Malononitrile-based endoplasmic reticulum hypochlorous acid fluorescent probe, preparation method and application thereof

Abstract

This invention discloses an endoplasmic reticulum hypochlorous acid fluorescent probe based on malononitrile, its preparation method, and its application. The method includes: dissolving 2-(3,5,5-trimethylcyclohexyl-2-en-1-methylene)malononitrile, 3-fluoro-4-hydroxybenzaldehyde, and piperidine in acetonitrile; removing the solvent after the reaction to obtain 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-methylene)malononitrile; dissolving 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-methylene)malononitrile, triethylamine, and 4-dimethylaminopyridine in N,N-dimethylformamide; adding N,N-dimethylcarbamoyl chloride; and extracting after the reaction to obtain methyl O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate. The endoplasmic reticulum hypochlorous acid fluorescent probe of the present invention exhibits almost no fluorescence in the absence of hypochlorous acid, but displays strong fluorescence at 668 nm after the addition of hypochlorous acid. It combines the advantages of high sensitivity to changes in hypochlorous acid, good selectivity, and large Stokes shift.

Description

Technical Field

[0001] This invention belongs to the field of endoplasmic reticulum hypochlorous acid fluorescent probe technology. More specifically, this invention relates to an endoplasmic reticulum hypochlorous acid fluorescent probe based on malononitrile, its preparation method, and its application. Background Technology

[0002] The endoplasmic reticulum (ER) is the largest membrane-bound organelle in eukaryotic cells, performing a variety of key cellular functions, including protein synthesis and processing, lipid synthesis, and calcium (Ca) storage and release. The large amount of membrane in the ER makes it susceptible to oxidative stress, which in turn affects cell structure and function. Hypochlorous acid (HOCl) is one of the important reactive oxygen species, formed by myeloperoxidase and related enzymes from H₂O₂ and chloride / pseudochloride ions (Cl₂O₂). - Hypochlorous acid (HCO3) is a reactive species produced in the endoplasmic reticulum. At normal concentrations, HCO3 serves as part of the immune defense process by eliminating invading bacteria and pathogens. However, improperly located or excessive HCO3 can alter nucleic acids, proteins, and lipids through oxidation and / or chlorination, leading to tissue damage. Furthermore, HCO3 can be released extracellularly, making these species and their products potential secondary signaling mediators. Most tissue damage caused by abnormal HCO3 levels is associated with a variety of diseases, such as atherosclerosis, neurodegenerative diseases, Alzheimer's disease, and even cancer. Therefore, accurate measurement of HCO3 levels in the endoplasmic reticulum is crucial for understanding disease formation and prevention.

[0003] In recent years, some small organic molecule fluorescent probes have been reported in the literature for monitoring hypochlorous acid levels in vivo and in vitro. However, most of these probes cannot cross the blood-brain barrier (BBB), and therefore cannot be used for hypochlorous acid imaging in the brain. Therefore, it is necessary to develop a fluorescent probe that can cross the blood-brain barrier and target the endoplasmic reticulum for detecting hypochlorous acid levels in intracellular and nervous system-related diseases. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0005] To achieve these and other advantages according to the present invention, an endoplasmic reticulum hypochlorous acid fluorescent probe based on malononitrile is provided, the chemical name of which is methyl O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate, and the structural formula of which is: .

[0006] A method for preparing an endoplasmic reticulum hypochlorous acid fluorescent probe based on malononitrile, comprising: 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonitrile, triethylamine, and 4-dimethylaminopyridine were dissolved in N,N-dimethylformamide, and then N,N-dimethylcarbamoyl chloride was added to carry out the reaction. The product was extracted with water / ethyl acetate and purified by silica gel column chromatography to obtain pure O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate.

[0007] Preferably, the molar equivalent ratio of 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malononitrile, triethylamine, 4-dimethylaminopyridine and N,N-dimethylformamide is 1 : 3~8 : 0.05~0.15 : 30~60; The molar equivalent of N,N-dimethylcarbamoyl chloride is 1.5 times that of 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile.

[0008] Preferably, the reaction temperature is 20~45 ℃ and the reaction time is 3~16 h.

[0009] Preferably, the volume ratio of the product, water, and ethyl acetate is 1:10:10.

[0010] Preferably, the structural formula of 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malononitrile is: .

[0011] Preferably, the method for preparing 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malononitrile includes: 2-(3,5,5-trimethylcyclohexyl-2-en-1-methylene)malonitrile and 3-fluoro-4-hydroxybenzaldehyde were dissolved in acetonitrile, piperidine was added, and the reaction was carried out. The solvent was removed to obtain crude 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-methylene)malonitrile, which was then separated by silica gel column chromatography to obtain pure 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-methylene)malonitrile.

[0012] Preferably, the molar equivalent ratio of 2-(3,5,5-trimethylcyclohexyl-2-ene-1-methylene)malonitrile, 3-fluoro-4-hydroxybenzaldehyde, piperidine and acetonitrile is 1 : 0.8~1.2 : 0.8~5 : 25~70.

[0013] Preferably, the reaction temperature is 50~90℃ and the reaction time is 0.5~8 h.

[0014] An application of a malononitrile-based fluorescent probe for endoplasmic reticulum hypochlorous acid, wherein the fluorescent probe is used for fluorescence imaging of hypochlorous acid in the endoplasmic reticulum of live cells and the brain of live mice.

[0015] The present invention includes at least the following beneficial effects: Compared with existing hypochlorous acid fluorescent probes, the probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate synthesized in the present invention has the following advantages: (1) The maximum fluorescence emission of the endoplasmic reticulum hypochlorous acid fluorescent probe synthesized in this invention is located at around 668 nm, which is in the near-infrared region. This can effectively reduce the interference of autofluorescence in biological samples and improve tissue penetration. (2) The endoplasmic reticulum hypochlorous acid fluorescent probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate has a large Stokes shift (~168 nm), which can effectively reduce interference from excitation light; (3) The endoplasmic reticulum hypochlorous acid fluorescent probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate has good selectivity for hypochlorous acid response and is not affected by common anions, cations, amino acids, other reactive oxygen species (ROS) and reactive nitrogen species (RNS); (4) The endoplasmic reticulum hypochlorous acid fluorescent probe has excellent endoplasmic reticulum targeting and localization capabilities, and can be used to achieve imaging detection of endoplasmic reticulum hypochlorous acid using laser confocal imaging technology; (5) The synthesis steps of the endoplasmic reticulum hypochlorous acid fluorescent probe of the present invention are simple and low in cost, and have potential commercial application value.

[0016] The endoplasmic reticulum hypochlorous acid fluorescent probe prepared in this invention exhibits almost no fluorescence in the absence of hypochlorous acid, but displays strong fluorescence at 668 nm upon the addition of hypochlorous acid. This probe combines high sensitivity to changes in hypochlorous acid (detection limit 1.4676 μM), good selectivity, and a large Stokes shift (168 nm). Fluorescence imaging confirmed good co-localization of the probe with commercially available endoplasmic reticulum-specific selective dyes, and it was successfully applied to monitor changes in endoplasmic reticulum hypochlorous acid in human cervical cancer cells (HeLa). In vivo imaging in mice confirmed that the probe can penetrate the blood-brain barrier to image the hypochlorous acid content in the brain, and imaging using this probe revealed that the hypochlorous acid content in the brain / cerebellum of BTBR autistic mice (ASD) was lower than that in normal mice.

[0017] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0018] Figure 1 This is a graph showing the UV-Vis absorption spectrum of the probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate in Example 1 of the present invention as the concentration of hypochlorous acid increases. Figure 2 This is the fluorescence emission spectrum of probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate as the concentration of hypochlorous acid increases in Example 1 of the present invention. Figure 3 This is a graph showing the fluorescence intensity of probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate at 668 nm as a function of hypochlorous acid concentration in Example 1 of this invention. Figure 4 In Example 1 of this invention, the fluorescence intensity of probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate at 668 nm showed a linear range of 0-400 μM with the concentration of hypochlorous acid, and the detection limit was 268 nM. Figure 5 This is a graph showing the change in fluorescence intensity at 668 nm of probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate over time before and after the addition of hypochlorous acid in Example 1 of this invention. Figure 6The selectivity of probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate to hypochlorous acid in the presence of common anions / cations and some common amino acids, reactive oxygen species, and reactive nitrogen species in organisms in Example 1 of this invention. Figure 7 The photostability of probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate in the presence and absence of hypochlorous acid in Example 1 of this invention; Figure 8 This is a co-localization imaging image of the probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate with the commercially available endoplasmic reticulum-specific selective dye ER Tracker Green in human cervical cancer cells (HeLa) in Example 1 of the present invention. Figure 9 Laser confocal imaging images of HeLa cells after treatment with blank, hypochlorous acid (5 μM), phorbol-12-myristate-13-acetate (PMA, 10 μM), and N-acetylcysteine ​​(NAC, 2 mM) / PMA (10 μM) for 30 min, respectively, followed by co-incubation with the probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate from Example 1 for 30 min. Figure 10 This is a statistical graph of the average intracellular fluorescence intensity in Embodiment 9 of the present invention; Figure 11 This is a real-time in vivo fluorescence image of the probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate injected into the head of a hairless C57 mouse via the tail vein in Example 1 of this invention. Figure 12 This is a graph showing the change in fluorescence intensity of the mouse head over time in Example 10 of this embodiment of the invention; Figure 13 The images show fluorescence images of various isolated tissues and organs after the probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate was injected into C57 mice via the tail vein in Example 1 of this invention. Figure 14 This is a statistical graph of the fluorescence intensity of various isolated tissues and organs in Example 11 of the present invention; Figure 15The images show in vivo fluorescence images of C57 mice (left) and BTBR autistic mice (ASD, right) after the probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate was injected into the head via the tail vein in Example 1 of this invention. Figure 16 This is a statistical graph of fluorescence intensity in the mouse head in Example 12 of the present invention; Figure 17 This is an in situ fluorescence imaging image of a mouse after craniotomy in Example 12 of this invention; Figure 18 This is a statistical graph of fluorescence intensity of mouse brain organs in Example 12 of the present invention. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0020] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof. Example 1 The preparation routes for compounds 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malononitrile and probe O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate are as follows: The specific preparation method includes the following steps: (1) To a solution of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile (1 g, 5.37 mmol) and 3-fluoro-4-hydroxybenzaldehyde (752.27 mg, 5.37 mmol) in acetonitrile (10 mL), piperidine (914.32 mg, 10.74 mmol, 1.06 mL) was added. The mixture was stirred at 80 °C for 1 h. After the reaction was complete, the reaction mixture was concentrated under reduced pressure to remove ethanol. The residue was diluted with 1 M hydrochloric acid (50 mL) and extracted with ethyl acetate (50 mL × 2). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by rapid silica gel chromatography, using hexane containing 0–45% ethyl acetate as a gradient eluent to give 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonadionitrile (1.2 g, 3.89 mmol, yield 72.48%) as a red solid.

[0021] The 1H NMR spectrum of the product is as follows: 1 H NMR (400 MHz, DMSO) δ 10.41 (s, 1H), 7.59 (dd, J =12.7, 2.1 Hz, 1H), 7.32 (dd, J = 8.4, 2.0Hz, 1H), 7.26 (d, J = 16.1 Hz, 1H), 7.18(d, J = 16.2 Hz, 1H), 6.96 (t, J = 8.7 Hz, 1H), 6.82 (s, 1H), 2.59 (s, 2H), 2.50(d, J = 1.9 Hz, 2H), 1.01 (s, 6H).

[0022] The carbon NMR spectrum of the product is as follows: 13 C NMR (101 MHz, DMSO) δ 170.75, 156.64, 152.89,150.49, δ 147.14 (d, J = 12.5 Hz, 1C), 137.44 (d, J = 2.6 Hz, 1C), 128.53 (d, J =6.5 Hz, 1C), 128.26, 126.09 (d, J = 2.8 Hz, 1C), 122.51, 118.35 (d, J = 3.2 Hz, 1C), 115.30, 115.12, 114.46, 113.65, 76.02, 42.75, 38.62, 32.14, 27.90.

[0023] The calculated high-resolution mass spectrometry value of the product is: MS: 307.12522 [MH] - The discovery value (ESI) is: 307.12534 [MH] - .

[0024] (2) To a solution of 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonidium (500 mg, 1.62 mmol), triethylamine (820.41 mg, 8.11 mmol, 1.13 mL), and 4-dimethylaminopyridine (19.81 mg, 162.15 μmol) in N,N-dimethylformamide (5 mL), N,N-dimethylcarbamoyl chloride (300.64 mg, 2.43 mmol) was added. The mixture was stirred at 25°C for 12 hours. After the reaction was complete, the reaction mixture was partitioned between water (50 mL) and ethyl acetate (50 mL). The organic phase was separated, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by preparative high-performance liquid chromatography (column: CD18-Welch Ultimate C18 150×40×7μm; mobile phase: [water (containing 0.225% formic acid)-acetonitrile]; gradient: acetonitrile content increased from 9% to 39% within 17 minutes) to obtain O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate (260 mg, 654.52 μmol, yield 40.36%), as a yellow solid.

[0025] The 1H NMR spectrum of the product is as follows: 1 H NMR (400 MHz, CDCl3) δ 7.30 (ddd, J = 9.8, 5.0, 2.1Hz, 2H), 7.15 (t, J = 8.2 Hz, 1H), 6.98 (d, J = 16.1 Hz, 1H), 6.92 (d, J = 16.2 Hz, 1H), 6.85 (d, J = 1.3 Hz, 1H), 3.47 (s, 3H), 3.37 (s, 3H), 2.61 (s, 2H), 2.45(d, J = 1.4 Hz, 2H), 1.08 (s, 6H).

[0026] The carbon NMR spectrum of the product is as follows: 13 C NMR (101 MHz, CDCl3) δ 186.46, 169.09, 155.92,153.43, 153.05,142.07 (d, J =12.4 Hz, 1C), 135.21 (d, J = 6.9 Hz, 1C), 134.85 (d,J = 2.5 Hz), 130.36, 125.60, 124.26, 123.58 (d, J = 3.2 Hz), 115.13, 114.94,113.35, 112.55, 79.48, 43.65, 42.99, 39.20, 38.95, 32.07, 28.03.

[0027] The calculated high-resolution mass spectrometry value of the product is: 396.15404 [M+H] + The ESI (Expert Sense Index) value is: 396.15375 [M+H] + .

[0028] Example 2 The concentration of probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate in Example 1 was maintained at 10 μM, and it was tested in V containing different concentrations of hypochlorous acid. PBS V 乙腈 The absorption spectrum was measured in a 7:3 mixture. Figure 1 As the concentration of hypochlorous acid increased from 0 μM to 900 μM, the absorption peak at 392 nm gradually decreased, while the absorption peak at 500 nm correspondingly increased.

[0029] Example 3 The concentration of probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate in Example 1 was maintained at 10 μM, and it was tested in V containing different concentrations of hypochlorous acid. PBS V 乙腈 The fluorescence emission spectrum was measured in a 7:3 mixture system with a fixed excitation wavelength of 500 nm. Figure 2 In the absence of hypochlorous acid, the fluorescence of the solution is negligible; as the concentration of hypochlorous acid increases from 0 μM to 900 μM, a fluorescence emission peak appears at 668 nm and gradually intensifies. The relationship between the fluorescence intensity of the probe at 668 nm and the concentration of hypochlorous acid is as follows: Figure 3 The linear response range of hypochlorous acid is 0~400μM. Figure 4 The linear regression equation is: F 668 = 8.647×c HClO +182.9, correlation coefficient R 2 = 0.9883; detection limit is 268.4 nM.

[0030] Example 4 In V PBS V 乙腈 In a 7:3 mixture, the probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate from Example 1 was added to a final concentration of 10 μM. The fluorescence excitation and emission wavelengths were fixed at 500 nm and 668 nm, respectively. Real-time data acquisition was performed at 1-second intervals to record the change in fluorescence intensity before and after the addition of 900 μM (final concentration) hypochlorous acid, thus examining the probe's response time to hypochlorous acid. Figure 5 As shown, the fluorescence intensity of the probe reaches its maximum value and remains stable within 1 second after the addition of hypochlorous acid. Therefore, the response speed of the probe to changes in the concentration of hypochlorous acid is less than 1 second.

[0031] Example 5 The concentration of probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate in Example 1 was maintained at 10 μM, and V PBS V 乙腈 The response of the probe to common anions and cations, as well as some amino acids, ROS, and RNS from living organisms, was investigated in a 7:3 mixture system. Figure 7 As shown, the probe showed almost no response to the above substances, demonstrating that the probe has excellent selectivity for hypochlorous acid. Figure 6 The order and concentration of the substances in the solution are as follows: 1. Endoplasmic reticulum hypochlorous acid fluorescent probe; 2. HClO (1 mM); 3. Na+ + (10mM); 4, Cu 2+ (1mM); 5,Mg 2+ (1mM); 6, Fe 2+ (1mM); 7, Ba 2+ (1mM); 8, Ag + (1mM); 9, K + (140mM); 10, Co 2+ (1mM); 11, Zn 2+ (1mM); 12, SO4 2- (1mM); 13, NO3 - (1mM); 14,Br - (1mM); 15, CO3 2- (1mM); 16, HCO3 - (1mM); 17, I - (1mM); 18, HPO4 2-(1mM); 19, PO4 3- (1mM); 20, S2O3 2- (1mM); 21, S 2- (1mM); 22, HS 2- (1mM); 23, Fe 3+ (1mM); 24, H2O2 (1mM); 25, ONOO - (1mM); 26, O2 - (1mM); 27, 1 O2 (1mM); 28, Cl - (10mM); 29, GSH (1mM); 30, Cys (1mM); 31, Hcy (1mM); 32β-Alu (1mM); 33, L-Pro (1mM).

[0032] Example 6 In two V PBS V 乙腈 In a 7:3 mixture, the probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate and the probe / hypochlorous acid were added to the solutions in Example 1, resulting in concentrations of 10 μM and 10 μM / 900 μM, respectively. Both solutions were continuously irradiated under 500 nm excitation light, and the fluorescence intensity at 668 nm was recorded in real time to evaluate the photostability of the probe. Figure 7 As shown, the decrease in fluorescence intensity of the two solutions within 60 minutes of continuous irradiation is negligible, demonstrating that the probe has good photostability.

[0033] Example 7 To confirm whether the probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate in Example 1 possesses endoplasmic reticulum (ER) targeting capability, we conducted a co-localization experiment with the commercially available ER-specific selective fluorescent dye ER Tracker Green. Adherent HeLa cells were co-incubated with the probe (final concentration 10 μM) at 37°C and 5% CO2 for 30 min. Afterward, the cells were gently washed three times with PBS to remove excess probe. Then, ER Tracker Green (final concentration 2 μM) was added, and incubation continued for another 5 min. The co-localization was observed under a laser confocal microscope. The excitation wavelengths for both the probe and ER Tracker Green were set to 488 nm. The green acquisition channel (490–530 nm) was used for ER Tracker Green emission, and the dark red channel was used to acquire probe emission. Figure 8 The deep red fluorescence (red pseudocolor) of the probe is distributed in the cytoplasm, indicating that the probe has good cell membrane permeability and low toxicity. Furthermore, the deep red fluorescence of the probe overlaps well with the green fluorescence of ER Tracker Green, and after software processing, yellow fluorescence is obtained, indicating that the probe and ER Tracker Green have significant co-localization imaging and can target the endoplasmic reticulum.

[0034] Example 8 To demonstrate that the probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate in Case Study 1 has the ability to respond to changes in endoplasmic reticulum hypochlorous acid in living cells, we treated HeLa cells with hypochlorous acid, phorbol-12-myristate-13-acetate (PMA), and N-acetylcysteine ​​(NAC) to alter intracellular hypochlorous acid levels through both exogenous and endogenous mechanisms. Adherent HeLa cells were washed three times with PBS and then divided into four groups. The first group was cultured in DMEM (Dulbecco modified high-glucose medium) for 30 min; the second group was cultured in DMEM containing 5 μM HClO for 30 min; the third group was cultured in DMEM containing 10 μM PMA for 1 h; and the fourth group was first cultured in DMEM containing 2 mM NAC for 30 min and then further cultured in DMEM containing 10 μM PMA for 30 min. Subsequently, these mediums were removed, and the cells were further cultured for 30 min in DMEM containing 10 μM of the probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate from Example 1. All cultures were incubated at 37 ℃ in a 5% CO2 incubator. The cells were then gently washed three times with PBS to remove excess probe. The cells were then observed under a confocal microscope. Fluorescence emission was collected in the deep red channel (590–750 nm) with a fixed excitation wavelength of 488 nm. Only weak fluorescence was observed in the control group cells. Figure 9 Cells incubated with hypochlorous acid showed a significant increase in fluorescence intensity, and elevated fluorescence was also observed in cells treated with PMA and NAC / PMA. The increase in fluorescence intensity was: hypochlorous acid > PMA > NAC / PMA. Figure 10 These results indicate that the probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate in Case 1 can accurately respond to changes in the hypochlorous acid content of the endoplasmic reticulum.

[0035] Example 9 To demonstrate the ability of the probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate from Case 1 to cross the blood-brain barrier and to explore its in vivo metabolism, we performed real-time in vivo imaging in small animals. Before the imaging experiment, healthy C57 mice underwent head hair removal. The probe stock solution was then diluted to 400 μM with PBS, and 50 μL (1 nM / g; 345 μg / kg) was injected into the mice via the tail vein. The control group received the same volume of PBS into another mouse. Fluorescence was then simultaneously acquired from both mice in real time. Excitation and emission filters were fixed at 480 nm and 680 nm, respectively, with an exposure time of 200 ms. Compared with control mice (… Figure 11 Compared to mice injected with the probe (left), mice injected with the probe (left) Figure 11 A clear fluorescent signal was collected from the right head, indicating that the probe in Case 1 could effectively penetrate the blood-brain barrier. 25-30 minutes prior to the event (…), Figure 12 As the injection time increased, the signal in the mouse head gradually increased, and then gradually decreased after 25-30 minutes. This phenomenon indicates that the optimal window for observing changes in hypochlorous acid in the mouse brain using this probe is 25-30 minutes.

[0036] Example 10 To further demonstrate the ability of the probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate from Case 1 to cross the blood-brain barrier and its metabolic distribution in vivo, we further performed in vitro imaging of major organs in C57 mice. The probe stock solution was diluted to 400 μM with PBS, and 50 μL (1 nM / g; 345 μg / kg) was injected into mice via the tail vein. Mice were then euthanized by cervical dislocation at 27 min. Subsequently, the brain, cerebellum, heart, liver, spleen, lungs, and kidneys of the mice were isolated, washed with cold PBS, and then imaged using a live imaging system. Excitation and emission filters were fixed at 480 nm and 680 nm, respectively, with an exposure time of 200 ms. Figure 13 The results showed that significant fluorescence was observed in the brain, cerebellum, liver, lungs, and kidneys of mice, with the strongest fluorescence observed in the lungs. Figure 14 The liver was the next most affected organ. This result further demonstrates that the probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-2-fluorophenyl) dimethyl thiocarbamate in Implementation Case 1 has the ability to cross the blood-brain barrier, and that it is metabolized by the liver and kidneys, with the liver being the primary metabolic organ.

[0037] Example 11 The hypochlorous acid content in the brain / cerebellum of BTBR autistic mice (ASD) was imaged using the probe O-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)-2-fluorophenyl)dimethylthiocarbamate from Example 1. Before the imaging experiment, healthy C57 mice and ASD model mice underwent head hair removal. The probe stock solution was diluted to 400 μM with PBS, and 50 μL (1 nM / g; 345 μg / kg) was injected into each mouse via tail vein. Imaging was performed on these mice at 27 min. Excitation and emission filters were fixed at 480 nm and 680 nm, respectively, with an exposure time of 200 ms. Figure 15 and Figure 16 The results showed that, compared with normal C57 mice, the ASD model mice had significantly reduced fluorescence signals collected from their heads (*** p <0.001). This phenomenon indicates that the hypochlorous acid content in the brain tissue of ASD model mice is lower than that in normal mice.

[0038] Example 12 The mice in Case 11 were immediately euthanized by cervical dislocation after imaging. In situ craniotomy was performed to expose the brain tissue, and imaging was repeated to eliminate the influence of skin and skull on the signal. The excitation and emission filters remained fixed at 480nm and 680nm, respectively, with an exposure time of 200ms. Figure 17 and Figure 18 The results show that the results are consistent with Figure 15 and Figure 16 The results were consistent, indicating that the hypochlorous acid content in the brain tissue of ASD model mice was lower than that in normal mice, and that the skin and skull showed signal attenuation in the imaging results ( Figure 16 and Figure 17 However, it will not interfere with the evaluation conclusions.

[0039] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0040] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. An endoplasmic reticulum hypochlorite fluorescent probe based on malononitrile, characterized by, The chemical name of the endoplasmic reticulum hypochlorous acid fluorescent probe is methyl O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate, and the structural formula of the endoplasmic reticulum hypochlorous acid fluorescent probe is: 。 2. A method of preparing a malononitrile-based endoplasmic reticulum hypochlorite fluorescent probe according to claim 1, characterized by, include: 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonitrile, triethylamine, and 4-dimethylaminopyridine were dissolved in N,N-dimethylformamide, and then N,N-dimethylcarbamoyl chloride was added to carry out the reaction. The product was extracted with water / ethyl acetate and purified by silica gel column chromatography to obtain pure O-(4-(2-(3-(dicyanomethylene))-5,5-dimethylcyclohexyl-1-enyl)vinyl)-2-fluorophenyl)dimethylcarbamate.

3. The method of preparing a malononitrile-based endoplasmic reticulum hypochlorite fluorescent probe according to claim 2, wherein, The molar equivalent ratio of 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonitrile, triethylamine, 4-dimethylaminopyridine and N,N-dimethylformamide is 1 : 3~8 : 0.05~0.15 : 30~60; The molar equivalent of N,N-dimethylcarbamoyl chloride is 1.5 times that of 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile.

4. The method of preparing a malononitrile-based endoplasmic reticulum hypochlorite fluorescent probe according to claim 2, wherein, The reaction temperature is 20~45℃, and the reaction time is 3~16h.

5. The method of preparing a malononitrile-based endoplasmic reticulum hypochlorite fluorescent probe according to claim 2, wherein, The volume ratio of the product, water, and ethyl acetate was 1:10:

10.

6. The method of preparing a malononitrile-based endoplasmic reticulum hypochlorite fluorescent probe according to claim 2, wherein, The structural formula of 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile is: 。 7. The method for preparing the endoplasmic reticulum hypochlorous acid fluorescent probe based on malononitrile as described in claim 2, characterized in that, The preparation methods of 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-ethylene)malononitrile include: 2-(3,5,5-trimethylcyclohexyl-2-en-1-methylene)malonitrile and 3-fluoro-4-hydroxybenzaldehyde were dissolved in acetonitrile, piperidine was added, and the reaction was carried out. The solvent was removed to obtain crude 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-methylene)malonitrile, which was then separated by silica gel column chromatography to obtain pure 2-(3-(3-fluoro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-methylene)malonitrile.

8. The method for preparing the endoplasmic reticulum hypochlorous acid fluorescent probe based on malononitrile as described in claim 7, characterized in that, The molar equivalent ratio of 2-(3,5,5-trimethylcyclohexyl-2-ene-1-methylene)malonitrile, 3-fluoro-4-hydroxybenzaldehyde, piperidine and acetonitrile is 1:0.8~1.2:0.8~5:25~70.

9. The method for preparing the endoplasmic reticulum hypochlorous acid fluorescent probe based on malononitrile as described in claim 7, characterized in that, The reaction temperature is 50~90℃, and the reaction time is 0.5~8h.

10. An application of the endoplasmic reticulum hypochlorous acid fluorescent probe based on malononitrile as described in claim 1, characterized in that, The malononitrile-based endoplasmic reticulum hypochlorous acid fluorescent probe was used for fluorescence imaging of hypochlorous acid in the endoplasmic reticulum of live cells and the brain of live mice.

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