Endoplasmic reticulum targeted lipid peroxidation fluorescent indicator and application thereof

Abstract

The invention discloses an endoplasmic reticulum targeted lipid peroxidation fluorescent indicator and application thereof, and the indicator introduces a conjugated diolefin structure as a lipid peroxidation recognition unit to obtain a series of BODIPY derivatives with the maximum emission wavelength of 620 nm. A conjugated diene part of the fluorescent probe can be subjected to oxidative cracking under the action of reactive oxygen species, and a conjugated system of the fluorescent probe becomes smaller due to breakage of double bonds, which shows that absorption and emission wavelengths are obviously shortened. Therefore, the LPO process can be monitored through the change of the fluorescence ratio. Meanwhile, hydrophilic succinimide ester sulfonic acid group NHS is introduced as a group reacting with protein, the oil-water coefficient of the compound is moderate, the compound easily penetrates through a cell membrane and reacts with adjacent protein free amino in a short time, the probe can be fixed on the protein of an endoplasmic reticulum through a chemical bond, and the probe can be used for long-time fluorescence imaging research. And long-time real-time tracking is carried out on the movement and form change of the endoplasmic reticulum.

Description

Technical Field

[0001] The invention relates to an endoplasmic reticulum-targeted lipid peroxidation fluorescent indicator and application thereof, and belongs to the field of fine chemicals. Background Art

[0002] Lipid peroxidation is a chain oxidation reaction initiated by reactive oxygen species (ROS) attacking polyunsaturated fatty acids in biological membranes. This process produces toxic products such as malondialdehyde (MDA) and 4-hydroxynonenoic acid (4-HNE), leading to membrane disruption, protein dysfunction, and organelle damage. The endoplasmic reticulum (ER) plays a crucial role in protein synthesis and folding, as well as maintaining intracellular calcium homeostasis. The ER membrane is rich in unsaturated fatty acids and is highly sensitive to oxidative damage. A growing body of research has demonstrated that ER oxidative stress is closely associated with various metabolic diseases, such as diabetes and obesity. Excessive ROS further exacerbate ER stress, leading to cellular dysfunction and even apoptosis. The ER is highly dynamic, constantly changing its morphology and spatial distribution. Visualizing and tracking the ER and monitoring its lipid peroxidation processes not only contributes to understanding the molecular mechanisms of ER involvement in biological processes but also offers important insights into disease treatment. Therefore, universal monitoring methods are urgently needed.

[0003] Fluorescence detection technology based on fluorescent dyes offers the advantages of high sensitivity and real-time performance, making it suitable for monitoring microstructural and material changes within living cells. The development of endoplasmic reticulum-targeted fluorescent probes for lipid peroxidation has significant practical value. Summary of the Invention

[0004] The present invention provides a class of endoplasmic reticulum-targeted lipid peroxidation fluorescent indicators based on BODIPY and their applications. This indicator can rapidly target and fixate to the endoplasmic reticulum of living cells, identifying lipid peroxidation processes in situ in the endoplasmic reticulum, generating a fluorescent signal that changes from red to green, and then indicating lipid peroxidation occurring on the endoplasmic reticulum through a fluorescence ratio value. The design of the probe molecule takes into account both ratio quantification and stable endoplasmic reticulum targeting. Using the BODIPY dye as the matrix, a conjugated diene structure is introduced as the response group of LPO, and an active group R3 that can react with proteins is designed. The probe can be fixed to the protein of the endoplasmic reticulum through a chemical bond, achieving long-term stable labeling. The compound has the following general structural formula:

[0005]

[0006] Wherein, n is 1, 2, 3, 4 or 5.

[0007] R1 and R2 are each independently H, Cl, an alkyl group having 1 to 10 carbon atoms, or an alkyl group substituted with an alkoxycarbonyl group having 2 to 15 carbon atoms.

[0008] R3 is or

[0009] R4 is or

[0010] Y is a phenyl group or a phenyl group having at least one substituent.

[0011] The substituent is selected from C1-C10 alkyl, C1-C10 alkoxy, hydroxy, nitro, and amino.

[0012] In some specific compounds, R1 and R2 are independently H, Cl, an alkyl group having 1 to 5 carbon atoms, or an alkyl group substituted with an alkoxycarbonyl group having 2 to 10 carbon atoms.

[0013] The substituent is selected from C1-C5 alkyl, C1-C5 alkoxy, hydroxy, nitro, and amino.

[0014] In some specific compounds, R1 and R2 are independently H, Cl, an alkyl group having 1 to 3 carbon atoms, or an alkyl group substituted with an alkoxycarbonyl group having 3 to 6 carbon atoms.

[0015] The substituent is selected from C1-C5 alkyl, C1-C5 alkoxy, hydroxy, nitro, and amino.

[0016] In some specific compounds, R1 and R2 are independently H, Cl, or an alkyl group having 1 to 3 carbon atoms; and Y is a phenyl group or a phenyl group having 1 to 2 substituents.

[0017] The substituent is selected from C1-C5 alkyl and C1-C5 alkoxy.

[0018] In some specific compounds, R1 and R2 are independently H, Cl, CH3, or C2H5.

[0019] R3 is R4 is Y is

[0020] In some specific compounds, R1 and R2 are independently H, Cl, or CH3.

[0021] R3 is R4 is Y is

[0022] The compound is used in the preparation of drugs for targeting the endoplasmic reticulum and monitoring endoplasmic reticulum lipid peroxidation. The compound is used in the preparation of drugs for detecting and tracking the endoplasmic reticulum.

[0023] The compound is used in preparing medicine for tracking the location of bacterial infection and inflammation in the endoplasmic reticulum.

[0024] The compound is used in the preparation of endoplasmic reticulum-targeting drugs or materials.

[0025] The compound is used in preparing a lipid peroxidation fluorescent indicator.

[0026] The method for preparing the endoplasmic reticulum-targeted lipid peroxidation fluorescent indicator includes the following reaction formula and reaction steps:

[0027] 1. Synthesis steps of intermediate F

[0028]

[0029] i) Compound b was dissolved in water, triethylamine was added, R3-Cl was dissolved in acetonitrile, and an aqueous solution of compound a was slowly added. After the reaction was completed, the mixture was washed three times with saturated aqueous ammonium chloride solution. The solvent was removed under vacuum, and the crude product was separated and purified by silica gel column chromatography to obtain compound F-Boc.

[0030] ii) Compound F-Boc is dissolved in dichloromethane, trifluoroacetic acid is added thereto, and the mixture is reacted at room temperature. After the reaction is completed, the solvent is removed under vacuum to obtain Compound F.

[0031] 2. Synthesis steps of endoplasmic reticulum-targeted lipid peroxidation fluorescent indicator

[0032]

[0033] i) removing water with a water separator, heating compounds A, B, piperidine and glacial acetic acid in toluene to react, concentrating the reaction mixture and extracting with dichloromethane, washing the organic phase with brine, drying, concentrating, and purifying on a silica gel column to obtain compound C;

[0034] ii) Compound C, Zn powder, and ammonium chloride were dissolved in a mixture of water and ethanol, glacial acetic acid was added, and the mixture was reacted at 60°C. After the reaction, the zinc powder was removed by filtration, and the mixture was extracted with a saturated sodium bicarbonate solution and dichloromethane. The solvent was removed under vacuum. The crude product was purified by silica gel column chromatography to obtain Compound D.

[0035] iii) Dissolve thiophosgene and triethylamine in dichloromethane, add the dichloromethane solution of Compound D under ice-cooling, and react at room temperature. After the reaction, extract with water and dichloromethane, and purify the crude product by silica gel column chromatography to obtain Compound E;

[0036] iv) Compound F and Compound E were dissolved in acetone, and triethylamine was added thereto. The reaction was carried out in an ice bath. After completion of the reaction, the mixture was extracted with water and dichloromethane, the solvent was removed under vacuum, and the crude product was purified by silica gel column chromatography to obtain Compound ER-B;

[0037] v) Compound ER-B was dissolved in a mixture of tetrahydrofuran and acetonitrile. Compound R4-H was then added sequentially under ice-cooling, with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as a catalyst. The reaction was carried out at room temperature. After completion of the reaction, the solvent was removed under vacuum, the mixture was repeatedly washed with n-hexane, the supernatant was removed by centrifugation, and the mixture was dried with argon to obtain ER-BN without further purification.

[0038] Wherein, the definitions of R1, R2, R3, R4 and Y are the same as those in the general structural formula.

[0039] The beneficial effects of the present invention include: a type of endoplasmic reticulum-targeted lipid peroxidation fluorescent indicator and its application, wherein the endoplasmic reticulum-targeted lipid peroxidation fluorescent indicator introduces a conjugated diene structure as a lipid peroxidation recognition unit, and successfully obtains a series of BODIPY derivatives with a maximum emission wavelength of 620 nm. The conjugated diene portion of the fluorescent probe undergoes oxidative cleavage under the action of reactive oxygen species, and the rupture of the double bond causes its conjugated system to become smaller, which is manifested as a significant shortening of the absorption and emission wavelengths. Therefore, the probe molecule can monitor the LPO process through changes in the fluorescence ratio. At the same time, a hydrophilic succinimidyl ester sulfonic acid group NHS is introduced as a group that reacts with proteins. This type of compound has a moderate oil-water coefficient, is easy to penetrate the cell membrane, and reacts with adjacent protein free amino groups in a short time. The probe can be fixed to the protein of the endoplasmic reticulum through a chemical bond, and can be used for long-term fluorescence imaging studies, and long-term real-time tracking of the movement and morphological changes of the endoplasmic reticulum. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 These are the absorption and emission spectra of compound ER-B6 in a solution simulating intracellular lipid peroxidation. Figure 2 This is the endoplasmic reticulum co-localization imaging of compound ER-B-N6 in living cells.

[0041] Figure 3 These are images of the endoplasmic reticulum of compound ER-B-N6 before and after fixation of cells with paraformaldehyde.

[0042] Figure 4 This is an image of the endoplasmic reticulum of living cells under lipopolysaccharide stimulation conditions of compound ER-B-N6. DETAILED DESCRIPTION

[0043] To make the technical solution of the present invention clearer, the following detailed description of the specific embodiments of the present invention is given in conjunction with the technical solution and the accompanying drawings. The present invention is illustrated by the following examples but is not limited thereto. Unless otherwise specified, all parts and percentages are by weight.

[0044] Example 1

[0045]

[0046] A (1.27 mmol) and B (1.27 mmol) were stirred in 30 mL of dry toluene (dehydrated with molecular sieves before use). 1 mL of piperidine and 1 mL of glacial acetic acid were added as catalysts. The reaction was heated to reflux at 150°C. The water produced during the reaction was removed using a water separator. The reaction was monitored by TLC until completion. After cooling to room temperature, most of the toluene in the reaction solution was removed under reduced pressure. The resulting liquid was diluted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 10) to obtain solid C2.

[0047]

[0048] Compound C1 (0.25 mmol), Zn powder (3.04 mmol), and NH4Cl (2.03 mmol) were dissolved in 12 mL of a 1:4 mixture of water and ethanol. 24 μL of glacial acetic acid was added and the reaction was allowed to proceed at 60°C. The reaction was monitored by TLC. After completion of the reaction, the zinc powder was removed by filtration, and the mixture was extracted with a saturated sodium bicarbonate solution and dichloromethane. The solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to obtain Compound D1.

[0049]

[0050] Thiophosgene (1.86 mmol) and triethylamine (0.93 mmol) were dissolved in 5 mL of dichloromethane. Compound D1 (0.16 mmol) in 5 mL of dichloromethane was added under ice-cooling. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1) to obtain Compound E1.

[0051]

[0052] i) Compound b1 (1.18 mmol) was dissolved in 5 mL of water, and triethylamine (300 μL) was added. Compound a1 (1.18 mmol) was dissolved in 10 mL of acetonitrile, and the aqueous solution containing compound b1 was slowly added. The reaction was monitored by TLC. After completion of the reaction, the mixture was washed three times with saturated aqueous ammonium chloride. The collected organic phase was dried over anhydrous sodium sulfate, filtered to remove the anhydrous sodium sulfate, and the solvent was removed under vacuum. The crude product was isolated and purified by silica gel column chromatography (dichloromethane / methanol = 40 / 1) to obtain compound F1-Boc.

[0053] ii) Compound F1-Boc (0.5 mmol) was dissolved in 5 mL of dichloromethane, and 5 mL of trifluoroacetic acid was added thereto, and the mixture was reacted at room temperature. After the reaction was completed, the solvent was removed under vacuum to obtain Compound F1.

[0054]

[0055] Compound F1 (0.11 mmol) and compound E1 (0.09 mmol) were dissolved in 5 mL of acetone, and triethylamine (300 μL) was added. The reaction was carried out in an ice bath. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to obtain ER-B1.

[0056]

[0057] Compound ER-B1 (1.21 μmol) was dissolved in 0.5 mL of a 10:1 tetrahydrofuran:acetonitrile mixture. G1 (1.45 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride catalyst (1.45 μmol) were added sequentially under an ice bath. The reaction was carried out at room temperature and monitored by TLC. The solvent was removed under vacuum, and the mixture was repeatedly washed with n-hexane. The supernatant was removed by centrifugation and dried with argon to obtain compound ER-B-N1, which did not require purification.

[0058] Example 2

[0059]

[0060] A2 (1.27 mmol) and B2 (1.27 mmol) were stirred in 30 mL of dry toluene (dehydrated with molecular sieves before use). 1 mL of piperidine and 1 mL of glacial acetic acid were added as catalysts. The reaction was heated to reflux at 150°C. The water produced during the reaction was removed using a water separator. The reaction was monitored by TLC until completion. After cooling to room temperature, most of the toluene in the reaction solution was removed under reduced pressure. The resulting liquid was diluted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 10) to obtain solid C3.

[0061]

[0062] Compound C2 (0.25 mmol), Zn powder (3.04 mmol), and NH4Cl (2.03 mmol) were dissolved in 12 mL of a 1:4 mixture of water and ethanol. 24 μL of glacial acetic acid was added and the reaction was allowed to proceed at 60°C. The reaction was monitored by TLC. After completion of the reaction, the zinc powder was removed by filtration, and the mixture was extracted with a saturated sodium bicarbonate solution and dichloromethane. The solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to obtain compound D2.

[0063]

[0064] Dissolve thiophosgene (1.86 mmol) and triethylamine (0.93 mmol) in 5 mL of dichloromethane. Add 5 mL of a dichloromethane solution containing compound D2 (0.16 mmol) under ice-cooling. Monitor the reaction by TLC. After completion of the reaction, extract with water and dichloromethane. The crude product is purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1) to obtain compound E2.

[0065]

[0066] i) Compound b2 (1.18 mmol) was dissolved in 5 mL of water, and triethylamine (300 μL) was added. Compound a2 (1.18 mmol) was dissolved in 10 mL of acetonitrile, and the aqueous solution containing compound b2 was slowly added. The reaction was monitored by TLC. After the reaction was completed, the mixture was washed three times with saturated aqueous ammonium chloride. The collected organic phase was dried over anhydrous sodium sulfate, filtered to remove the anhydrous sodium sulfate, and the solvent was removed under vacuum. The crude product was separated and purified by silica gel column chromatography (dichloromethane / methanol = 40 / 1) to obtain compound F2-Boc.

[0067] ii) Compound F2-Boc (0.5 mmol) was dissolved in 5 mL of dichloromethane, and 5 mL of trifluoroacetic acid was added thereto, and the mixture was reacted at room temperature. After the reaction was completed, the solvent was removed under vacuum to obtain Compound F2.

[0068]

[0069] Compound F2 (0.11 mmol) and compound E2 (0.09 mmol) were dissolved in 5 mL of acetone, and triethylamine (300 μL) was added. The reaction was carried out in an ice bath. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to obtain ER-B3.

[0070]

[0071] Compound ER-B2 (1.21 μmol) was dissolved in 0.5 mL of a 10:1 tetrahydrofuran:acetonitrile mixture. G2 (1.45 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride catalyst (1.45 μmol) were added sequentially under an ice bath. The reaction was carried out at room temperature and monitored by TLC. The solvent was removed under vacuum, and the mixture was repeatedly washed with n-hexane. The supernatant was removed by centrifugation and dried with argon to obtain compound ER-B-N2, which did not require further purification.

[0072] Example 3

[0073]

[0074] A3 (1.27 mmol) and B3 (1.27 mmol) were stirred in 30 mL of dry toluene (dehydrated with molecular sieves before use). 1 mL of piperidine and 1 mL of glacial acetic acid were added as catalysts. The reaction was heated to reflux at 150°C. The water produced during the reaction was removed using a water separator. The reaction was monitored by TLC until completion. After cooling to room temperature, most of the toluene in the reaction solution was removed under reduced pressure. The resulting liquid was diluted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 10) to obtain solid C3.

[0075]

[0076] Compound C3 (0.25 mmol), Zn powder (3.04 mmol), and NH4Cl (2.03 mmol) were dissolved in 12 mL of a 1:4 mixture of water and ethanol. 24 μL of glacial acetic acid was added and the reaction was allowed to proceed at 60°C. The reaction was monitored by TLC. After completion of the reaction, the zinc powder was removed by filtration, and the product was extracted with a saturated sodium bicarbonate solution and dichloromethane. The solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to obtain compound D3.

[0077]

[0078] Thiophosgene (1.86 mmol) and triethylamine (0.93 mmol) were dissolved in 5 mL of dichloromethane. Compound D3 (0.16 mmol) in 5 mL of dichloromethane was added under ice-cooling. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1) to obtain Compound E3.

[0079]

[0080] i) Compound b3 (1.18 mmol) was dissolved in 5 mL of water, triethylamine (300 μL) was added, p-toluenesulfonyl chloride (1.18 mmol) was dissolved in 10 mL of acetonitrile, and the aqueous solution containing compound b3 was slowly added. The reaction was monitored by TLC. After the reaction was completed, the mixture was washed three times with saturated aqueous ammonium chloride. The collected organic phase was dried over anhydrous sodium sulfate, filtered to remove the anhydrous sodium sulfate, and the solvent was removed under vacuum. The crude product was separated and purified by silica gel column chromatography (dichloromethane / methanol = 40 / 1) to obtain compound F3-Boc.

[0081] ii) Compound F2-Boc (0.5 mmol) was dissolved in 5 mL of dichloromethane, and 5 mL of trifluoroacetic acid was added thereto, and the mixture was reacted at room temperature. After the reaction was completed, the solvent was removed under vacuum to obtain Compound F3.

[0082]

[0083] Compound F3 (0.11 mmol) and compound D3 (0.09 mmol) were dissolved in 5 mL of acetone, and triethylamine (300 μL) was added. The reaction was carried out in an ice bath. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to obtain ER-B3.

[0084]

[0085] Compound ER-B3 (1.21 μmol) was dissolved in 0.5 mL of a 10:1 tetrahydrofuran:acetonitrile mixture. G3 (1.45 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride catalyst (1.45 μmol) were added sequentially under an ice bath. The reaction was carried out at room temperature and monitored by TLC. The solvent was removed under vacuum, and the mixture was repeatedly washed with n-hexane. The supernatant was removed by centrifugation and dried with argon to obtain compound ER-B-N3, which did not require purification.

[0086] Example 4

[0087]

[0088] A4 (1.27 mmol) and B4 (1.27 mmol) were stirred in 30 mL of dry toluene (dehydrated with molecular sieves before use). 1 mL of piperidine and 1 mL of glacial acetic acid were added as catalysts. The reaction was heated to reflux at 150°C. Water produced during the reaction was removed using a water separator. The reaction was monitored by TLC until completion. After cooling to room temperature, most of the toluene in the reaction solution was removed under reduced pressure. The resulting liquid was diluted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 10) to obtain solid C4.

[0089]

[0090] Compound C4 (0.25 mmol), Zn powder (3.04 mmol), and NH4Cl (2.03 mmol) were dissolved in 12 mL of a 1:4 mixture of water and ethanol. 24 μL of glacial acetic acid was added and the reaction was allowed to proceed at 60°C. The reaction was monitored by TLC. After completion of the reaction, the zinc powder was removed by filtration, and the mixture was extracted with a saturated sodium bicarbonate solution and dichloromethane. The solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to obtain compound D4.

[0091]

[0092] Dissolve thiophosgene (1.86 mmol) and triethylamine (0.93 mmol) in 5 mL of dichloromethane. Add 5 mL of a dichloromethane solution containing compound D4 (0.16 mmol) under ice-cooling. Monitor the reaction by TLC. After completion of the reaction, extract with water and dichloromethane. The crude product is purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1) to obtain compound E4.

[0093]

[0094] i) Compound b4 (1.18 mmol) was dissolved in 5 mL of water, triethylamine (300 μL) was added, p-toluenesulfonyl chloride (1.18 mmol) was dissolved in 10 mL of acetonitrile, and the aqueous solution containing compound b4 was slowly added. The reaction was monitored by TLC. After the reaction was completed, the mixture was washed three times with saturated aqueous ammonium chloride. The collected organic phase was dried over anhydrous sodium sulfate, filtered to remove the anhydrous sodium sulfate, and the solvent was removed under vacuum. The crude product was separated and purified by silica gel column chromatography (dichloromethane / methanol = 40 / 1) to obtain compound F4-Boc.

[0095] ii) Compound F4-Boc (0.5 mmol) was dissolved in 5 mL of dichloromethane, and 5 mL of trifluoroacetic acid was added thereto, and the mixture was reacted at room temperature. After the reaction was completed, the solvent was removed under vacuum to obtain Compound F4.

[0096]

[0097] Compound F4 (0.11 mmol) and compound D4 (0.09 mmol) were dissolved in 5 mL of acetone, and triethylamine (300 μL) was added. The reaction was carried out in an ice bath. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to obtain ER-B4.

[0098]

[0099] Compound ER-B4 (1.21 μmol) was dissolved in 0.5 mL of a 10:1 tetrahydrofuran:acetonitrile mixture. G4 (1.45 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride catalyst (1.45 μmol) were added sequentially under an ice bath. The reaction was carried out at room temperature and monitored by TLC. The solvent was removed under vacuum, and the mixture was repeatedly washed with n-hexane. The supernatant was removed by centrifugation and dried with argon to obtain compound ER-B-N4 without further purification.

[0100] Example 5

[0101]

[0102] A5 (1.27 mmol) and B5 (1.27 mmol) were stirred in 30 mL of dry toluene (dehydrated with molecular sieves before use). 1 mL of piperidine and 1 mL of glacial acetic acid were added as catalysts. The reaction was heated to reflux at 150°C. The water produced during the reaction was removed using a water separator. The reaction was monitored by TLC until completion. After cooling to room temperature, most of the toluene in the reaction solution was removed under reduced pressure. The resulting liquid was diluted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 10) to obtain solid C5.

[0103]

[0104] Compound C5 (0.25 mmol), Zn powder (3.04 mmol), and NH4Cl (2.03 mmol) were dissolved in 12 mL of a 1:4 mixture of water and ethanol. 24 μL of glacial acetic acid was added and the reaction was allowed to proceed at 60°C. The reaction was monitored by TLC. After completion of the reaction, the zinc powder was removed by filtration, and the mixture was extracted with a saturated sodium bicarbonate solution and dichloromethane. The solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to obtain compound D5.

[0105]

[0106] Thiophosgene (1.86 mmol) and triethylamine (0.93 mmol) were dissolved in 5 mL of dichloromethane. Compound D5 (0.16 mmol) in 5 mL of dichloromethane was added under ice-cooling. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1) to obtain compound E5.

[0107]

[0108] i) Compound b1 (1.18 mmol) was dissolved in 5 mL of water, and triethylamine (300 μL) was added. Compound a2 (1.18 mmol) was dissolved in 10 mL of acetonitrile, and the aqueous solution containing compound b1 was slowly added. The reaction was monitored by TLC. After the reaction was completed, the mixture was washed three times with saturated aqueous ammonium chloride. The collected organic phase was dried over anhydrous sodium sulfate, filtered to remove the anhydrous sodium sulfate, and the solvent was removed under vacuum. The crude product was separated and purified by silica gel column chromatography (dichloromethane / methanol = 40 / 1) to obtain compound F5-Boc.

[0109] ii) Compound F2-Boc (0.5 mmol) was dissolved in 5 mL of dichloromethane, and 5 mL of trifluoroacetic acid was added thereto, and the mixture was reacted at room temperature. After the reaction was completed, the solvent was removed under vacuum to obtain Compound F5.

[0110]

[0111] Compound F5 (0.11 mmol) and compound E5 (0.09 mmol) were dissolved in 5 mL of acetone, and triethylamine (300 μL) was added. The reaction was carried out in an ice bath. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to obtain ER-B5.

[0112]

[0113] Compound ER-B5 (1.21 μmol) was dissolved in 0.5 mL of a 10:1 tetrahydrofuran:acetonitrile mixture. G5 (1.45 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride catalyst (1.45 μmol) were added sequentially under an ice bath. The reaction was carried out at room temperature and monitored by TLC. The solvent was removed under vacuum, and the mixture was repeatedly washed with n-hexane. The supernatant was removed by centrifugation and dried with argon to obtain compound ER-B-N5, which did not require purification.

[0114] Example 6

[0115]

[0116] i) Compound b4 (1.18 mmol) was dissolved in 5 mL of water, and triethylamine (300 μL) was added. Compound a1 (1.18 mmol) was dissolved in 10 mL of acetonitrile, and the aqueous solution containing compound b4 was slowly added. The reaction was monitored by TLC. After the reaction was completed, the mixture was washed three times with saturated aqueous ammonium chloride. The collected organic phase was dried over anhydrous sodium sulfate, filtered to remove the anhydrous sodium sulfate, and the solvent was removed under vacuum. The crude product was separated and purified by silica gel column chromatography (dichloromethane / methanol = 40 / 1) to obtain compound F6-Boc.

[0117] ii) Compound F4-Boc (0.5 mmol) was dissolved in 5 mL of dichloromethane, and 5 mL of trifluoroacetic acid was added thereto, and the mixture was reacted at room temperature. After the reaction was completed, the solvent was removed under vacuum to obtain Compound F6.

[0118]

[0119] Compound F6 (0.11 mmol) and compound D1 (0.09 mmol) were dissolved in 5 mL of acetone, and triethylamine (300 μL) was added. The reaction was carried out in an ice bath. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to obtain ER-B6.

[0120]

[0121] Compound ER-B6 (1.21 μmol) was dissolved in 0.5 mL of a 10:1 tetrahydrofuran:acetonitrile mixture. G1 (1.45 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride catalyst (1.45 μmol) were added sequentially under an ice bath. The reaction was carried out at room temperature and monitored by TLC. The solvent was removed under vacuum, and the mixture was repeatedly washed with n-hexane. The supernatant was removed by centrifugation and dried with argon to obtain compound ER-B-N6 without further purification.

[0122] Example 7

[0123]

[0124] A2 (1.27 mmol) and B7 (1.27 mmol) were stirred in 30 mL of dry toluene (dehydrated with molecular sieves before use). 1 mL of piperidine and 1 mL of glacial acetic acid were added as catalysts. The reaction was heated to reflux at 150°C. The water produced during the reaction was removed using a water separator. The reaction was monitored by TLC until completion. After cooling to room temperature, most of the toluene in the reaction solution was removed under reduced pressure. The resulting liquid was diluted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 10) to obtain solid C7.

[0125]

[0126] Compound C7 (0.25 mmol), Zn powder (3.04 mmol), and NH4Cl (2.03 mmol) were dissolved in 12 mL of a 1:4 mixture of water and ethanol. 24 μL of glacial acetic acid was added and the reaction was allowed to proceed at 60°C. The reaction was monitored by TLC. After completion of the reaction, the zinc powder was removed by filtration, and the mixture was extracted with a saturated sodium bicarbonate solution and dichloromethane. The solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to obtain compound D7.

[0127]

[0128] Thiophosgene (1.86 mmol) and triethylamine (0.93 mmol) were dissolved in 5 mL of dichloromethane. A solution of compound D2 (0.16 mmol) in 5 mL of dichloromethane was added under ice-cooling. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1) to obtain compound E7.

[0129]

[0130] Compound E7 (0.11 mmol) and compound F2 (0.09 mmol) were dissolved in 5 mL of acetone, and triethylamine (300 μL) was added. The reaction was carried out in an ice bath. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to obtain ER-B7.

[0131]

[0132] Compound ER-B7 (1.21 μmol) was dissolved in 0.5 mL of a 10:1 tetrahydrofuran:acetonitrile mixture. G7 (1.45 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride catalyst (1.45 μmol) were added sequentially under an ice bath. The reaction was carried out at room temperature and monitored by TLC. The solvent was removed under vacuum, and the mixture was repeatedly washed with n-hexane. The supernatant was removed by centrifugation and dried with argon to obtain compound ER-B-N7, which did not require purification.

[0133] Example 8

[0134]

[0135] A3 (1.27 mmol) and B8 (1.27 mmol) were stirred in 30 mL of dry toluene (dehydrated with molecular sieves before use). 1 mL of piperidine and 1 mL of glacial acetic acid were added as catalysts. The reaction was heated to reflux at 150°C. The water produced during the reaction was removed using a water separator. The reaction was monitored by TLC until completion. After cooling to room temperature, most of the toluene in the reaction solution was removed under reduced pressure. The resulting liquid was diluted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 10) to obtain solid C8.

[0136]

[0137] Compound C8 (0.25 mmol), Zn powder (3.04 mmol), and NH4Cl (2.03 mmol) were dissolved in 12 mL of a 1:4 mixture of water and ethanol. 24 μL of glacial acetic acid was added and the reaction was allowed to proceed at 60°C. The reaction was monitored by TLC. After completion of the reaction, the zinc powder was removed by filtration, and the mixture was extracted with a saturated sodium bicarbonate solution and dichloromethane. The solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to obtain compound D8.

[0138]

[0139] Thiophosgene (1.86 mmol) and triethylamine (0.93 mmol) were dissolved in 5 mL of dichloromethane. Compound D8 (0.16 mmol) in 5 mL of dichloromethane was added under ice-cooling. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1) to obtain compound E8.

[0140]

[0141] Compound F3 (0.11 mmol) and compound D8 (0.09 mmol) were dissolved in 5 mL of acetone, and triethylamine (300 μL) was added. The reaction was carried out in an ice bath. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to obtain ER-B8.

[0142]

[0143] Compound ER-B8 (1.21 μmol) was dissolved in 0.5 mL of a 10:1 tetrahydrofuran:acetonitrile mixture. G3 (1.45 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride catalyst (1.45 μmol) were added sequentially under an ice bath. The reaction was carried out at room temperature and monitored by TLC. The solvent was removed under vacuum, and the mixture was repeatedly washed with n-hexane. The supernatant was removed by centrifugation and dried with argon to obtain compound ER-B-N8, which did not require purification.

[0144] Example 9

[0145]

[0146] A4 (1.27 mmol) and B9 (1.27 mmol) were stirred in 30 mL of dry toluene (dehydrated with molecular sieves before use). 1 mL of piperidine and 1 mL of glacial acetic acid were added as catalysts. The reaction was heated to reflux at 150°C. Water produced during the reaction was removed using a water separator. The reaction was monitored by TLC until completion. After cooling to room temperature, most of the toluene in the reaction solution was removed under reduced pressure. The resulting liquid was diluted with dichloromethane, washed with water, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 10) to obtain solid C9.

[0147]

[0148] Compound C9 (0.25 mmol), Zn powder (3.04 mmol), and NH4Cl (2.03 mmol) were dissolved in 12 mL of a 1:4 mixture of water and ethanol. 24 μL of glacial acetic acid was added and the reaction was allowed to react at 60°C, monitored by TLC. After completion of the reaction, the zinc powder was removed by filtration, and the mixture was extracted with a saturated sodium bicarbonate solution and dichloromethane. The solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to obtain compound D9.

[0149]

[0150] Thiophosgene (1.86 mmol) and triethylamine (0.93 mmol) were dissolved in 5 mL of dichloromethane. Compound D4 (0.16 mmol) in 5 mL of dichloromethane was added under ice-cooling. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1) to obtain compound E9.

[0151]

[0152] Compound F4 (0.11 mmol) and compound D9 (0.09 mmol) were dissolved in 5 mL of acetone, and triethylamine (300 μL) was added. The reaction was carried out in an ice bath. The reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with water and dichloromethane, and the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to obtain ER-B9.

[0153]

[0154] Compound ER-B9 (1.21 μmol) was dissolved in 0.5 mL of a 10:1 tetrahydrofuran:acetonitrile mixture. G9 (1.45 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride catalyst (1.45 μmol) were added sequentially under an ice bath. The reaction was carried out at room temperature and monitored by TLC. The solvent was removed under vacuum, and the mixture was repeatedly washed with n-hexane. The supernatant was removed by centrifugation and dried with argon to obtain compound ER-B-N9 without further purification.

[0155] Example 10

[0156]

[0157] Compound ER-B5 (1.21 μmol) was dissolved in 0.5 mL of a 10:1 tetrahydrofuran:acetonitrile mixture. G10 (1.45 μmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride catalyst (1.45 μmol) were added sequentially under an ice bath. The reaction was carried out at room temperature and monitored by TLC. The solvent was removed under vacuum, and the mixture was repeatedly washed with n-hexane. The supernatant was removed by centrifugation and dried with argon to obtain compound ER-B-N10, which did not require further purification.

[0158] Example 11

[0159] The properties of compound ER-B6 were studied by dissolving ER-B6 in a PBS solution (containing 70% MeOH) containing 1 μM hemin and 1 mM cumene hydroperoxide, and scanning the changes in UV absorption and fluorescence emission wavelengths over time.

[0160] like Figure 1 As shown, the conjugated diene structure in ER-B6 results in a maximum absorption peak at 580 nm, with strong red fluorescence at 620 nm. Due to the oxidative cleavage of the conjugated diene portion under the action of reactive oxygen species, the cleavage of the double bond leads to a decrease in the conjugated system, manifested as a significant decrease in the absorption and emission wavelengths. 1 μM hemin and 1 mM cumene hydroperoxide were added to PBS (containing 70% MeOH) to simulate the in vivo LPO process. Figure 1 As shown in the figure, the maximum absorption wavelength of ER-B6 decreases from 580nm to 500nm, and the maximum emission peak decreases from 620nm to 510nm. At the same time, as time goes by, the absorption peak at 580nm gradually weakens while the absorption peak intensity at 500nm gradually increases, the emission peak intensity at 620nm gradually weakens, and the emission peak intensity at 510nm gradually increases. The ratio of the two peak intensities is 510 / 620 The significant ratio change from 0.81 to 8.52 can improve the accuracy of ER-B6 quantitative monitoring of LPO process.

[0161] Example 12

[0162] Fluorescence imaging experiments were performed on the compound ER-B-N6. A549 cells, Hela cells, and RAW 264.7 cells were incubated in a cell incubator at 37°C and 5% CO2 for 24 hours. The cells were then incubated with 3.5 μM ER-BN and 1 μM commercial endoplasmic reticulum probe ER-Tracker Green for 1 hour, washed three times with PBS, and then confocal imaging was performed.

[0163] like Figure 2 As shown, the red channel of the probe ER-B-N6 almost completely overlaps with the green channel of the commercial endoplasmic reticulum probe. ER-BN has high specificity for the endoplasmic reticulum in these three cell types, with Pearson coefficients all above 0.85. In addition, in A549 cells and Hela cells, the reticular structure of the endoplasmic reticulum is clearly visible in the red channel of ER-B-N6. These experimental results indicate that ER-B-N6 can achieve endoplasmic reticulum-specific staining in different types of living cells.

[0164] Example 13

[0165] After incubating A549 cells in a 37°C, 5% CO2 incubator for 24 hours, the cells were stained with 3.5 μM E-BN for 1 hour, the culture medium in the culture dish was aspirated, and the cells were washed twice with PBS before imaging. 1 mL of 4% paraformaldehyde solution (PFA) was then added to the culture dish, and the cells were fixed for 20 minutes at room temperature. The cells were then washed again three times with PBS buffer to remove excess PFA before confocal imaging.

[0166] Imaging results such as Figure 3 As shown in the figure, there is no obvious change in the fluorescence intensity of ER-B-N6 before and after cell fixation, and the ER reticular outline labeled with ER-B-N6 is still clearly visible, indicating that the active ester part in ER-B-N6 successfully reacts with the protein on the ER and fixes the probe molecule on the ER. Therefore, it is feasible to use ER-B-N6 to image the ER for a long time.

[0167] Example 14

[0168] After incubating A549 cells in a 37°C, 5% CO2 cell incubator for 24 hours, 3.5 μM E-B-N6 was used to stain the A549 cells. The stained cells were stimulated with LPS (1 μg / mL) and confocal imaging was performed at different time periods after drug stimulation. The imaging results are shown in Figure 2. Figure 4 As shown in the figure, as the drug stimulation time increases, the fluorescence intensity of the green channel gradually increases, while the fluorescence intensity of the red channel gradually decreases. At the same time, the degree of LPO can be intuitively described by the change in the fluorescence intensity ratio. This result effectively proves that ER-B-N6 can monitor the LPO process in the endoplasmic reticulum in inflammatory cells.

Claims

1. A compound having the following general structural formula: in, n is 1, 2, 3, 4 or 5; R1 and R2 are each independently H, Cl, an alkyl group having 1 to 10 carbon atoms, or an alkyl group substituted with an alkoxycarbonyl group having 2 to 15 carbon atoms; R3 is or R4 is or Y is a phenyl group or a phenyl group containing at least one substituent; The substituent is selected from C1-C10 alkyl, C1-C10 alkoxy, hydroxy, nitro, and amino.

2. The compound according to claim 1, characterized in that R1 and R2 are each independently H, Cl, an alkyl group having 1 to 5 carbon atoms, or an alkyl group substituted with an alkoxycarbonyl group having 2 to 10 carbon atoms; The substituent is selected from C1-C5 alkyl, C1-C5 alkoxy, hydroxy, nitro, and amino.

3. The compound according to claim 1, characterized in that R1 and R2 are each independently H, Cl, an alkyl group having 1 to 3 carbon atoms, or an alkyl group substituted with an alkoxycarbonyl group having 3 to 6 carbon atoms; The substituent is selected from C1-C5 alkyl, C1-C5 alkoxy, hydroxy, nitro, and amino.

4. The compound according to claim 1, characterized in that R1 and R2 are each independently H, Cl, or an alkyl group having 1 to 3 carbon atoms; Y is a phenyl group or a phenyl group containing 1-2 substituents; The substituent is selected from C1-C5 alkyl and C1-C5 alkoxy.

5. The compound according to claim 1, characterized in that R1 and R2 are each independently H, Cl, CH3, or C2H5; R3 is R4 is Y is 6. The compound according to claim 1, characterized in that R1 and R2 are each independently H, Cl, or CH3; R3 is R4 is Y is 7. Use of the compound according to any one of claims 1 to 6 in the preparation of a drug that targets the endoplasmic reticulum and monitors endoplasmic reticulum lipid peroxidation.

8. Use of the compound according to any one of claims 1 to 6 in the preparation of drugs for detecting, tracking or targeting the endoplasmic reticulum.

9. Use of the compound according to any one of claims 1 to 6 in the preparation of a drug for tracking the location of bacterial infection or inflammation in the endoplasmic reticulum.

10. Use of the compound according to any one of claims 1 to 6 in the preparation of a lipid peroxidation fluorescent indicator.

Images