probe lifespan barely changes with the increase of HOCl / ClO concentration, indicating that the probe lifespan is almost unaffected by the HOCl / ClO - impact.

[0086] Example 12: Add HOCl / ClO to pure water containing 90% glycerol by volume for the probe of the present invention. -The fluorescence lifetime spectrum that changes with pH is shown in the following figure ( Fig.18 ).

[0087] Considering that the probe will be used to detect the complex physiological environment of cells, the fluorescence signal of the probe in different pH environments is monitored. Consistent with the above experiment, it is only necessary to replace the above aqueous solution with different pH solutions. It can be found that although the pH changes from 3 to 10, its fluorescence lifetime intensity does not change basically, and remains at about 0.84ns, which indicates that the probe lifetime is not affected by the environmental pH.

[0088] Example 13: Fluorescence spectrum of the above-mentioned probe prodrug MIT-FP in a 50% ethanol aqueous solution showing the change in fluorescence intensity over time ( Fig.19 ).

[0089] In order to explore the reaction time of the probe, the addition of HOCl / ClO - The time-dependent fluorescence spectrum of the probe is shown in Figure 2. When the probe is exposed to saturated concentrations of HOCl / ClO - During the reaction, the strong fluorescence emission signal at 590 nm reached the maximum value after about 15 min, indicating that the probe reacted with HOCl / ClO - The reaction time is about 15min.

[0090] Example 14: Fluorescence intensity change spectra of the above-mentioned probe prodrug MIT-FP in solutions of different polarity ( Fig. 20 ).

[0091] In order to explore the effect of solvent polarity on the probe, the recognition of HOCl / ClO in solutions with different polarity was studied. - Fluorescence spectra. Compared with the more viscous solvent glycerol, the probe reacts with HOCl / ClO in a more polar solvent such as DMSO. - During the reaction, the fluorescence intensity of the probe did not change significantly; similarly, when reacting in a less polar solution such as CH2Cl2, the probe did not change significantly. The above results show that the probe can react with HOCl / ClO in different solvents. - There is a good response and the reaction product has good recognition only for highly viscous solvents.

[0092] Example 15: Reaction of the above-mentioned probe prodrug MIT-FP with HOCl / ClO in acetonitrile / water mixed solution - Mass spectrometry mechanism ( Fig.21 ).

[0093] To further verify the sensing mechanism of MIT-FP, HOCl / ClO was added to the solution of MIT-FP in a mixture of acetonitrile / water = 1:1. -HRMS monitoring was performed under the condition of . From the results, it can be seen that the solution after the reaction has an ion peak at 506.1422, which can be attributed to the peak corresponding to FP, so it can be considered that the probe FP is generated after the reaction.

[0094] Example 16: The recognition effect of the above-mentioned probe prodrug MIT-FP on OX-LDL in RAW264.7 cells ( Fig. 22 ).

[0095] The probe prodrug MIT-FP can be activated and converted into the probe FP in the cell mitochondria, and the changes of OX-LDL in the cell can be monitored dynamically in real time. Different concentrations of oxidized low-density lipoprotein (OX-LDL, 0μg / mL, 2μg / mL, 10μg / mL, 50μg / mL) were added to the 4-well plate inoculated with RAW264.7 cells and grown to the logarithmic phase, and cells without OX-LDL were used as controls. After 10 hours of co-incubation, different degrees of atherosclerosis cell models can be preliminarily established, and then the same concentration of probes are added to them, and the cells are placed in a cell culture incubator for another 10 hours before imaging under a fluorescence confocal microscope.

[0096] It can be found that in cells without the addition of OX-LDL, FP showed a very weak green fluorescence signal. However, in the established atherosclerotic cell model, it showed a more obvious signal enhancement. Moreover, as the model became more and more diseased, that is, as the amount of exogenous OX-LDL increased, the fluorescence signal of the probe changed significantly, indicating that MIT-FP can achieve dynamic monitoring of different degrees of cell pathology.

[0097] Example 17: Recognition effect of the above-mentioned probe prodrug MIT-FP in M0, M1 and M2 cells ( Fig.23 ).

[0098] The probe prodrug MIT-FP can be activated and converted into the probe FP in the cell mitochondria, which can dynamically distinguish M1 and M2 cells in real time. In a 4-well plate inoculated with M1 and M2 and grown to the logarithmic phase, undifferentiated M0 cells were used as a control. The probe prodrug MIT-FP was co-incubated with the cells for 10 hours and then imaged under a fluorescence confocal microscope.

[0099] It can be found that in undifferentiated M0 cells, the probe prodrug FP shows a clear green fluorescence signal, but in cells differentiated into M1, the cell fluorescence intensity is significantly enhanced, and a plaque-like signal appears. For the M2 type, the cell fluorescence intensity is very weak. It can be seen that as the model becomes more and more pathological, the fluorescence signal of the probe prodrug MIT-FP is significantly enhanced, and dynamic monitoring of different pathological types of M0, M1 and M2 cells can be achieved.

[0100] Example 18: Fluorescence imaging of the probe prodrug MIT-FP in atherosclerotic blood vessels ( Fig.24 ).

[0101] The above-mentioned probe prodrug was co-incubated with the blood vessels of early atherosclerotic mice in PBS culture medium, and then placed under a small animal in vivo imaging device for imaging. It can be found that compared with the control group, the fluorescence intensity of the probe in the experimental group at both ends of the blood vessels is stronger and the fluorescence intensity reaches the maximum value at about 2 hours. This shows that the probe prodrug MIT-FP can effectively detect vascular plaques in atherosclerotic mice.

[0102] Example 19: Confocal fluorescence imaging of the probe prodrug MIT-FP in atherosclerotic plaque tissue ( Fig.25 ).

[0103] The above-mentioned probe prodrug and plaque tissue were co-incubated in PBS culture medium, and then placed under a confocal microscope for imaging of the plaque. Under bright field, it can be seen that in normal tissue, smooth and flat plaque cardiac artery tissue is displayed, while a more obvious black block area can be found at the plaque location ( Fig.26 ). Under a fluorescence microscope, it can be found that the probe shows a relatively weak red fluorescence in normal tissue, while the probe shows an obvious green fluorescence signal in the lesion tissue. It is particularly noteworthy that the yellow superposition signal is shown at the edge of normal tissue and lesion tissue, indicating that the plaques of early atherosclerosis show a tendency to spread outward. The probe prodrug MIT-FP can effectively distinguish this process, realize the distinction between lesions and normal tissues, and contribute to the early diagnosis of atherosclerosis.

[0104] Example 20: Prodrug MIT-FP or probe FP can be prepared into lyophilized powder.

[0105] The probe prodrug MIT-FP is synthesized according to the method of Example 1. After the reaction is completed and purified, it is freeze-dried and stored at low temperature. When used, the freeze-dried powder of the required mass can be prepared.

[0106] Example 21: The prodrug MIT-FP or the probe FP can be made into a spray liquid, and the prescription is as follows (based on 1000 prescriptions):

[0107] Take 100 mg of the probe prodrug MIT-FP in Example 1, dissolve it in 100 mL of dimethyl sulfoxide to prepare a prodrug MIT-FP solution, add water for injection to 1000 mL, mix well and filter, and dispense the resulting solution into ampoules under sterile conditions to prepare a spray solution of 100 mL / bottle, with an active ingredient content of about 10 mg / bottle.

[0108] Example 22: Probe prodrug MIT-FP or probe FP can be made into a commercial kit.

[0109] The above-mentioned prodrug MIT-FP and probe FP were synthesized according to the methods of Examples 1 and 2, respectively. After the reaction was completed and purified, they were freeze-dried and stored at low temperature. When used, for the probe prodrug MIT-FP, 1.58 mg of solid powder was weighed and 1 mL of DMSO was added to make a 2 mmol mother solution, and ultrasonic dispersion was performed to make a kit. When the reagent is used, 10 μL of solution is taken from the kit, added to 2 mL of culture medium and diluted to 10 μmol as a working solution, the sample is stained, and the inflammatory site is stained and evaluated using a live imager or a fluorescent confocal microscope; for the probe FP, 1.01 mg of solid powder was weighed and 1 mL of DMSO was added to make a 2 mmol mother solution, and ultrasonic dispersion was performed to make a kit. When the reagent is used, 10 μL of solution is taken from the kit, added to 2 mL of culture medium and diluted to 10 μmol as a working solution, and the lipoprotein oxidation level in the cell is stained and evaluated using a fluorescent confocal microscope.

[0110] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent replacement are within the protection scope required by the present invention.

Claims

1. A probe prodrug, characterized in that: It has the following structure:

2. A probe molecule, characterized in that: It has the following structure:

3. The method for preparing the probe prodrug MIT-FP according to claim 1, characterized in that: The following steps are involved: Glycine tert-butyl ester hydrochloride is dispersed in tert-butyl alcohol, and N-ethylcarbazole-3-carboxaldehyde is added to continue the reaction; then the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester is prepared and added, and stirring is continued; after the reaction is completed, the mixture is extracted with water and CH2Cl2; the organic layer is dried over anhydrous sodium sulfate, and then the solvent is removed by a rotary evaporator; the residue is purified by column chromatography to obtain a yellow solid; The intermediate tert-butyl 2-(1-isopropoxymethyleneamino)acetate needs to be prepared and used immediately, and is synthesized by the following method: dissolving glycine tert-butyl hydrochloride, ethyl acetimidate hydrochloride, and sodium carbonate in a mixed solution of water / ether, followed by high-speed stirring. After the reaction is completed, the ether phase is collected, dried over anhydrous sodium sulfate, and the solvent is removed to obtain a white oily liquid; Then, the yellow solid was mixed with pyridine-4-carboxaldehyde, and refluxed for 10-15 hours under the catalysis of a catalyst. The obtained red solution was extracted with water and CH2Cl2, dried over anhydrous magnesium sulfate, and purified by silica gel column chromatography to obtain a red solid. Finally, the red solid was mixed with 4-bromomethylphenylboronic acid pinacol ester, and acetonitrile was used as a solvent at room temperature to react overnight to obtain a purple solution, which was filtered and washed with acetonitrile, petroleum ether, and cyclohexane to obtain a dark purple solid; The reaction route is as follows: 。 4. The method for preparing the probe molecule FP according to claim 2, characterized in that: The following steps are involved: Glycine tert-butyl ester hydrochloride is dispersed in tert-butyl alcohol, and N-ethylcarbazole-3-carboxaldehyde is added to continue the reaction; then the intermediate 2-(1-isopropoxymethyleneamino)acetic acid tert-butyl ester is prepared and added, and stirring is continued; after the reaction is completed, the mixture is extracted with water and CH2Cl2; the organic layer is dried over anhydrous sodium sulfate, and then the solvent is removed by a rotary evaporator; the residue is purified by column chromatography to obtain a yellow solid; The intermediate tert-butyl 2-(1-isopropoxymethyleneamino)acetate needs to be prepared and used immediately, and is synthesized by the following method: dissolving glycine tert-butyl hydrochloride, ethyl acetimidate hydrochloride, and sodium carbonate in a mixed solution of water / ether, followed by high-speed stirring. After the reaction is completed, the ether phase is collected, dried over anhydrous sodium sulfate, and the solvent is removed to obtain a white oily liquid; Then, the yellow solid was mixed with pyridine-4-carboxaldehyde, and refluxed for 10-15 hours under the catalysis of a catalyst. The obtained red solution was extracted with water and CH2Cl2, dried over anhydrous magnesium sulfate, and purified by silica gel column chromatography to obtain a red solid. The reaction route is as follows: 。 5. A composition comprising the probe prodrug MIT-FP according to claim 1 or the probe molecule FP according to claim 2, characterized in that: The active ingredient is the probe prodrug MIT-FP or the probe molecule FP and a pharmaceutically acceptable pharmaceutical excipient combination to prepare a clinically applicable spray, lyophilized powder injection or commercial kit.

6. A composition comprising the probe prodrug MIT-FP according to claim 1 or the probe molecule FP according to claim 2, characterized in that: It comprises the probe prodrug MIT-FP or probe FP and a pharmaceutically acceptable carrier.

7. Use of the probe prodrug MIT-FP according to claim 1 or the probe molecule FP according to claim 2 in the preparation of early diagnostic reagents and therapeutic drugs for atherosclerosis.

8. Use of the probe prodrug MIT-FP according to claim 1 or the probe molecule FP according to claim 2 in the preparation of a reagent or drug for evaluating the oxidation of lipoproteins HDL and LDL.

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