CN115322165A

CN115322165A - Near-infrared fluorescence probe imaging dynamic fluctuation of ferrous ions in hepatic fibrosis

Info

Publication number: CN115322165A
Application number: CN202211206104.9A
Authority: CN
Inventors: 赵飞翔
Original assignee: Zhenjiang Baidan Medical Biotechnology Co ltd
Current assignee: Zhenjiang Baidan Medical Biotechnology Co ltd
Priority date: 2022-09-30
Filing date: 2022-09-30
Publication date: 2022-11-11

Abstract

The invention relates to a malononitrile isophorone derivative fluorescent molecular probe and a preparation method and application thereof, wherein the name of the compound is (E) -4- (6- (2- (3- (dicyanoethylene) -5, 5-dimethylcyclohexyl 1-1-acylethylene) naphthalene-2-acyl morpholine 4-oxide, the compound has the advantages of small molecular weight and simple structure, can be used as a molecular sensor, sensitively and selectively detect ferrous ions in living cells, and starts a fluorescent reaction.

Description

Near-infrared fluorescence probe imaging dynamic fluctuation of ferrous ions in hepatic fibrosis

Technical Field

The invention relates to a malononitrile isophorone derivative fluorescent molecular probe, a preparation method thereof and application thereof in detecting dynamic fluctuation of ferrous ions in hepatic fibrosis and displaying that oleanolic acid can reversely induce iron death.

Background

Hepatic fibrosis is a pathophysiological process, which refers to abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver injury has liver fibrosis in the process of liver repair and healing, and if the injury factor cannot be removed for a long time, the fibrosis process can be continuously developed into liver cirrhosis for a long time. In recent years, researches find that the activation of Hepatic Stellate Cells (HSCs) is a core link of the development of hepatic fibrosis, and the targeted induction of hepatic stellate cell death is an effective measure for preventing and treating hepatic fibrosis. Iron death (Ferroptosis) is a novel cell death mode discovered in recent years, is iron-dependent, and is different from apoptosis, cell necrosis and autophagy. Therefore, the target induction of HSC iron death is expected to become a new strategy for treating hepatic fibrosis. However, there is currently less connection between liver fibrosis and iron death, and therefore a highly efficient fluorescent probe is needed to further reveal the relationship between the two.

An effective, sensitive and low-toxicity fluorescent molecular probe is obtained by preparing the malononitrile isophorone derivative, and a series of experiments in both genders show that the fluorescent molecule has good performance and high potential practical application value.

Disclosure of Invention

The invention aims to provide a malononitrile isophorone derivative fluorescent molecular probe, a preparation method thereof and application thereof in detecting dynamic fluctuation of ferrous ions in hepatic fibrosis.

The technical scheme of the invention is as follows:

a malononitrile isophorone derivative fluorescent molecular probe is characterized in that the probe has the following structure:

a process for preparing the above malononitrile isophorone derivative, which comprises the steps of:

step 1, dissolving cesium carbonate, 1 '-binaphthyl-2, 2' -bis-diphenylphosphine and lead acetate in toluene, and stirring for 10-15 minutes under the conditions of room temperature and nitrogen protection until the solution turns red. 6-bromo-2-naphthaldehyde was dissolved in 2mL of a toluene solution and added to a morpholine solution as a reaction solution. The reaction solution was stirred for 6 hours at 100 ℃ under nitrogen protection. After the reaction is finished, naturally cooling the mixed solution to room temperature, quenching the reaction by using a saturated sodium bicarbonate solution, extracting by using an ethyl acetate-water system, decompressing and rotary-distilling to remove the solution, and purifying the crude product by silica gel column chromatography to obtain a white solid which is a product in the first step;

and 2, adding the 2- (3, 5-trimethylcyclohex-2-eneylidene) malononitrile and the product of the first step into an eggplant-shaped reaction bottle dissolved with absolute ethyl alcohol, adding piperidine into the reaction bottle, and heating the reaction bottle to 80 ℃ for reaction for 12 hours. After the reaction is finished, cooling the reaction product to room temperature and filtering the reaction product to obtain a dark red solid which is a second step product;

and 3, dissolving the second step product in dry dichloromethane, and adding m-chloroperoxybenzoic acid into the solution under the ice bath condition. And (3) detecting by TLC, adding saturated sodium bicarbonate solution to quench the reaction, extracting an organic layer, washing with saturated salt water for three times, drying with anhydrous sodium sulfate, and purifying the crude product by silica gel column chromatography to obtain brick red solid which is a final product.

The invention has the advantages that: the compound has very sensitive effect on detecting dynamic fluctuation of ferrous ions in hepatic fibrosis, and has the advantages of quick detection process, stable performance, low toxicity and the like. Experiments show that the fluorescent molecule can effectively and quickly detect ferrous ions of internal and external sources, and most importantly, the fluorescent molecule has very short cell membrane permeation time; the malononitrile isophorone derivative is used as a lead compound to synthesize a probe molecule with fluorescence characteristics, so that the content of ferrous ions in cells can be effectively detected, and the method has a wide application prospect.

Detailed Description

The present invention is further illustrated in detail by the following examples, but it should be noted that the scope of the present invention is not limited by these examples at all.

The first embodiment is as follows:

(E) Preparation of (E) -4- (6- (2- (3- (dicyanoethylene) -5, 5-dimethylcyclohexyl 1-1-acylethylene) naphthalene-2-acylmorpholine 4-oxide

4.2 g, 13 mmol of cesium carbonate, 100mg, 3% of 1,1 '-binaphthyl-2, 2' -bisdiphenylphosphine and 2 mg, 1% of lead acetate are dissolved in toluene and stirred at room temperature under nitrogen protection for 10-15 minutes until the solution turns red. 2.34g, 10 mmol of 6-bromo-2-naphthaldehyde was dissolved in 2mL of a toluene solution, and the solution was added to a solution of 1.00 g, 13.75 mmol of morpholine. The reaction solution was stirred for 6 hours at 100 ℃ under nitrogen protection. After the reaction is finished, naturally cooling the mixed solution to room temperature, quenching the reaction by using a saturated sodium bicarbonate solution, extracting by using an ethyl acetate-water system, decompressing, carrying out rotary evaporation to remove the solution, and purifying a crude product by using a silica gel column chromatography to obtain a white solid; 1.5g,6.25mmol and 1.2g,6.25mmol of (3, 5-trimethylcyclohex-2-enylidene) malononitrile were added to the obtained product in an eggplant-shaped reaction flask containing 25mL of absolute ethanol, and after adding 300. Mu.L of piperidine thereto, the mixture was heated to 80 ℃ for 12 hours. After the reaction is finished, cooling the mixture to room temperature and filtering the mixture to obtain a dark red solid; 100mg of the obtained product and 0.244mmol of the obtained product were dissolved in 15mL of anhydrous dichloromethane, and 64mg of 0.367mmol of m-chloroperoxybenzoic acid was added thereto under ice-bath conditions. And (3) after the TLC detection reaction is finished, adding saturated sodium bicarbonate solution to quench the reaction, extracting an organic layer, washing the organic layer with saturated saline water for three times, drying the organic layer with anhydrous sodium sulfate, and purifying a crude product by silica gel column chromatography to obtain a target product brick red solid of 95mg with the yield of 92%. Fully dissolving the mixture, adding 73.5 mmol of crotonaldehyde and 6 mL of crotonaldehyde, magnetically stirring to uniformly mix, reacting at normal temperature for 1 h, detecting the reaction progress degree by TLC (thin layer chromatography), wherein the mass ratio of the materials is 4-N, N-dimethylaniline: crotonaldehyde =1:2, adding 35 mL of toluene into the reaction solution, further carrying out reflux reaction at 115 ℃ overnight, cooling to room temperature, removing a toluene layer, neutralizing a water layer with a saturated sodium hydroxide solution, extracting the obtained solution with dichloromethane, washing with a saturated sodium chloride solution twice, drying, filtering with anhydrous sodium sulfate, concentrating under reduced pressure, and purifying the crude product by silica gel column chromatography to obtain a brown yellow solid product; selenium dioxide is added to dioxane/water volume ratio is dioxane: water =10:1, i.e. 140 mL of dioxane and 40 mL of waterHeating the solution at 80 ℃ for 30min, adding 18.8 mmol and 3.5 g of the obtained product, magnetically stirring to mix uniformly, reacting at 80 ℃ for 4 h, cooling to room temperature, filtering through diatomite, washing filter residue with a small amount of dichloromethane, concentrating the filtrate under reduced pressure, and separating by silica gel column chromatography, wherein an eluent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 6; dissolving 3.0 mmol,0.6 g of the obtained product and 3.3 mmol,0.37 g of ethyl cyanoacetate in an ethanol solution, stirring for 1 h at room temperature, washing the obtained ethanol mixture with cold ethanol for 3 times to obtain a solid, recrystallizing the solid in a mixed solution of ethanol and acetone, and carrying out volume ratio of ethanol: acetone =9:1, 0.73 g of the title compound was obtained as a red powder in 83% yield. ¹ H NMR (600 MHz, DMSO-d6) δ 8.70 (s, 1H), 8.30 – 8.20 (m, 2H), 8.02 (t, J = 7.9 Hz, 3H), 7.59 (d, J = 16.1 Hz, 1H), 7.46 (d, J = 16.2 Hz, 1H), 6.94 (s, 1H), 4.46 (t, J = 11.4 Hz, 2H), 4.13 (td, J = 11.6, 3.6 Hz, 2H), 3.88 – 3.83 (m, 2H), 3.08 (d, J = 11.2 Hz, 2H), 2.60 (d, J = 15.3 Hz, 4H), 1.03 (s, 6H). ¹³ C NMR (151 MHz, DMSO) δ 170.72, 156.03, 137.52, 135.30, 133.08, 133.01, 131.12, 129.86, 129.79, 128.68, 125.51, 123.73, 120.43, 119.60, 114.26, 113.47, 77.22, 66.78, 62.02, 42.73, 38.59, 32.16, 27.90. HRMS (ESI-TOF) m/z: [M+H] ⁺ Calcd for C ₂₇ H ₂₇ N ₃ O ₂ 426.2182, Found 426.2177.

The property and activity of the fluorescent molecular compound are tested by applying experiments, the fluorescent molecular probe prepared in the first embodiment is tested in the second to tenth embodiments, and specific data and analysis are as follows:

example two:

FIG. 1: in PBS solution, the fluorescent molecular probe is associated with Fe ²⁺ Fluorescence spectra and fluorescence change profiles of concentration changes

mu.M fluorescent molecular probe was dissolved in PBS (pH 7.4, 10 mM, 1% DMSO,2Mm CTAB) and incubated at 37 ℃ for 5min, then separately in different Fe ²⁺ Measuring the fluorescence spectrum characteristic of Fe under the concentration ^2+- The concentration range is 0-50μM, detection is carried out on an Edinburgh FLS980 instrument, and the excitation wavelength is475 nm, and the slit width is 15 nm.

The results show that Fe is reduced at 750 nm ²⁺ Increasing the concentration from 0 to 50. Mu.M (corresponding to 5 equivalents of probe) gave a standard curve; fe at 750 nm ²⁺ The concentration of the nano-particles shows a strong linear relation between 0 and 15 mu M, and the correlation coefficient is 0.9861. It is thus understood that with Fe ²⁺ Increasing concentration, increasing fluorescence intensity, fe ²⁺ When the concentration reaches about 5 equivalents, the fluorescence intensity reaches the maximum and remains stable.

Example three:

FIG. 2: specific selectivity and interference experimental diagram of the fluorescent molecular probe in PBS solution

Dissolving 10 μ M fluorescent molecular probe in PBS (pH 7.4, 10 mM, 1% DMSO,2mM CTAB), incubating at 37 deg.C for 5min, and detecting its selectivity, fe, with different analytes ²⁺ Was measured on an Edinburgh FLS980 instrument at an excitation wavelength of 475 nm and a slit width of 15 nm, with a concentration of 50 μ M and other substances tested.

The fluorescent molecular probe can be specific to Fe ²⁺ In response, other substances tested were sodium, potassium, magnesium, calcium and other cations. The fluorescent molecular probe is directed to Fe compared to other cations ²⁺ Has good selectivity. The fluorescent molecule can be used as Fe ²⁺ The probe of (1).

Example four:

FIG. 3: in PBS (phosphate buffer solution), the fluorescence spectrum of the fluorescent molecular probe responding to pH

mu.M fluorescent molecular probe was dissolved in PBS (pH 7.4, 10 mM, 1% DMSO,2Mm CTAB) and after incubation for 5min at 37 ℃ its performance was measured at different pH values, respectively, ranging from: 4-12. Detection was carried out on an Edinburgh FLS980 instrument with an excitation wavelength of 475 nm and a slit width of 15 nm.

As can be seen from the figure, the fluorescent molecular probe itself is hardly affected by pH. The fluorescent molecular probe is mixed with 50 mu M Fe ²⁺ Upon co-incubation, it can be seen that the fluorescence intensity of the molecule is zero at pH 4, whereas at pH 5-8, the fluorescent property phaseFor stable, stable fractions sufficient for in vivo experiments.

Example five:

FIG. 4: fluorescence spectrum of the fluorescent molecular probe in PBS solution with the change of stability with time

10 μ M fluorescent molecular probe was dissolved in PBS (pH 7.4, 10 mM, 1% CH) ₃ CN,2Nm CTAB) solution, and then adding 50 mu M Fe ²⁺ Incubating at 37 ℃, and respectively detecting the performance of the samples under different incubation times, wherein the incubation time ranges are as follows: 0-48 h. Detection was carried out on an Edinburgh FLS980 instrument with an excitation wavelength of 475 nm and a slit width of 15 nm.

The fluorescent molecular probe has stable self-fluorescence property, and Fe is added ²⁺ Then, the fluorescence intensity is enhanced, and the fluorescence performance is stable. By precisely controlling the response time, the detection system can be used in a short incubation time while ensuring that it lasts more than 48 h, which indicates the stability of the fluorescent molecular probe detection system.

Example six:

FIG. 5: the fluorescence molecular probe is used for cytotoxicity test

The cytotoxicity of the fluorescent molecular probe is evaluated by a CCK-8 method. The test cells were LX-2 cells (human hepatic stellate cells). The cell culture medium is complete medium (DMEM) containing 10% FBS and 1% double antibody, and the cells are planted in 96-well plate and cultured at 37 deg.C and 5% CO ₂ In the incubator, the cell density is 5X 10 ³ one/mL.

It can be concluded from the figure that the fluorescent molecular probe is less cytotoxic, even at higher probe concentrations, demonstrating that the fluorescent molecular probe can be further tested clinically.

Example seven:

FIG. 6: in living cells, the fluorescent molecular probe is used for time-dependent experimental fluorescence confocal picture of entering cells

10 mu M of the fluorescent molecular probe and LX-2 cells are subjected to 5% CO at 37 DEG C ₂ Co-incubation in incubator, cell culture in complete medium containing 10% fetal bovine serum FBS,1% double antibody, co-incubation time of each group with probe set to 30min, and then 100 µM Fe ²⁺ Incubating with cells for 0-36 min. Analysis of cellular fluorescence imaging (lambda) _ex = 475 nm，λ _em = 700-800 nm), scale bar: 25. and (5) mu M.

As can be seen from the figure, the fluorescent molecular probe has completely entered into LX-2 cells at the incubation time of 12 min, and the rapid cell permeation process is very excellent and important, which can shorten the whole detection period.

Example eight:

FIG. 7: in living cells, detecting the exogenous Fe of the fluorescent molecular probe along with the cells ²⁺ Fluorescence confocal images of concentration changes

HeLa cells were cultured in complete medium containing 10% FBS,1% double antibody, cells at 37 ℃ and 5% CO ₂ After culturing for 12 h in an incubator, adding 10 mu M of the fluorescent molecular probe into the cells, and adding Fe with different concentrations into the cells after 30min ²⁺ And (5) carrying out incubation on the solution (0 muM, 20 muM, 40 muM, 60 muM, 80 muM and 100 muM) for 30min, and then taking a fluorescence confocal picture.

The fluorescence intensity and Fe of the fluorescent molecular probe can be obtained by analyzing the image ²⁺ The increase in concentration is positively correlated. This shows that the fluorescent molecular probe can be used for detecting the Fe of the overproof external source ²⁺ And has better performance in the aspects of cell infiltration and living thin imaging.

Example nine:

FIG. 8: in living cells, detecting the endogenous Fe of the fluorescent molecular probe to the cells under different conditions ²⁺ Fluorescence confocal images of concentration changes

The intracellular imaging performance of the fluorescent molecular probe is further detected subsequently. LX-2 cells were cultured in complete medium containing 10% FBS,1% double antibody, cells at 37 deg.C, 5% CO ₂ After 12 h of culture in an incubator, adding 10 mu M of the fluorescent molecular probe into the cells, and respectively adding 10 mu M of an iron death inducer (ilastin, erastin) into the cells after 30 min; 10. mu M sorafenib. At this time, under confocal fluorescence microscope, the first two groups with the iron death inducer added have obvious fluorescence enhancement, and the second group with the iron death inducer inhibitor added has obvious fluorescence enhancementThe fluorescence intensity of the three groups is obviously reduced. The result also can show that the fluorescent molecular probe can effectively detect the endogenous Fe of the cells ²⁺ . Adding 10 mu M bipyridyl into the cells after the probe incubation; 10. mu M Fer-1 and 10 mu M ilastin and Fer-1. Incubating for 0.5 h by using bipyridine; incubation for 6 h by Fer-1; the last group was incubated with ilastin for 12 h and then with Fer-1 for 6 h. After 30min of incubation, the fluorescence intensity is significantly reduced. Therefore, the fluorescence is enhanced by the endogenous Fe in the living cells ²⁺ Is relevant to the generation of (2).

The result shows that the compound can be used as a molecular sensor and can be sensitive. Selective detection of Fe in living cells ²⁺ And the fluorescence reaction is started, the fluorescent probe has the characteristic of high selectivity, and more particularly, the fluorescent molecular probe is almost non-toxic to cells and has very good cell membrane permeation effect, and can penetrate through living cell membranes within 12 min to start the fluorescence reaction. It provides a new method for detecting endogenous Fe in living cells ²⁺ The method has very important practical significance.

Example ten:

FIG. 9: in living cells, the fluorescent molecular probe is detected and compared with other commercial dye organelle location staining fluorescence confocal images

The organelle localization of the fluorescent probe is then detected. LX-2 cells were cultured in complete medium containing 10% FBS,1% double antibody, cells at 37 ℃,5% CO ₂ After culturing for 12 hours in an incubator, adding 10 mu M of the fluorescent molecular probe into the cells, incubating for 30min, and then respectively tracking green dyes with 10 mu M endoplasmic reticulum; tracking the green dye by 10 mu M mitochondria; and (5) co-incubation for 30min by tracking green dye with 10 mu M lysosome. Analysis of cellular fluorescence imaging (lambda) _ex = 475 nm，λ _em = 700-800 nm), scale bar: 25. and (5) mu M.

As can be seen in the figure, the fluorescent probe has a strong correlation with lysosome tracking green dye, indicating that the probe is mainly localized in the lysosome of the cells.

Example eleven:

FIG. 10: in vivo, detecting the living body image formed by the fluorescent probe

The in vivo imaging ability of the fluorescent probe was examined. Nude mice were selected without any treatment. After intraperitoneal injection of LZP (100 μ M in 200 μ L normal saline), fe was injected at the same site ²⁺ (500. Mu.M in 500. Mu.L normal saline) solution. Fluorescence images were collected at 0,5, 10, 15, 20, 30 minutes. The in vivo imaging fluorescence intensity of the mouse is gradually enhanced along with the increase of the time, which shows that the in vivo imaging effect of the probe is excellent.

Example twelve:

FIG. 11: detecting the imaging effect of the probe in a hepatic fibrosis mouse model

And detecting the imaging effect of the probe in a hepatic fibrosis mouse model. To establish a liver fibrosis (HF) mouse model, female BALB/c mice (4-6 weeks) were selected and randomly divided into two groups. One group of mice had no other treatment as a blank mouse; group of CCl administration by intraperitoneal injection ₄ A mixed solution of (10 mL/kg body weight) and olive oil (1 (w/v)) was injected intraperitoneally once every two days for four weeks.

The imaging effect is shown in the figure, the fluorescence intensity and the fluorescence range in the model mouse are increased along with the increase of time, and the blank group is unchanged, which indicates that the probe can be used for the fluorescence imaging of the hepatic fibrosis mouse model.

Description of the drawings:

FIG. 1 shows the fluorescent molecular probe with Fe in PBS solution ²⁺ Fluorescence spectra of concentration changes and fluorescence change plots.

FIG. 2 is a graph of the selectivity and interference of the fluorescent molecular probe in PBS.

FIG. 3 is a graph of the fluorescence spectrum of the fluorescent molecular probe in PBS solution in response to pH.

FIG. 4 is a graph of the fluorescence spectrum of the fluorescent molecular probe in PBS solution with increasing stability over time.

FIG. 5 is a graph showing the cytotoxicity test of the fluorescent molecular probe.

FIG. 6 is a time-dependent experimental fluorescence confocal map of the fluorescent molecular probe into a cell in a living cell.

FIG. 7 shows that in living cells, the fluorescent molecular probe is detected along with exogenous Fe of cells ²⁺ Fluorescence confocal images of concentration changes

FIG. 8 shows the detection of endogenous Fe in living cells by the fluorescent molecular probe under different conditions ²⁺ Fluorescence confocal plot of concentration change.

FIG. 9 is a comparison of fluorescence confocal images of the fluorescent molecular probe and other commercial dye organelle localization staining in living cells.

FIG. 10 is an image of a living body in which the fluorescence probe was detected.

FIG. 11 is a graph showing the effect of detecting the probe in imaging a mouse model of liver fibrosis.

Claims

1. A malononitrile isophorone derivative fluorescent molecular probe is characterized in that the fluorescent molecular probe is composed of the following structural formula:

2. the method for preparing a malononitrile isophorone derivative fluorescent molecular probe according to claim 1, which comprises the following steps:

step 1: dissolving cesium carbonate, 1 '-binaphthyl-2, 2' -bis-diphenylphosphine and lead acetate in toluene, and stirring for 10-15 minutes under the conditions of room temperature and nitrogen protection until the solution turns red; dissolving 6-bromo-2-naphthaldehyde in 2mL of toluene solution, and adding the solution into morpholine solution; stirring for 6 hours at 100 ℃ under the protection of nitrogen. After the reaction is finished, naturally cooling the mixed solution to room temperature, quenching the reaction by using a saturated sodium bicarbonate solution, extracting by using an ethyl acetate-water system, distilling under reduced pressure to remove the solvent, and purifying the crude product by using a silica gel column chromatography to obtain a white solid which is a product in the first step;

step 2: adding 2- (3, 5-trimethylcyclohex-2-enylidene) malononitrile and the product of the first step into absolute ethyl alcohol, adding piperidine, and heating to 80 ℃ for reaction for 12 hours; after the reaction is finished, cooling to room temperature, and filtering to obtain a dark red solid which is a second step product;

step 3, dissolving the product of the second step in dry dichloromethane, and adding m-chloroperoxybenzoic acid under the condition of ice-water bath; and (3) detecting by thin-layer chromatography, adding a saturated sodium bicarbonate solution after the reaction is finished, quenching the reaction, extracting an organic layer, washing the organic layer with saturated salt water for three times, drying the organic layer by using anhydrous sodium sulfate, and purifying the crude product by silica gel column chromatography to obtain brick red solid which is a final product.

3. The spectral characterization, in vivo imaging and use in dynamic fluctuation detection of ferrous ions in liver fibrosis of a malononitrile isofrenone derivative fluorescent molecular probe according to claims 1 and 2.