CN114890915B - Mitochondrial targeting fluorescent probe for detecting temperature and preparation method and application thereof - Google Patents

Mitochondrial targeting fluorescent probe for detecting temperature and preparation method and application thereof Download PDF

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CN114890915B
CN114890915B CN202210372149.7A CN202210372149A CN114890915B CN 114890915 B CN114890915 B CN 114890915B CN 202210372149 A CN202210372149 A CN 202210372149A CN 114890915 B CN114890915 B CN 114890915B
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CN114890915A (en
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朱勍
卢奇
王凯
薛建苑
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Zhejiang University of Technology ZJUT
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
    • C07C255/42—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by singly-bound nitrogen atoms, not being further bound to other hetero atoms
    • C07C255/43—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by singly-bound nitrogen atoms, not being further bound to other hetero atoms the carbon skeleton being further substituted by singly-bound oxygen atoms
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    • G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
    • G01K11/00—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00
    • G01K11/32—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00 using changes in transmittance, scattering or luminescence in optical fibres
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Abstract

The invention aims to provide a mitochondrial targeting fluorescent probe for detecting temperature, and a preparation method and application thereof. The invention also relates to application of the near-mitochondrial targeting temperature probe in preparation of a fluorescent probe for temperature detection. Specifically, the fluorescent probe is used for measuring the intracellular temperature, the detection temperature is 25-60 ℃, and the maximum fluorescence intensity can be changed by about 27 times.

Description

Mitochondrial targeting fluorescent probe for detecting temperature and preparation method and application thereof
Field of the art
The invention belongs to the technical field of fluorescent probes, and particularly relates to a mitochondrial targeting fluorescent probe for detecting temperature and a preparation method and application thereof.
(II) background art
Mitochondria play a vital role in eukaryotic cell metabolism, and activities such as intracellular energy metabolism, apoptosis, free radical generation and the like have a very important effect on the temperature of mitochondria, which often causes a large difference between the temperature of mitochondria and other organelles in cells. Therefore, the abnormal temperature state of mitochondria is often used as an important index for over-expression of proteins and disturbance of intracellular environment, and the index can reflect the working state of mitochondria, so that the index is used as diagnosis and treatment means for early diseases, such as diseases in aspects of various metabolic dysfunctions, such as liver cirrhosis and the like. However, the detection of mitochondrial temperature in the market still has many defects, such as inability to precisely mark mitochondria, low sensitivity to temperature, and great damage to cells caused by external equipment. Therefore, development of a novel temperature detection means which can be applied to a cell layer and takes mitochondria as a target is important.
In recent years, by virtue of higher sensitivity, effective specificity and the minimally invasive nature of organisms, small molecule fluorescent probes are becoming important tools for real-time detection imaging at the cellular level. Therefore, the temperature detection function of mitochondria targeting is endowed with a small molecular fluorescent probe, and the application value is great. The patent designs a mitochondrion targeting fluorescent probe with high sensitivity to temperature, and the probe can provide an effective tool for researching the temperature of mitochondria and other organelles and other scientific and technical fields, and provides early warning for the clinical diagnosis field.
(III) summary of the invention
In order to solve the problems of low positioning accuracy, low temperature sensitivity and the like in the existing detection of the temperature of mitochondria, the invention provides a mitochondrial targeting fluorescent probe for detecting the temperature, and a preparation method and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a compound of formula (1),
Figure BDA0003588996780000021
the invention also relates to a preparation method of the compound shown in the formula (1), which comprises the following steps:
adding trimethylamine water solution and the compound (2) into ethanol E, and refluxing at 80-83 ℃ for 8-12h (preferably 10h at 80 ℃), and obtaining a compound (3) after post-treatment A of the obtained reaction solution A; the ratio of the amount of the compound (2) to the substance of trimethylamine contained in the aqueous solution of trimethylamine is 1: 90-100 (preferably 1:100);
Figure BDA0003588996780000022
(2) Adding malononitrile, p-diethylaminoacetophenone, acetic acid and ammonium acetate into toluene, and carrying out reaction reflux for 15-20h at 110-112 ℃ (preferably 110 ℃ for 18 h), and carrying out post-treatment on the obtained reaction solution B to obtain a compound (4); the mass ratio of the malononitrile, the p-diethylaminoacetophenone, the acetic acid and the ammonium acetate is 13-15: 4 to 5:5 to 5.5:1 (preferably 15:5:5.5:1);
Figure BDA0003588996780000023
(3) Adding the compound (3), the compound (4) and piperidine into ethanol F, carrying out reflux reaction for 2-4h at 80-83 ℃ (preferably, carrying out reflux reaction for 2h at 80 ℃), and carrying out post-treatment C on the obtained reaction solution C to obtain a compound shown in a formula (1); the ratio of the amounts of the substances of the compound (3), the compound (4) and the piperidine is 1:1 to 1.2:0.2 to 0.4 (preferably 1:1:0.2);
Figure BDA0003588996780000031
further, the mass fraction of the trimethylamine aqueous solution in the step (1) is 30%.
Still further, the volume of ethanol E in step (1) is 43-46mL/g (preferably 45.45 mL/g) based on the mass of compound (2).
Further, the post-treatment a in the step (1) is: and concentrating the reaction solution A under reduced pressure, performing column chromatography purification by using a mixed solution of dichloromethane and methanol with the volume ratio of 10:1 as an eluent, collecting an eluent containing a target product, and concentrating under reduced pressure to obtain the compound (3) as a light brown oily substance.
Further, the volume of toluene in step (2) is 30-32mL/g (preferably 30.92 mL/g) based on the mass of the p-diethylaminoacetophenone.
Further, the post-treatment B in step (2) is: and (3) cooling the reaction liquid B to room temperature, performing suction filtration, and performing silica gel column chromatography purification on the obtained filter cake by using a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1 as an eluent, and concentrating under reduced pressure to obtain the compound (4). As a pale yellow solid.
Further, the volume of the ethanol F in the step (3) is 30 to 31mL/g (preferably 30.76 mL/g) based on the mass of the compound (4).
Further, the post-treatment C in step (3) is: and extracting the reaction liquid C by using dichloromethane, concentrating an organic phase under reduced pressure, performing silica gel column chromatography by using a mixed solution of dichloromethane and petroleum ether with a volume ratio of 1:1 as an eluent, collecting an eluent containing a target product, and concentrating under reduced pressure to obtain the compound shown in the formula (1).
In a third aspect, the invention also provides an application of the compound shown in the formula (1) as a fluorescent probe for targeting mitochondria in temperature detection.
Further, the compound shown in the formula (1) is used as a fluorescent probe in a cell pyrosis model.
Further, the compound shown in the formula (1) is applied to a fluorescent probe liver injury cell model.
The letters A-F are only used for distinguishing the same steps or substances in different stages, are convenient to describe and have no other special meaning.
The compound (2) of the present invention is a disclosed compound, and the method for its synthesis and preparation can be referred to in the literature: (Garam Han, prof. Dr. Dongwok Kim, prof. Dr. Young Park, prof. Dr. Jean Bouffard, prof. Dr. Young Kim, excimers Beyond Pyrene: a Far-Red Optical Proximity Reporter and its Application to the Label-Free Detection of DNA, angel Chem, (2015) 3912-3916).
Compared with the small molecular fluorescent probe in the prior art, the invention has the following main beneficial effects:
compound (c) as described in reference (Fukui Shen, wen Yang, jin Cui, small-Molecule Fluorogenic Probe for the Detection of Mitochondrial Temperature In Vivo, ACS,2021,93,40,13417-13420):
Figure BDA0003588996780000051
the compound (a) can detect the mitochondrial temperature at the cell level, but has low sensitivity to temperature; in PBS, the temperature is raised from 20 ℃ to 42 ℃, the fluorescence intensity is not changed by three times, the Stokes shift is not more than 100nm, and the temperature of mitochondria is difficult to accurately detect in a complex environment.
After a series of screens, the invention retains the temperature sensitive nitrogen diethyl during the design of compound (1), adding long chain quaternary ammonium salts that help target the mitochondrial membrane. According to detection, the compound (1) is excited to be ex=490 nm, em=660 nm, when the temperature is increased from 20 ℃ to 42 ℃, the fluorescence intensity changes approximately 4 times, the temperature is gradually increased from 25 ℃ to 60 ℃, the fluorescence intensity is different approximately 27 times, and the compound (1) has good linear relation and good mitochondrial targeting. Thus, the compound (1) can be applied to fluorescence detection of mitochondrial temperature. The fluorescence quantitative detection principle of the temperature is as follows: the compound (1) rotates the carbon-carbon single bond structure which is limited to rotate in the process of detecting the increase of the ambient temperature, so that the non-radiation end energy of the probe is increased, and the fluorescent probe is turned-off. Meanwhile, the quaternary ammonium salt provides mitochondria targeting capability for the fluorescent probe. The change in fluorescence intensity of the probe at 660nm was detected at 490nm excitation, thereby obtaining the relationship between temperature and fluorescence intensity.
The compound (1) can be used as a fluorescent probe for detecting mitochondrial temperature, has the fluorescence performance excited to ex=490nm and em=660 nm, has larger Stokes displacement, and has the advantages of low background interference, small photodamage to biological samples and the like; the compound (1) has high temperature sensitivity, targets mitochondria and has higher temperature sensitivity, and an effective research tool is provided for researching the physiological effect of mitochondrial temperature in cells.
(IV) description of the drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of the compound (1) prepared in example 3 of the present invention.
FIG. 2 is a nuclear magnetic resonance spectrum of the compound (1) prepared in example 3 of the present invention.
FIG. 3 is a graph showing fluorescence absorption spectra of the compound (1) prepared in example 3 of the present invention, added to PBS buffer.
FIG. 4 is a graph showing fluorescence emission spectra of the compound (1) prepared in example 3 of the present invention, added to PBS buffer.
FIG. 5 shows the fluorescence emission spectra (pH=7.4) of the compound (1) prepared in example 3 of the present invention, added to PBS buffer at different stages of temperature 25℃to 60 ℃. The excitation wavelength is 490nm and the emission wavelength is 660nm.
FIG. 6 is a graph showing the linear relationship between the different phases of the compound (1) prepared in example 3 of the present invention, added to PBS buffer, at a temperature ranging from 25℃to 60℃and having a pH=7.4. The excitation wavelength is 490nm and the emission wavelength is 660nm.
FIG. 7 shows the fluorescence intensity of the compound (1) prepared in example 3 of the present invention, which was added to a glycerol/water buffer (v/v=10:0 to 1:9), and the fluorescence intensity of the compound (1) was measured at different viscosities, respectively. The excitation wavelength was 490nm and the emission wavelength was 660nm.
Fig. 8 is a fluorescence chart showing the selectivity result of the compound (1) prepared in example 3 of the present invention under the condition of PBS buffer (ph=7.4). 1-15 are Cu respectively 2+ 、Ca 2+ 、CO 3 2- 、Mn + 、HSO 3 - 、NH 4 + 、Zn 2+ 、Mg + 、HCO 3 - 、ClO - 、Fe 3+ 、Na + 、K + 、PO 4 3- 、Cl - . The excitation wavelength was 490nm and the emission wavelength was 660nm.
FIG. 9 is a fluorescent chart of the compound (1) prepared in example 3 of the present invention under PBS conditions of different pH buffers.
FIG. 10 is a co-localized fluorescence image of compound (1) prepared in example 3 of the present invention in Huh7 cells.
FIG. 11 shows the temperature measurement at the cell level of the compound (1) produced in example 3 of the present invention.
FIG. 12 shows the detection of compound (1) prepared in example 3 of the present invention in a cell scorch model.
FIG. 13 shows the detection of compound (1) prepared in example 3 of the present invention in a liver injury cell model.
FIG. 14 is a nuclear magnetic resonance hydrogen spectrum of the compound (3) produced in example 1 of the present invention.
FIG. 15 is a nuclear magnetic resonance hydrogen spectrum of the compound (4) produced in example 2 of the present invention.
FIG. 16 is a nuclear magnetic resonance hydrogen spectrum of comparative example (a) prepared in example 13 of the present invention.
FIG. 17 is a nuclear magnetic resonance hydrogen spectrum of comparative example (b) prepared in example 14 of the present invention.
FIG. 18 is a graph of fluorescence emission spectra (pH=7.4) at various stages of 25℃to 60℃in PBS buffer, according to comparative example (a) prepared in example 13 of the present invention.
FIG. 19 is a graph of fluorescence emission spectra (pH=7.4) at various stages of 25℃to 60℃in PBS buffer added to comparative example (b) prepared in example 14 of the present invention.
(fifth) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
example 1: preparation of Compound (3)
Compound (2) (0.33 g,1.0 mmol) and an aqueous solution of trimethylamine (6.0 g,100.0mmol of trimethylamine, mass fraction of 30%) were added to a round-bottomed flask containing 15mL of ethanol, and refluxed at 80 ℃ for 10 hours. The reaction solution was concentrated under reduced pressure, and the residual liquid was purified by column chromatography (eluent: dichloromethane/methanol=10/1). The eluent containing the target compound was collected and concentrated under reduced pressure to obtain 0.28g of pure product of light brown oily compound (3) with a product yield of 94%. The nuclear magnetic hydrogen spectrum is shown in figure 14.1H NMR (500 MHz, chloroform-d) δ9.91 (s, 1H), 7.55 (d, J=8.9 Hz, 1H), 6.27 (dd, J=8.9, 2.2Hz, 1H), 6.03 (d, J=2.2 Hz, 1H), 4.13 (t, J=5.7 Hz, 2H), 3.89-3.85 (m, 2H), 3.43 (d, J=7.2 Hz, 2H), 3.41 (s, 9H), 2.49 (s, 2H), 2.10-2.01 (m, 2H), 1.98 (p, J=6.9, 6.4Hz, 2H), 1.20 (t, J=7.1 Hz, 6H).
Example 2: preparation of Compound (4)
P-diethylaminoacetophenone (0.97 g,5.0 mmol) and malononitrile (0.99 g,15.0 mmol) were completely dissolved in a round-bottomed flask containing 30mL of toluene. Then, ammonium acetate (0.77 g,1.0 mmol) and acetic acid (0.33 g,5.5 mmol) were added to the reaction system, and the mixture was refluxed at 110℃for 18 hours. After the reaction system was cooled to room temperature, a part of solid was obtained by suction filtration. Purification by column chromatography on silica gel eluting with petroleum ether/ethyl acetate (5:1 v/v) and concentration of the eluate containing the target compound under reduced pressure gave compound (4) as a pure product, 1.14g, in 95% yield as a pale yellow solid. The nuclear magnetic hydrogen spectrum is shown in figure 15.1H NMR (500 MHz, chloroform-d) delta 7.71 (s, 1H), 7.69 (s, 1H), 6.69 (s, 1H), 6.67 (s, 1H), 3.45 (t, J=7.1 Hz, 4H), 2.58 (s, 3H), 1.23 (t, J=7.1 Hz, 6H).
Example 3: preparation of Compound (1)
Compound (3) (0.10 g,0.42 mmol), compound (4) (0.13 g,0.42 mmol) and piperidine (7.1 mg,0.084 mmol) were added to a 4ml ethanol solution, the reaction was refluxed at 80 ℃ for 2 hours, after the reaction was completed by TLC, the reaction solution was extracted with methylene chloride, the organic phase was concentrated under reduced pressure, and the volume ratio of methylene chloride to petroleum ether was 1 by silica gel column chromatography: the mixed solvent of 1 is eluent, the eluent containing the target product is collected, and the pure product of 37.69mg of the compound (1) is obtained by decompression and concentration, the product yield is 17 percent, and the solid is orange red. The nuclear magnetic hydrogen spectrum is shown in figure 1, and the nuclear magnetic carbon spectrum is shown in figure 2.1HNMR (500 MHz, chloroform-d) delta 7.70 (d, J=15.1 Hz, 1H), 7.30 (d, J=8.6 Hz, 2H), 7.06-6.98 (m, 2H), 6.70 (d, J=8.6 Hz, 2H), 6.23-6.15 (m, 2H), 4.22 (t, J=4.9 Hz, 2H), 3.76 (t, J=8.1 Hz, 2H), 3.43 (q, J=6.8 Hz, 8H), 3.35 (s, 9H), 2.27-2.11 (m, 4H), 1.22 (td, J=7.0, 4.6Hz, 12H) 13C NMR (126 MHz, chloroform-d) delta 161.31,151.89,148.17,136.68,131.87,120.62,119.89,116.71,110.63,104.74,94.79,67.70,66.09,53.64,44.87,44.44,25.79,21.14,12.71,12.59.
Example 4: fluorescence absorption emission spectrum of Compound (1) (5. Mu.M) in PBS detection System (excitation wavelength 490nm, emission wavelength 660 nm.)
An amount of the compound (1) of example 3 was weighed, a probe mother solution having a concentration of 1mM was prepared by using dimethyl sulfoxide, and 2. Mu.L of the mother solution was extracted and added to 398. Mu.L of LPBS buffer. This was transferred to a 96-well plate, and the fluorescence absorption and emission spectra of compound (1) were measured.
The fluorescence spectra are shown in fig. 3 and 4.
Example 5: the fluorescence emission intensity of the compound (1) in ph=7.4 was varied with temperature (excitation wavelength 490nm, emission wavelength 660 nm) at different temperature gradients (20 ℃,25 ℃, 30 ℃, 35 ℃,40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃).
An amount of the compound (1) of example (3) was weighed, a probe mother solution having a concentration of 1mM was prepared by using dimethyl sulfoxide, and 2. Mu.L of the mother solution was extracted and added to 398. Mu.L of LPBS buffer. Transferring the sample to a 96-well plate, continuously heating the small hole to be detected by laser, respectively detecting fluorescence intensity at 20 ℃,25 ℃, 30 ℃, 35 ℃,40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃ and preparing a relevant linear curve.
The fluorescence spectrum is shown in fig. 5 and 6. The results show that the fluorescence intensity of the compound (1) gradually decreases with the increase of the temperature, the fluorescence intensity at the temperature of 60 ℃ is about 27 times different from the fluorescence intensity at the temperature of 25 ℃, and the temperature and the fluorescence intensity have better linear relation. The compound (1) was shown to have a strong temperature sensitivity.
Example 6: the relationship between the fluorescence intensity and the viscosity of the probe (excitation wavelength: 490nm, emission wavelength: 660 nm) was observed in the compound (1) in a buffer of glycerol and water at pH 7.4.
An amount of the compound (1) of example 3 was weighed, a probe mother solution having a concentration of 1mM was prepared using dimethyl sulfoxide, 398. Mu.L of the mother solution was added to glycerol/water solutions of different viscosities (glycerol: water: 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0, respectively), and the mixture was added to a 96-well plate at 37℃to determine a fluorescence spectrum of the compound (1).
The fluorescence spectrum is shown in FIG. 7. The results show that the maximum viscosity glycerol: water 9: fluorescent intensity and minimum viscosity of 1 glycerol: water 0:1, the effect of viscosity on the fluorescence intensity of the compound (1) is almost negligible compared with temperature.
Example 7: the compound (1) (5. Mu.M) was selectively detected by fluorescence spectrum (excitation wavelength 490nm, emission wavelength 660 nm) under PBS detection system (pH 7.4).
Weighing a certain amount of the compound (1) in the example 3, preparing a probe mother solution with 1mM concentration by using dimethyl sulfoxide, extracting 2 mu L of the mother solution, adding into 398 mu L of the prepared aqueous solution respectively (preparing a solution containing biologically relevant active micromolecules 1-20 by using copper chloride, calcium chloride, potassium carbonate, manganese chloride, sodium bisulphite, ammonium chloride, zinc chloride, magnesium chloride, sodium bicarbonate, sodium hypochlorite, ferric chloride and sodium phosphate, cu respectively) 2+ 、Ca 2+ 、CO 3 2- 、Mn + 、HSO 3 - 、NH 4 + 、Zn 2+ 、Mg + 、HCO 3 - 、ClO - 、Fe 3+ 、Na + 、K + 、PO 4 3- 、Cl - ) The fluorescence spectra of compound (1) at 25℃and 60℃were measured at 25℃by adding the same to a 96-well plate.
The fluorescence spectrum is shown in FIG. 8. The results show that the biological related active small molecules have no effect on the fluorescence intensity of the compound (1), and only the change of temperature can change the fluorescence intensity of the compound (1).
Example 8: the compound (1) (5. Mu.M) was detected spectroscopically under the influence of pH (excitation wavelength 490nm, emission wavelength 660 nm).
A certain amount of the compound (1) of example 3 was weighed, a probe mother liquor having a concentration of 1mM was prepared by using dimethyl sulfoxide, 2. Mu.L of the mother liquor was extracted and added to buffers having different pH values, respectively (pH 3-13, buffers were prepared by using 0.2mol/L disodium hydrogen phosphate and 0.1mol/L citric acid, respectively; pH11-13, buffers were prepared by using 0.05mol/L disodium hydrogen phosphate, 0.1mol/L sodium hydroxide and 0.2mol/L potassium chloride, respectively). The mixture was added to a 96-well plate, and the fluorescence spectrum was measured.
The fluorescence spectrum is shown in FIG. 9. As a result, the fluorescence intensity of the compound (1) was found to be unable to account for the pH relationship.
Example 9: cell co-localization imaging of compound (1).
An amount of the compound (1) of example 3 was weighed, a probe mother solution having a concentration of 1mM was prepared by using dimethyl sulfoxide, and 2. Mu.L was aspirated and added to 1.998 mM LDMEM medium. 1mL of a culture solution containing the compound (I) was added to HeLa cells, incubated at 37℃for 0.5h, washed twice with DMEM medium, then added with Molecular mitochondrial Green fluorescent Probe (C1048), incubated at 37℃for 20min, washed twice with PBS, and finally subjected to fluorescence imaging with a Olympus Fluoview FV 1200 confocal microscope.
The fluorescence spectrum is shown in FIG. 10. Experimental results show that the compound (1) can target mitochondria in cells.
Example 10: temperature detection of Compound (1) (5. Mu.M) at the cell level.
An amount of the compound (1) of example 3 was weighed, a probe mother solution having a concentration of 1mM was prepared by using dimethyl sulfoxide, and 2. Mu.L was aspirated and added to 1.998 mM LDMEM medium. 1mL of culture solution containing the compound (1) is added into Huh7 cells, and the cells are stained for 15min at 37 ℃ and washed twice by PBS; 2. Mu.L of the same concentration of compound (1) was aspirated into DMEM, 2. Mu.L of mitochondrial uncoupling agent (concentration of 10mM in dimethyl sulfoxide) was added for raising the cell temperature, and the cells were co-stained at 37℃for 15min, washed twice with PBS, and fluorescence imaged with a Olympus Fluoview FV 1200 confocal microscope.
The fluorescence spectrum is shown in FIG. 11. Experimental results show that the compound can be used for temperature detection at the cell level.
Example 11: use of the temperature sensitivity of compound (1) in a model of apoptosis.
An amount of the compound (1) of example 3 was weighed, a probe mother solution having a concentration of 1mM was prepared by using dimethyl sulfoxide, and 2. Mu.L was aspirated and added to 1.998 mM LDMEM medium. 1mL of culture solution containing the compound (1) is added into Miha cells, and the cells are stained for 15min at 37 ℃ and washed twice by PBS; the same concentration of compound (1) was pipetted into 2 μl to DMEM, added to Miha cells previously treated with lipopolysaccharide (100 ng/mL) and ATP (2.5 mM) for 3h (the lipopolysaccharide and ATP combination is known to cause scorch of cells), stained for 15min at 37 ℃, and washed twice with pbs; two groups of cells were imaged for fluorescence using a Olympus Fluoview FV 1200 confocal microscope.
The fluorescence spectrum is shown in FIG. 12, and the results show that the fluorescence intensity of the cells added with only the compound (1) is remarkable, while the fluorescence intensity of the cell group added with lipopolysaccharide and ATP is weak. It is illustrated that compound (1) can find application in the apoptosis of cells.
Example 12: use of the temperature sensitivity of compound (1) in a liver injury cell model.
An amount of the compound (1) of example 3 was weighed, a probe mother solution having a concentration of 1mM was prepared by using dimethyl sulfoxide, and 2. Mu.L was aspirated and added to 1.998 mM LDMEM medium. 1mL of culture solution containing the compound (1) is added into Huh7 cells, and the cells are stained for 15min at 37 ℃ and washed twice by PBS; the same concentration of compound (1) was pipetted into 2 μl to DMEM, added to two groups of Huh7 cells previously treated with paracetamol, which is a known liver injury drug, and paracetamol (NAC), respectively, incubated for 3h and washed twice with pbs. Two groups of cells were imaged for fluorescence using a Olympus Fluoview FV 1200 confocal microscope.
The fluorescence spectrum is shown in FIG. 13, and the result shows that the cell group added with the compound (1) only has strong fluorescence intensity; and the cell group added with paracetamol has weak fluorescence intensity due to the rise of temperature; the fluorescence intensity of the cell group added with the liver repairing agent is stronger. The temperature sensitivity of the compound (1) was demonstrated for the detection of liver injury.
Example 13: preparation of comparative example (a).
Figure BDA0003588996780000101
1-Ethyl-2, 3-trimethyl-3H-indole-1-ium (188.29 mg,1.0 mmol), 4-diethylaminobenzaldehyde (177.24 mg,1.0 mmol) and piperidine (85.15 mg,0.2 mmol) were added to 4ml ethanol, reflux was conducted at 80℃for 2H, TLC was followed by extraction of the reaction solution with dichloromethane, concentration of the organic phase under reduced pressure, column chromatography on silica gel, and volume ratio of dichloromethane to petroleum ether was 1:1 as eluent, collecting the eluent containing the target product, concentrating under reduced pressure to obtain 46mg of pure product of comparative example (a), wherein the product yield is 28% and the pure product is dark red solid. Nuclear magnetic hydrogen spectra are shown in fig. 16.1H NMR (400 mhz, chloroform-d) delta 8.07 (d, j=15.3 hz, 2H), 7.48 (dd, j=7.9, 1.3hz, 2H), 7.43-7.36 (m, 2H), 7.25 (dd, j=15.3, 2.5hz, 1H), 6.79 (d, j=8.9 hz, 2H), 4.68 (q, j=7.2 hz, 2H), 3.51 (q, j=7.1 hz, 4H), 1.78 (s, 6H), 1.54 (t, j=7.3 hz, 3H), 1.24 (t, j=7.1 hz, 6H).
Example 14: preparation of comparative example (b).
Figure BDA0003588996780000102
Will be
4- (5, 5-Difluoroo-1, 3,7, 9-tetramethy-5H-4 l4,5l 4-dipyrrroo [1,2-c:2',1' -f ] [1,3,2] diazaborin-10-yl) -N, N-diethylelanine (188.29 mg,1.0 mmol), 4-diethylaminobenzaldehyde (39.88 mg,0.225 mmol) and acetic acid (27.02 mg,0.45 mmol) were added to 4ml ethanol, refluxed at 80℃for 2H, TLC was examined, the reaction solution was extracted with methylene chloride, the organic phase was concentrated under reduced pressure, and the silica gel column chromatography was carried out with a volume ratio of methylene chloride to petroleum ether of 1:1 as eluent, collecting eluent containing target product, concentrating under reduced pressure to obtain 23mg of pure product of comparative example (b), and obtaining 13% of product yield as dark green solid. The nuclear magnetic hydrogen spectrum is shown in figure 17.1H NMR (400 MHz, chloride-d) delta 7.51 (dt, J=13.6, 7.7Hz, 3H), 7.21 (d, J=16.1 Hz, 1H), 7.06 (q, J=9.9, 9.1Hz, 2H), 6.76 (d, J=8.3 Hz, 2H), 6.68 (dd, J=12.4, 8.7Hz, 2H), 6.62 (s, 1H), 5.99 (d, J=6.3 Hz, 1H), 3.42 (q, J=6.9 Hz, 8H), 2.59 (d, J=12.9 Hz, 3H), 1.56 (d, J=15.5 Hz, 6H), 1.24-1.18 (m, 12H).
Example 15: the fluorescence spectra (20 ℃,25 ℃, 30 ℃, 35 ℃,40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃) of comparative example (a) in example 13 were measured at different temperatures, respectively.
A certain amount of comparative example (a) of example 13 was weighed, and a probe mother solution having a concentration of 1mM was prepared using dimethyl sulfoxide as a solvent, and 2. Mu.L of the mother solution was extracted and added to 398. Mu.L of LPBS buffer, followed by detection in a 96-well plate. The fluorescence intensities were measured at 20 ℃,25 ℃, 30 ℃, 35 ℃,40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ by continuously increasing the temperature. The fluorescence spectrum is shown in FIG. 18.
Example 16: the fluorescence spectra (20 ℃,25 ℃, 30 ℃, 35 ℃,40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃) of comparative example (b) in example 14 were measured at different temperatures, respectively.
An amount of comparative example (b) of example 14 was weighed, and prepared into a probe mother solution having a concentration of 1mM using dimethyl sulfoxide as a solvent, and 2. Mu.L of the mother solution was extracted and added to 398. Mu.L of LPBS buffer, followed by detection in a 96-well plate. The fluorescence intensities were measured at 20 ℃,25 ℃, 30 ℃, 35 ℃,40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ by continuously increasing the temperature. The fluorescence spectrum is shown in FIG. 19.
Comparative example (a) ex=550 nm, em=620 nm, the fluorescence intensity differs less than 2 times from 25 ℃ to 45 ℃ with stokes shift of only 70nm;
comparative example (b) ex=640 nm, em=800 nm, and the fluorescence intensity differs by less than 1 time when the temperature is raised from 25 ℃ to 45 ℃;
although both compounds are capable of labeling mitochondria, they are less sensitive to temperature and are difficult to apply in complex cell detection environments.

Claims (10)

1. A compound represented by the formula (1),
Figure FDA0004152797020000011
2. a process for the preparation of a compound of formula (1) as claimed in claim 1, characterized in that the process comprises the steps of:
adding trimethylamine water solution and the compound (2) into ethanol E, and refluxing at 80-83 ℃ for 8-12 hours to obtain a reaction solution A, and performing aftertreatment A to obtain a compound (3); the ratio of the amount of the compound (2) to the substance of trimethylamine contained in the aqueous solution of trimethylamine is 1: 90-100;
Figure FDA0004152797020000012
(2) Adding malononitrile, p-diethylaminoacetophenone, acetic acid and ammonium acetate into toluene, and carrying out reaction reflux for 15-20h at 110-112 ℃, wherein the obtained reaction liquid B is subjected to post-treatment B to obtain a compound (4); the mass ratio of the malononitrile, the p-diethylaminoacetophenone, the acetic acid and the ammonium acetate is 13-15: 4 to 5:5 to 5.5:1, a step of;
Figure FDA0004152797020000013
(3) Adding the compound (3), the compound (4) and piperidine into ethanol F, carrying out reflux reaction for 2-4h at 80-83 ℃, and carrying out post-treatment on the obtained reaction solution C to obtain a compound shown in a formula (1); the ratio of the amounts of the substances of the compound (3), the compound (4) and the piperidine is 1:1 to 1.2:0.2 to 0.4;
Figure FDA0004152797020000021
3. a process for producing a compound represented by the formula (1) according to claim 2, characterized in that: the mass fraction of the trimethylamine aqueous solution in the step (1) is 30%.
4. A process for producing a compound represented by the formula (1) according to claim 2, characterized in that: the volume of the ethanol E in the step (1) is 43-46mL/g based on the mass of the compound (2).
5. A process for producing a compound represented by the formula (1) according to claim 2, characterized in that: the post-treatment A in the step (1) is as follows: concentrating the reaction solution A under reduced pressure, performing column chromatography purification by using a mixed solution of dichloromethane and methanol with the volume ratio of 10:1 as an eluent, collecting an eluent containing a target product, and concentrating under reduced pressure to obtain a compound (3).
6. A process for producing a compound represented by the formula (1) according to claim 2, characterized in that: the volume of the toluene in the step (2) is 30-32mL/g based on the mass of the diethylaminoacetophenone.
7. A process for producing a compound represented by the formula (1) according to claim 2, characterized in that: the post-treatment B in the step (2) is as follows: and (3) cooling the reaction liquid B to room temperature, performing suction filtration, and performing silica gel column chromatography purification on the obtained filter cake by using a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1 as an eluent, and concentrating under reduced pressure to obtain the compound (4).
8. A process for producing a compound represented by the formula (1) according to claim 2, characterized in that: the volume of the ethanol F in the step (3) is 30-31mL/g based on the mass of the compound (4).
9. A process for producing a compound represented by the formula (1) according to claim 2, characterized in that: the post-treatment C in the step (3) is as follows: and extracting the reaction liquid C by using dichloromethane, concentrating an organic phase under reduced pressure, performing silica gel column chromatography by using a mixed solution of dichloromethane and petroleum ether with a volume ratio of 1:1 as an eluent, collecting an eluent containing a target product, and concentrating under reduced pressure to obtain the compound shown in the formula (1).
10. The use of a compound of formula (1) according to claim 1 for the preparation of a temperature detection fluorescent probe targeting mitochondria.
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