Lipid peroxidation detection probe based on perylene imide-triphenylphosphine structure, preparation method therefor, and use thereof

Abstract

The present invention belongs to the technical fields of lipid peroxidation fluorescent probe design synthesis and visualization imaging. Provided are a lipid peroxidation detection probe based on a perylene imide-triphenylphosphine structure, a preparation method therefor and the use thereof. The structural formula of the lipid peroxidation detection probe is shown as formula (I). The lipid peroxidation detection probe is based on structural optimization of perylene imide-triphenylphosphine compound Liperfluo, thereby greatly improving the stability thereof. The present invention has good sensitivity and selectivity to a ferroptosis marker lipid hydroperoxides, involves simple pretreatment of test samples, can be used for detecting ferroptosis endogenously and exogenously caused by fibrosarcoma cells, and can be used for studying specific regulation mechanisms and biological functions for ferroptosis-related metabolic diseases. The detection probe is a solid powder, is easy to store and use, involves a simple synthesis method, has a high yield and a low cost, and has good prospects in market application.

Description

A lipid peroxidation detection probe based on peryleneimide-triphenylphosphine structure, its preparation method and application Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a lipid peroxidation detection probe based on a peryleneimide-triphenylphosphine structure, its preparation method, and its application. Background Technology

[0002] Ferroptosis is a novel form of cell death discovered in recent years. It is an oxidative cell death that occurs under various inductions and is iron-dependent. Its occurrence is caused by an imbalance between the generation and degradation of intracellular lipid reactive oxygen species (ROS), among which the accumulation of lipid peroxides is the most direct cause of ferroptosis.

[0003] Phospholipids (PLs) are major components of biological membranes and lipoproteins, playing crucial biological functions. Glycerophospholipids are the most abundant phospholipids in the body, classified according to their phospholipid heads into phosphatidylcholines (PC), phosphatidylethanolamine (PE), phosphatidyl serines (PS), phosphatidyl inositols (PI), phosphatidic acid (PA), and cardiolipin (CL). Fatty acids are esterified at the sn-1 and sn-2 positions of glycerophospholipids, forming their hydrophobic tails. Polyunsaturated fatty acids (PUFAs), rich in diallyl carbon structures, are mostly located at the sn-2 position, forming unsaturated phospholipids (PUFA-PLs). The abundant diallyl carbon units in PUFAs are highly sensitive to reactive oxygen species (ROS), making PUFA-PLs readily oxidized into oxidized phospholipids (OxPLs) through enzymatic or non-enzymatic oxidation, accompanied by the formation of various other oxidation products. The oxidation of unsaturated phospholipids has a significant impact on their structure and biological activity, affecting the fluidity and permeability of biological membranes and altering the biological functions of lipids and proteins within the membrane.

[0004] Phospholipid peroxidation is an important outcome of oxidative stress. Phospholipid hydroperoxides (PLOOHs), as primary oxidation products, are at a critical juncture in the phospholipid oxidation process and are closely related to pathological processes such as neurodegenerative diseases, atherosclerosis, and ischemia-reperfusion injury. They are also a key characteristic of ferroptosis. Multiple studies have found specific accumulations of PSOOH and PIOOH in AD mouse models and post-mortem brain samples from AD patients, suggesting that both phospholipid hydroperoxides are involved in early neurological damage in AD. While research on the mechanisms of ferroptosis has developed rapidly, the development of ferroptosis-specific biomarkers and related efficient detection technologies has lagged behind, especially the lack of probes that can specifically, sensitively, and in real-time detect the process of cellular phospholipid peroxidation. Recent studies have confirmed that phosphatidylethanolamine (PE) is the preferred substrate for phospholipid oxidation in the ferroptosis process, and PEOOH is considered a major signaling molecule for ferroptosis. Therefore, the precise detection and regulation of various PLOOHs not only contributes to the biological basis of oxidative stress and the accurate diagnosis and treatment of related diseases, but also promotes the rapid development of precise detection technologies for novel ferroptosis biomarkers.

[0005] In recent years, high-sensitivity, high-resolution fluorescence detection technology has been favored by researchers and widely used in the detection of various substances. Compared with other monitoring methods, fluorescence imaging technology is sensitive, accurate, and can monitor in real time with high accuracy. Therefore, in ferroptosis-related research activities, fluorescent probes are often used to evaluate the process of ferroptosis and the level of action of ferroptosis inhibitors. The early-developed small molecule probe Liperfluo has been successfully applied to the detection of ferroptosis and cell imaging, but this probe has drawbacks such as structural instability, poor solubility, and weak specificity. Therefore, improving the stability and solubility of Liperfluo and designing and synthesizing small molecules that can be used to detect phospholipid hydroperoxides is of great significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a lipid peroxidation detection probe based on a peryleneimide-triphenylphosphine structure, its preparation method, and its applications. The compound provided by this invention exhibits significantly improved stability compared to Liperfluo, excellent selectivity for phospholipid hydroperoxides, and is suitable for preparing diagnostic reagents or drugs for detecting or treating ferroptosis-related diseases. Furthermore, it displays a significant fluorescence spectral change, demonstrating extremely strong selectivity for lipid hydroperoxides, with a clear and easily identifiable phenomenon.

[0007] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0008] On one hand, the present invention provides a lipid peroxidation detection probe molecule based on a peryleneimide-triphenylphosphine structure, the structural formula of which is shown below:

[0009] ,

[0010] In the formula, R is at least one of alkoxy, alkyl, halogen, and haloalkyl.

[0011] Furthermore, the R group is specifically selected from at least one of methoxy, methyl, fluorine, chlorine and trifluoromethyl groups.

[0012] Preferably, the compound is specifically compounds I-1 to I-5, and their structural formulas are as follows:

[0013] ,

[0014] ,

[0015] ,

[0016] ,

[0017] .

[0018] On the other hand, the present invention also provides a method for preparing the lipid peroxidation detection probe molecule compound based on the perylene imide-triphenylphosphine structure, which includes the following steps:

[0019] ,

[0020] (1) Compound 1, p-iodoaniline, CH3COOK, and Pd(OAc)2 were dissolved in DMA, protected with N2, and reacted at 130°C. After the reaction was completed, the mixture was separated and purified to obtain compound 2.

[0021] (2) The compound 2, N,N-dimethylaniline and trichlorosilane were dissolved in dry dioxane, protected with Ar, and reacted at 110°C. After the reaction was completed, the compound 3 was obtained by separation and purification.

[0022] (3) Dissolve compound 4 and KOH in H2O, add them to react at 90°C, add AcOH dropwise to the above reaction solution, continue the reaction, and after the reaction is completed, separate and filter to obtain compound 5;

[0023] (4) The compound 5 and aminotetraethylene glycol monomethyl ether were dissolved in H2O / EtOH (V / V=1:1), reacted at room temperature, and then heated to 90°C and refluxed overnight. After the reaction was completed, the compound 6 was obtained by separation and filtration.

[0024] (5) Compound 3, compound 6, Zn(OAc)2 and imidazole were mixed, protected with N2, and reacted at 130℃. After the reaction was completed, compound 7 was obtained by separation and purification, which is the lipid peroxidation detection probe.

[0025] Furthermore, in step (1), the molar ratio of compound 1: p-iodoaniline: CH3COOK: Pd(OAc)2 is 1-2: 1-3: 1-3: 0.001-0.1.

[0026] Preferably, in step (1), the molar ratio of compound 1: p-iodoaniline: CH3COOK: Pd(OAc)2 is 1:1.2:1.4:0.01.

[0027] Furthermore, in step (2), the molar ratio of compound 2: N,N-dimethylaniline: trichlorosilane is 1-1.1: 8-12: 8-12.

[0028] Preferably, in step (2), the molar ratio of compound 2: N,N-dimethylaniline:trichlorosilane is 1:10:10.

[0029] Furthermore, in step (3), the molar ratio of compound 4:KOH:AcOH is 0.8-1.2:20-30:30-40.

[0030] Preferably, in step (3), the molar ratio of compound 4:KOH:AcOH is 1:28:35.

[0031] Furthermore, in step (4), the molar ratio of compound 5 to aminotetraethylene monomethyl ether is 0.8-1.2:0.9-1.1.

[0032] Preferably, in step (4), the molar ratio of compound 5 to aminotetraethylene monomethyl ether is 1:1.

[0033] Furthermore, in step (5), the molar ratio of compound 6: compound 3: Zn(OAc)2 is 0.9-1.1: 1-1.3: 0.4-0.6.

[0034] Preferably, in step (5), the molar ratio of compound 6: compound 3: Zn(OAc)2 is 1:1.2:0.5.

[0035] Preferably, the specific method for separation and purification in step (1) is as follows: after the reaction is completed, the mixture is poured into 30 mL of water, the aqueous layer is extracted with dichloromethane, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain yellow oily compound 2.

[0036] Preferably, the specific method for separation and purification in step (2) is as follows: after the reaction is completed, the mixture is poured into 30 mL of water, the aqueous layer is extracted with dichloromethane, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain white solid compound 3.

[0037] Preferably, the specific method for separation and purification in step (3) is as follows: after the reaction is completed, the mixed reaction is cooled, filtered, washed with water, and dried to obtain reddish-brown solid compound 5.

[0038] Preferably, the specific method for separation and purification in step (4) is as follows: after the reaction is completed, the mixed reaction is cooled, filtered, washed with water, and dried to obtain reddish-brown solid compound 6.

[0039] Preferably, the specific method for separation and purification in step (5) is as follows: after the reaction is completed, the mixture is poured into 30 mL of water, the aqueous layer is extracted with ethyl acetate, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain red solid compound 7.

[0040] On the other hand, the present invention also provides the application of the lipid peroxidation detection probe in the preparation of detection reagents for detecting lipid peroxidation.

[0041] On the other hand, the present invention also provides the application of the lipid peroxidation detection probe in the preparation of detection reagents and imaging reagents for detecting ferroptosis-related diseases.

[0042] Furthermore, the iron death-related diseases include neurodegenerative diseases, tissue ischemia-reperfusion injury, stroke, cardiovascular diseases, liver and kidney failure, inflammation, and diabetic complications.

[0043] Furthermore, the drug contains a lipid peroxidation detection probe or a pharmaceutically acceptable salt, excipient, or carrier thereof.

[0044] On the other hand, the present invention also provides the application of the lipid peroxidation detection probe in the preparation of antioxidant fluorescent probes or antioxidant indicators.

[0045] Furthermore, the antioxidant fluorescent probe is used for highly sensitive detection of lipid hydroperoxides, and has the ability to specifically detect indicators of lipid peroxidation. Beneficial effects

[0046] 1. This invention optimizes the structure of the perylene imide-triphenylphosphine compound Liperfluo. Through experiments, the stability of the lipid peroxidation detection probe based on the perylene imide-triphenylphosphine structure is significantly improved. It has good sensitivity and selectivity for the ferroptosis biomarker lipid hydroperoxide, and can be used for sensitive detection of ferroptosis and for research on ferroptosis-related diseases.

[0047] 2. The preparation method of the lipid peroxidation detection probe based on the perylene imide-triphenylphosphine structure described in this invention is simple in steps, easy in sample pretreatment, and has a high yield, making it suitable for large-scale application.

[0048] 3. The lipid peroxidation detection probe based on peryleneimide-triphenylphosphine structure described in this invention can be used as a fluorescent probe, exhibiting significant changes in fluorescence spectrum. During the detection of ferroptosis, the monitoring effect can be evaluated in real time through changes in its own fluorescence spectrum.

[0049] The lipid peroxidation detection probe based on peryleneimide-triphenylphosphine structure provided by this invention can be used to detect ferroptosis caused by endogenous and exogenous factors in fibrosarcoma cells; and to study specific regulatory mechanisms and biological functions of ferroptosis-related metabolic diseases; moreover, the lipid peroxidation detection probe is a solid powder, which is easy to store and use, and the synthesis method is simple, with high yield and low cost, and has good market application prospects. Attached Figure Description

[0050] Figure 1 shows the 1H NMR spectrum of compound I-1;

[0051] Figure 2 shows the 1H NMR spectrum of compound I-2;

[0052] Figure 3 shows the 1H NMR spectrum of compound I-3;

[0053] Figure 4 shows the 1H NMR spectrum of compound I-4;

[0054] Figure 5 shows the 1H NMR spectrum of compound I-5;

[0055] Figure 6A shows the selectivity test results of compound I-1 in detecting lipid hydroperoxides;

[0056] Figure 6B shows the selectivity test results of compound I-2 in detecting lipid hydroperoxides;

[0057] Figure 6C shows the selectivity test results of compound I-3 in detecting lipid hydroperoxides;

[0058] Figure 6D shows the selectivity test results of compound I-4 for detecting lipid hydroperoxides;

[0059] Figure 7 shows the reaction spectra and linear relationships of compound I-1 with different concentrations of PEOOH;

[0060] Figure 8 shows the UV and fluorescence spectra of compound I-1 in its oxidized state;

[0061] Figure 9A shows the intrinsic stability of compound I-1;

[0062] Figure 9B shows the intrinsic stability of compound I-2;

[0063] Figure 9C shows the intrinsic stability of compound I-3;

[0064] Figure 9D shows the intrinsic stability of compound I-4.

[0065] Figure 9E shows the intrinsic stability of compound I-5;

[0066] Figure 9F shows the stability of the compound Liperfluo itself;

[0067] Figure 10 shows cell imaging of compound I-1;

[0068] Figure 11 shows cell imaging of compound I-2;

[0069] Figure 12 shows cell imaging of compounds I-1, I-2, Liperfluo, and I-3. Detailed Implementation

[0070] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0071] The overall synthetic route for the iron death detection probe based on the peryleneimide-triphenylphosphine structure provided by this invention is as follows:

[0072]

[0073] Among them, (a) DMA, CH3COOK, Pd(OAc) 2, (a) Drying 1,4-dioxane, N,N-dimethylaniline, trichlorosilane, and Ar at 110°C; (b) H2O, KOH at 90°C; AcOH at 90°C; (d) H2O / EtOH (V / V=1:1), stirred at room temperature; overnight at 90°C; (e) the corresponding triphenylphosphine, imidazole, and Zn(OAc). 2 , N2, 130℃.

[0074] (1) Step a: Compound 1 (1.589 mmol), p-iodoaniline (1.907 mmol), potassium acetate (0.826 mmol), and palladium acetate (0.01589 mmol) were dissolved in DMA (10 mL), substituted with N2 for protection, and reacted at 130 °C for 4 h. After the reaction was completed, the mixture was cooled to room temperature, the solvent was evaporated, the aqueous phase was extracted with dichloromethane, the organic layer was dried, and concentrated. The mixture was separated by column chromatography to obtain a yellow oily substance, namely compound 2.

[0075] (2) Step b: Compound 2 (0.436 mmol), N,N-dimethylaniline (4.358 mmol), and trichlorosilane (4.358 mmol) were dissolved in Dry 1,4-Dioxane (20 mL), under Ar protection, and reacted at 110 °C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, the solvent was evaporated, the aqueous phase was extracted with dichloromethane, the organic layer was dried, and concentrated. The mixture was separated by column chromatography to obtain a white solid, namely compound 3.

[0076] (3) Step c: Dissolve compound 4 (5.100 mmol) and KOH (200 mmol) in H2O and react at 90℃ for 5 h. The solution turns green. Add 10 ml AcOH (10 mL) to the above reaction solution and continue the reaction. After the reaction is completed, cool to room temperature, filter, wash with water, and dry to obtain a reddish-brown solid, which is compound 5.

[0077] (4) Step d: Dissolve compound 5 (1.040 mmol) in H2O / EtOH (V / V=1:1, 20 mL), add aminotetraethylene glycol monomethyl ether (1.040 mmol), react at room temperature for 5 h, then transfer to an oil pan at 90℃ overnight. After the reaction is complete, cool to room temperature, filter, wash with water, and dry to obtain a brick-red solid, which is compound 6.

[0078] (5) Step e: Compound 6 (0.5111 mmol), compound 3 (0.562 mmol), Zn(OAc)2 (0.266 mmol) were mixed with imidazole, and the mixture was substituted with N2 for protection. The reaction was carried out at 130 °C for 4 h. After the reaction was completed, the mixture was cooled to room temperature, the solvent was evaporated, the aqueous phase was extracted with ethyl acetate, the organic layer was dried, and the mixture was concentrated. The mixture was separated by column chromatography to obtain a red solid, which was compound 7.

[0079] Example 1: Preparation of compound I-1

[0080]

[0081] In this embodiment, compound I-1 was prepared using the experimental method described above. The proton NMR spectrum is shown in Figure 1. 1H NMR (400 MHz, Chloroform-d) δ 8.69 (d, J = 7.9 Hz, 2H), 8.63 – 8.51 (m, 6H), 7.47 – 7.40 (m, 2H), 7.39 – 7.31 (m, 6H), 6.93 (d, J = 8.9 Hz, 4H), 4.46 (t, J = 6.0 Hz, 2H), 3.87 (d, J = 5.1 Hz, 2H), 3.83 (s, 6H), 3.74 – 3.70 (m, 2H), 3.65 – 3.57 (m, 8H), 3.50 (dd, J = 5.8, 3.4 Hz, 2H), 3.34 (s, 3H).

[0082] Example 2: Preparation of compound I-2

[0083]

[0084] In this embodiment, compound I-2 was prepared using the experimental method described above. The proton NMR spectrum is shown in Figure 2. 1 H NMR (400 MHz, Chloroform-d) δ 8.67 (d, J = 7.9 Hz, 2H), 8.58 (d, J = 7.9 Hz, 2H), 8.53 (d, J = 8.1 Hz, 2H), 8.48 (d, J = 8.1 Hz, 2H), 7.47 (dd, J = 8.4, 6.8 Hz, 2H), 7.36 – 7.29 (m, 6H), 7.22 – 7.16 (m, 4H), 4.46 (t, J = 6.0 Hz, 2H), 3.86 (t, J = 5.9 Hz, 2H), 3.72 (dd, J = 5.9, 3.6 Hz, 2H), 3.65 – 3.57 (m, 8H), 3.53 – 3.47 (m, 2H), 3.34 (s, 3H), 2.37 (s, 6H).

[0085] Example 3: Preparation of compound I-3

[0086]

[0087] In this embodiment, compound I-3 was prepared using the experimental method described above. The proton NMR spectrum is shown in Figure 3. 1H NMR (400 MHz, Chloroform-d) δ 8.65 (d, J = 8.0 Hz, 2H), 8.55 (d, J = 8.0 Hz, 2H), 8.49 (d, J = 8.1 Hz, 2H), 8.43 (d, J = 8.1 Hz, 2H), 7.47 – 7.42 (m, 2H), 7.41 – 7.35 (m, 6H), 7.10 (t, J = 8.8 Hz, 4H), 4.45 (t, J = 5.9 Hz, 2H), 3.86 (t, J = 5.9 Hz, 2H), 3.74 – 3.70 (m, 2H), 3.65 – 3.57 (m, 8H), 3.52 – 3.49 (m, 2H), 3.34 (s, 3H).

[0088] Example 4: Preparation of compound I-4

[0089]

[0090] In this embodiment, compound I-4 was prepared using the experimental method described above. The proton NMR spectrum is shown in Figure 4. 1 H NMR (400 MHz, Chloroform-d) δ 8.68 (d, J = 8.0 Hz, 2H), 8.60 – 8.47 (m, 6H), 7.41 – 7.35 (m, 7H), 7.35 – 7.27 (m, 5H), 4.46 (t, J = 6.0 Hz, 2H), 3.86 (t, J = 6.0 Hz, 2H), 3.72 (dd, J = 5.9, 3.6 Hz, 2H), 3.66 – 3.56 (m, 8H), 3.50 (d, J = 9.2 Hz, 2H), 3.34 (s, 3H).

[0091] Example 5: Preparation of compound I-5

[0092]

[0093] In this embodiment, compound I-5 was prepared using the experimental method described above. The proton NMR spectrum is shown in Figure 5. 1H NMR (400 MHz, Chloroform-d) δ 8.66 (d, J = 8.0 Hz, 2H), 8.56 (d, J = 8.0 Hz, 2H), 8.51 (d, J = 8.1 Hz, 2H), 8.45 (d, J = 8.2 Hz, 2H), 7.65 (d, J = 7.9 Hz, 4H), 7.55 – 7.48 (m, 6H), 7.45 (dd, J = 8.5, 1.3 Hz, 2H), 4.45 (t, J = 6.0 Hz, 2H), 3.86 (t, J = 6.0 Hz, 2H), 3.72 (dd, J = 5.9, 3.6 Hz, 2H), 3.65 – 3.57 (m, 8H), 3.52 – 3.48 (m, 2H), 3.34 (s, 3H).

[0094] Example 6

[0095] Based on the peryleneimide-triphenylphosphine structure-based iron death detection probe molecule provided by this invention, the probe solution is prepared as follows: Weigh appropriate amounts of red solid powder probe compounds I-1 to I-5, dissolve them in a measured amount of dimethyl sulfoxide (DMSO) to obtain a 1 mM stock solution. Dilute the stock solution with methanol, measure the final concentration of the solution to be 1.0 μM, and record the UV and fluorescence spectra at 37°C.

[0096] Example 7

[0097] Preparation of analytes for selective experiments: The analytes required for the experiment include H₂O₂, HClO, t-BuOOH, CumOOH, and •O₂. - ONOO - Various reactive oxygen species (ROS), including •OH, PCOOH, PEOOH, and PSOOH, were prepared according to the following method. The H₂O₂ solution was diluted from a 30% H₂O₂ solution, and the concentration was determined by the absorbance at 240 nm (ε = 43.6 M⁻¹ cm⁻¹). A commercially available sodium hypochlorite solution was used as the source of hypochlorite, and the molar absorbance of hypochlorite (ClO₂) was determined at 292 nm using a molar absorbance of 350 M⁻¹ cm⁻¹. - The concentrations of t-BuOOH, CHP, and ONOO- are available for direct purchase. •O2 - Hydroxyl radicals (•OH) are generated by mixing FeSO4 with 10 equivalents of H2O2, according to Fe... 2+Concentration calculation of •OH concentration. PCOOH, PEOOH, and PSOOH are generated by the induction of the corresponding PC, PE, and PS via azobisisoheptanenitrile, respectively.

[0098] The experimental results are shown in Figures 6A-6D, where the horizontal axis represents the probe molecules (I-1~4) and the types of ROS added. The ROS include H₂O₂, HClO, t-BuOOH, CumOOH, and •O₂. - ONOO - •OH, PCOOH, PEOOH, PSOOH; Probe selectivity testing conditions: Ex = 488 nm, Em = 535 nm; As shown in Figures 6A-6D, these probe molecules exhibit good selectivity for phospholipid hydroperoxides compared to various reactive oxygen species, confirming their specific response to ferroptosis biomarkers. Furthermore, the probe molecules show a good linear relationship with phospholipid hydroperoxide concentration (Figure 7). Therefore, these molecules can be further studied as probes for ferroptosis biomarkers.

[0099] Example 8

[0100] Based on the peryleneimide-triphenylphosphine structure-based iron death detection probe molecule of the present invention, stability was assessed under the following conditions: room temperature, no light protection, and fluorescence intensity of the probe molecule was measured at equal time intervals. The stability of the probe molecule was evaluated by comparing the increase in fluorescence at 535 nm after oxidation with that of Liperfluo. The stock solution was diluted with methanol, and the final concentration of the solution used was measured to be 1.0 μM, Ex = 488 nm, Em = 535 nm.

[0101] After oxidation, the peak shape and position of Liperfluo and its derivatives remained unchanged. Excitation was performed at 488 nm, with a maximum emission peak at 535 nm (Figure 8). Long-term observation yielded experimental results, as shown in Figures 9A-9F. The line graphs indicate that the introduction of the electron-withdrawing group -CF3 significantly weakened the fluorescence intensity and made it almost unstable, with 67.6% oxidation occurring after 1 hour. Introducing electron-donating groups such as -OCH3 increased the fluorescence intensity. Furthermore, the introduction of -OCH3 / -CH3 significantly increased the stability of Liperfluo; at 48 h, the oxidation percentages were 30% and 36%, respectively, both lower than Liperfluo's 48.8%. Simultaneously, -OCH3 improved the probe solubility (Table 1).

[0102] Table 1 Solubility of different probes

[0103]

[0104] Note: Measurement at room temperature

[0105] Example 9

[0106] HT1080 cells were pretreated with RLS3 (2.5 μM) for 30 min to induce ferroptosis. Subsequently, the cells were co-incubated with the nuclear dye Hoechst and probe molecules (e.g., I-1, I-2) for 30 min, and the cell imaging of the probe molecules was observed using confocal microscopy. The experimental results are shown in Figures 10 and 11, indicating that the probe molecules have good ferroptosis imaging capabilities. Cells were incubated with Erastin (10 μM) for 3 h, followed by the addition of probe molecules I-1, I-2, Liperfluo, and I-3, respectively. After incubation for 30 min, cell imaging was performed using confocal microscopy (Figure 12). Compared with the commercial probe Liperfluo, probes I-1, I-2, and I-3 all exhibited good cell imaging capabilities. Therefore, I-1, I-2, and I-3 can be used for indicative imaging of ferroptosis in cells.

[0107] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A lipid peroxidation detection probe based on a peryleneimide-triphenylphosphine structure, characterized in that, The structural formula of the lipid peroxidation detection probe is shown below: ， In the formula, R is at least one of alkoxy, alkyl, halogen, and haloalkyl.

2. The lipid peroxidation detection probe according to claim 1, characterized in that, The R is selected from at least one of methoxy, methyl, fluorine, chlorine and trifluoromethyl.

3. The lipid peroxidation detection probe according to claim 1, characterized in that, The lipid peroxidation detection probes are specifically compounds I-1 to I-5, and their structural formulas are as follows: ， ， ， ， 。 4. The method for preparing the lipid peroxidation detection probe based on the perylene imide-triphenylphosphine structure according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: ， (1) Compound 1, p-iodoaniline, CH3COOK, and Pd(OAc)2 were dissolved in DMA, protected by N2, and reacted at high temperature. After the reaction was completed, the oily compound, namely compound 2, was obtained by separation and purification. (2) The compound 2, N,N-dimethylaniline and trichlorosilane were dissolved in dry dioxane, protected with Ar, and reacted at high temperature. After the reaction was completed, the compound 3 was obtained by separation and purification. (3) Dissolve compound 4 in H2O, add KOH, react at high temperature, add AcOH dropwise into the reaction solution, continue the reaction at high temperature, and after the reaction is completed, separate and filter to obtain compound 5; (4) Dissolve the compound 5 and aminotetraethylene glycol monomethyl ether in H2O / EtOH and react at room temperature. After the reaction is completed, separate and filter to obtain a brick-red solid, which is compound 6. (5) The compound 6, the compound 3, Zn(OAc)2 and imidazole are mixed, protected by N2, and reacted at high temperature. After the reaction is completed, the red solid compound is obtained by separation and purification, which is the lipid peroxidation detection probe.

5. The preparation method according to claim 4, characterized in that, In step (1), the molar ratio of compound 1: p-iodoaniline: CH3COOK: Pd(OAc)2 is 1-2: 1-3: 1-3: 0.001-0.

1.

6. The preparation method according to claim 4, characterized in that, In step (2), the molar ratio of compound 2: N,N-dimethylaniline:trichlorosilane is 1-1.1:8-12:8-12.

7. The preparation method according to claim 4, characterized in that, In step (3), the molar ratio of compound 4:KOH:AcOH is 0.8-1.2:20-30:30-40.

8. The preparation method according to claim 4, characterized in that, In step (4), the molar ratio of compound 5 to aminotetraethylene glycol monomethyl ether is 0.8-1.2:0.9-1.1, and the volume ratio of H2O / EtOH is V / V=1:1; in step (5), the molar ratio of compound 6 to compound 3 to Zn(OAc)2 is 0.9-1.1:1-1.3:0.4-0.

6.

9. The use of the lipid peroxidation detection probe according to any one of claims 1-3 in the preparation of a detection reagent for detecting lipid peroxidation and ferroptosis-related diseases.

10. The application according to claim 9, characterized in that, The iron death-related diseases include neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, liver and kidney failure, inflammation, and diabetic complications.

Images