Luminescent stable cation free radical compound based on methylene bridged pyrrole dimer and preparation and application thereof

Abstract

The invention belongs to the technical field of luminescent materials, and discloses a luminous stable cation free radical compound based on methylene bridging pyrrole dimer and preparation and application thereof. The structure of a cation free radical of the luminescence-stable cation free radical compound is shown in a formula II, and the cation free radical is stabilized by anions or a stabilizer. The preparation method comprises the following steps: oxidizing a compound shown as a formula I in a solvent through an oxidizing agent or under the action of lewis acid or protonic acid in a solid state, or oxidizing through ultraviolet irradiation in an oxygen atmosphere, thereby obtaining the cation free radical compound with stable luminescence. The cation free radical compound with stable luminescence has the advantages of high generation efficiency, good stability, high luminescence efficiency and the like, is used for cell fluorescence imaging and / or blood vessel fluorescence imaging, and has a good imaging effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical luminescent materials, and particularly relates to a luminescent stable cation radical compound based on a methylene bridged dimer of porphyrin and a preparation method and application thereof in biological imaging. BACKGROUND

[0002] Near-infrared fluorescence imaging has the advantages of strong penetration and small self-fluorescence interference. However, most of the existing near-infrared fluorescent groups have defects such as large molecular weight and poor water solubility, which easily interfere with the absorption of cells. Therefore, it is necessary to develop small near-infrared fluorescent groups. Compared with traditional closed-shell molecules, the energy gap between the excited state and the ground state of open-shell molecular cation radicals is much smaller, which provides the possibility for developing small-sized near-infrared fluorescent groups. However, cation radicals are usually unstable and prone to various subsequent reactions, and usually need to increase the conjugated system or add multiple electron-donating substituents to improve their stability, which limits their application in biomedicine.

[0003] It is necessary to develop a cation radical compound with simple synthesis, high luminescent efficiency, good stability and near-infrared luminescent properties and a preparation method thereof, so as to facilitate the application research of the cation radical compound in biological imaging. SUMMARY

[0004] In order to overcome the defects and shortcomings of the prior art, the primary purpose of the present application is to provide a luminescent stable cation radical compound based on a methylene bridged dimer of porphyrin, i.e. a near-infrared luminescent stable cation radical compound. The cation radical compound of the present application has the advantages of near-infrared luminescence, high luminescent efficiency and good stability.

[0005] Another purpose of the present application is to provide a preparation method of the above-mentioned cation radical compound.

[0006] Still another purpose of the present application is to provide the application of the above-mentioned luminescent stable cation radical compound. The luminescent stable cation radical compound is used for biological imaging. The biological imaging refers to cell mitochondrial fluorescence imaging and / or zebrafish vascular fluorescence imaging.

[0007] The purposes of the present application are achieved by the following technical solutions:

[0008] A luminescent stable cation radical compound based on a methylene bridged dimer of porphyrin, wherein the structure of the cation radical is formula II, and is stabilized by anions or stabilizers; and is obtained by oxidizing a compound of formula I;

[0009]

[0010] wherein R 1For substituted or unsubstituted alkyl groups, substituted or unsubstituted aryl groups; R 2 It is hydrogen, substituted or unsubstituted alkyl, alkyloxy, alkylamino; R 3 Alkyl groups, substituted alkyl groups;

[0011] R 1 In this context, the alkyl group is either straight-chain or branched; the substituted alkyl group refers to a group that substitutes hydrogen in the alkyl group to form a cyclic compound. The cyclic compound is preferably a cyclic compound formed from carbon, hydrogen, and one or more heteroatoms selected from N, S, and O. The cyclic compound is preferably tetrahydropyran. Tetrahydrothiam The groups formed by cyclic compounds are those formed by the loss of a hydrogen atom from a cyclic compound.

[0012] R 1 In this context, the alkyl group is C10. 1-10 alkyl;

[0013] R 1 In this context, the aryl group refers to a monocyclic or polycyclic aromatic group having 6-20 carbon atoms. Representative aryl groups include: phenyl, naphthyl, anthracene, and pyrene.

[0014] R 1 In this context, the substituted aryl group refers to the hydrogen atom on the aryl ring being substituted by one or more of alkoxy, alkylthio, amino, carboxyl, and cyano groups; for example, the hydrogen atom on a phenyl group is substituted by one or more of alkoxy, alkylthio, carboxyl, and cyano groups, and the substitution is monosubstituted, disubstituted, or trisubstituted.

[0015] R 2 In this context, the alkyl group is C10. 1-10 Alkyl, alkyloxy group is C 1-10 Alkyloxy and alkylamino are C 1-10 Alkylamino;

[0016] R 2 In this context, the substituted alkyl group refers to the alkyl group in which hydrogen atoms are replaced by groups such as hydroxyl, methoxy, carboxyl, or halogen.

[0017] R 3 In this context, the substituted alkyl group refers to the alkyl group whose hydrogen atoms are replaced by hydroxyl, methoxy, carboxyl, or halogen groups.

[0018] R 3 In this context, the alkyl group is C10. 1-10 Alkyl; the alkyl group in the substituted alkyl group is C10. 1-10 alkyl.

[0019] R 1 Preferably alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl), phenyl, naphthyl, or substituted aryl, R2 preferably alkyl (e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl); R 3 preferably substituted alkyl. The substituent in the substituted aryl is one or more of alkoxy (e.g. methoxy, ethoxy), alkylthio (e.g. methylthio, ethylthio), carboxyl, cyano, and the aryl is phenyl or naphthyl. The substituted alkyl is hydroxy-substituted C 1-5 alkyl (e.g. hydroxy-substituted methyl, hydroxy-substituted ethyl, hydroxy-substituted propyl, etc.) or methoxy-substituted C 1-5 alkyl (e.g. methoxy-substituted methyl, methoxy-substituted ethyl, methoxy-substituted propyl, etc.).

[0020] More preferably, R 1 is alkyl substituted by alkoxy, R 2 is C 1-10 alkyl; R 3 is -(CH2) n -OH (n is an integer of 1, 2, 3, 4, etc.).

[0021] The anion is preferably an anion provided by an oxidizing agent or a Lewis acid or a protonic acid. The stabilizer is silica gel.

[0022] The compound of formula I:

[0023] R 1 ~ R 3 as defined above.

[0024] The method for preparing the luminescent stable cationic radical compound comprises the following steps: oxidizing the compound of formula I in a solvent by an oxidizing agent or oxidizing in a solid state under the action of a Lewis acid or a protonic acid or oxidizing in an oxygen atmosphere by ultraviolet light.

[0025] The oxidizing agent is one or more of trifluoroacetic acid, sulfuric acid, p-bromo-triphenylamine radical cation hexachloroantimonate, tris(2,4-dibromo-triphenyl)amine hexachloroantimonate, tris(pentafluorophenyl)borane, sodium tetra(pentafluorophenyl)borate, nitrous hexafluoroantimonate, triethyloxonium hexachloroantimonate, [bis(trifluoroacetoxy)iodo]benzene, thallium trifluoroacetate, m-chloroperbenzoic acid, thallium trifluoroacetate, copper perchlorate, ferric trichloride, silver hexafluoroantimonate.

[0026] The solvent is an organic solvent, water or a PBS solution.

[0027] The organic solvent is one or more of acetonitrile, dichloromethane, trichloromethane, carbon tetrachloride, tetrahydrofuran or 1,4-dioxane.

[0028] The Lewis acid or protonic acid is one or more of tris(pentafluorophenyl)borane, citric acid monohydrate, phosphomolybdic acid, phosphotungstic acid, p-toluenesulfonic acid, pyruvic acid, 3-bromopyruvic acid, oxalic acid, 4-sulfonylcalix[4]arene, glycolic acid.

[0029] The solid state means that the compound of formula I is mixed with a Lewis acid or protonic acid and ground.

[0030] In the solvent, the concentration of the compound of formula I in the solvent is 1 μM to 1000 mM; and the molar ratio of the oxidizing agent to the compound of formula I is (0.001 to 10):1.

[0031] In the solvent, the reaction time is determined by detecting the radical signal or luminescence or fluorescence performance, for example, the reaction time is 1 to 120 min.

[0032] When the molar ratio of the oxidizing agent to the compound of formula I is (1.2 to 10):1, the reaction time is preferably 1 to 30 min.

[0033] When the molar ratio of the oxidizing agent to the compound of formula I is (0.001 to 1.1):1, the reaction time is 1 to 120 min.

[0034] In the solvent, the reaction temperature is room temperature.

[0035] In the solid state, the molar ratio of the compound of formula I to the Lewis acid or protonic acid is 0.01 to 10:1.

[0036] In the presence of ultraviolet light, the compound of formula I is in contact with silica gel.

[0037] Reaction equation:

[0038]

[0039] A complex containing a luminescent stable cationic radical compound is prepared by mainly wrapping the cationic radical compound with a polymer; specifically, the compound of formula I is oxidized; then the cationic radical compound is stirred with the polymer in an aqueous phase at high speed, and the solvent is removed. The compound of formula I is as defined in formula I above.

[0040] The polymer is one or more of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) copolymer (F68, F127, P123), polyoxyethylene castor oil (EL), distearoylphosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG), hyaluronic acid (HA), or polylactic acid-glycolic acid copolymer (PLGA); preferably, the polymer is poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) copolymer (F68, F127, P123). F127, P123), polyoxyethylene castor oil (EL), distearoylphosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG), hyaluronic acid (HA), or polylactic acid-glycolic acid copolymer (PLGA); preferably, the polymer is poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) copolymer (F68, F127, P123). F127.

[0041] The amount of the polymer and the compound of formula I is 0.01 mg:(10-30)mmol. The concentration of the polymer in the aqueous phase is 10-30 mg / mL. The speed of the high-speed stirring is 500-2000 rpm. The time of the high-speed stirring is 3-10 min.

[0042] The oxidation of the compound of formula I is carried out in a solvent by using an oxidant or by mixing the compound of formula I with a Lewis acid or a protonic acid, grinding, and dissolving the ground product in an organic solvent. The conditions are the same as those in the preparation of the cationic radical compound.

[0043] The organic solvent is one or more of dimethyl sulfoxide, acetonitrile, and ethanol.

[0044] In the preparation of the complex, preferably, the compound of formula I is oxidized by an oxidant in a solvent, and then the oxidation product is high-speed stirred with a polymer in an aqueous phase, all the solvent is removed, and the complex is obtained.

[0045] The luminescent stable cationic radical compound or the complex containing the luminescent stable cationic radical compound is used in cell fluorescence imaging, especially in organelle-specific fluorescence imaging, and is used as a fluorescence imaging agent. For example, it is used as an imaging agent for mitochondria-specific fluorescence imaging.

[0046] The complex containing the luminescent stable cationic radical compound is used in blood vessel imaging, especially in zebra fish blood vessel imaging, and is used as a fluorescence imaging agent for blood vessel imaging.

[0047] A fluorescence imaging probe comprises the luminescent stable cationic radical compound or the complex containing the luminescent stable cationic radical compound.

[0048] The fluorescence probe is prepared by oxidizing the compound of formula I. The oxidation conditions are the same as those of the luminescent stable cationic radical compound.

[0049] Alternatively, the preparation method of the fluorescence probe is the same as the preparation conditions of the complex containing the luminescent stable cationic radical compound.

[0050] The fluorescence probe is a fluorescence probe for organelle-specific fluorescence imaging and / or blood vessel imaging, i.e., a fluorescence imaging agent.

[0051] The luminescent stable cationic radical compound or the complex of the present application has excellent cell fluorescence imaging effect.

[0052] The present application has the following advantages and effects compared with the prior art:

[0053] (1) The cationic radical compound with near-infrared luminescence of the present application has the advantages of high generation efficiency, good stability and high luminescent efficiency.

[0054] (2) The luminescent stable cationic radical compound of the present application is generated by in-situ reaction, does not need to be separated, has high cell entry efficiency, and can be directly used for fluorescence imaging of living cells and fluorescence imaging of blood vessels, and has good imaging effect. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 UV-visible absorption spectrum and fluorescence emission spectrum of compound DP1 (100 μM) converted into cationic radical compound DP1 in dichloromethane with different molar equivalents of tris(pentafluorophenyl)borane B(C6F5)3: ·+ (A) UV-visible absorption spectrum; (B) fluorescence emission spectrum, excitation wavelength is 580 nm; (C) curve formed by absorption value and fluorescence intensity under different amounts of oxidant;

[0056] Figure 2 UV-visible absorption spectrum and fluorescence emission spectrum of compound DP1 (100 μM) converted into cationic radical compound DP1 in acetonitrile with different molar equivalents of anhydrous ferric chloride FeCl3: ·+ (A) UV-visible absorption spectrum; (B) fluorescence emission spectrum, excitation wavelength is 580 nm; (C) curve formed by absorption value and fluorescence intensity under different amounts of oxidant;

[0057] Figure 3 UV-visible absorption spectrum of compound DP1 (100 μM) converted into cationic radical compound DP1 by reaction with different oxidants (100 μM): ·+ (A) trifluoroacetic acid TFA; (B) nitrous hexafluoroantimonate NOSbF6; (C) sulfuric acid H2SO4; (D) [bis(trifluoroacetoxy)iodo]benzene PIFA; (E) triethyloxonium hexachloroantimonate Et3OSbCl6; (F) copper perchlorate Cu(ClO4)2;

[0058] Figure 4 Excitation spectrum, fluorescence emission spectrum and pictures before and after grinding of compound DP1 converted into cationic radical compound by solid-state grinding with equal molar equivalents of Lewis acid tris(pentafluorophenyl)borane B(C6F5)3 for 4 min: (A) excitation spectrum, emission wavelength is 790 nm; (B) fluorescence emission spectrum, excitation wavelength is 610 nm; (C) bright field pictures before and after grinding;

[0059] Figure 5The excitation spectrum, fluorescence emission spectrum, and images before and after grinding of compound DP1 with an equimolar amount of protic acid 3-bromopyruvic acid for 4 min to transform it into a cationic radical compound are shown below: (A) Excitation spectrum, emission wavelength 760 nm; (B) Fluorescence emission spectrum; (C) Bright field images before and after grinding.

[0060] Figure 6 To convert compound DP1 of different concentrations into cationic radical compound DP1 through in-situ oxidation on a silica gel plate (365nm light irradiation for 60s). ·+ The excitation and fluorescence emission spectra are as follows: (A) Excitation spectrum, emission wavelength 720 nm; (B) Fluorescence emission spectrum, excitation wavelength 560 nm.

[0061] Figure 7 The UV-Vis absorption and fluorescence emission spectra of compound DP2-DP7 (100 μM) and oxidant tris(pentafluorophenyl)borane B(C6F5)3 (100 μM) in dichloromethane are shown: (A) UV-Vis absorption spectrum; (B) Fluorescence emission spectrum, with an excitation wavelength of 580 nm.

[0062] Figure 8 The UV-Vis absorption and fluorescence emission spectra of compound DP1-OCH3 (500 μM) and oxidant tris(pentafluorophenyl)borane B(C6F5)3 (500 μM) in dichloromethane are shown: (A) UV-Vis absorption spectrum; (B) Fluorescence emission spectrum.

[0063] Figure 9 The excitation and fluorescence emission spectra of compounds DP2-DP7, DP1-OCH3 converted into corresponding cationic free radical compounds by solid grinding with an equimolar amount of oxidant tris(pentafluorophenyl)borane B(C6F5)3 for 4 min are as follows: (AG) are DP2, DP3, DP4, DP5, DP6, DP7 and DP1-OCH3, respectively. The fixed emission wavelength of the excitation spectrum is 790 nm and the fixed excitation wavelength of the fluorescence emission spectrum is 610 nm.

[0064] Figure 10 Bright field images showing the transformation of compounds DP2-DP7 and DP1-OCH3 into corresponding cationic free radical compounds by solid-state grinding with an equimolar amount of the oxidant tris(pentafluorophenyl)borane B(C6F5)3 for 4 min;

[0065] Figure 11 DP1 is a cationic radical compound. ·+UV-Vis absorption spectra, fluorescence emission spectra and relative fluorescence intensity change plots of DP1 (DP1 concentration is 100 μM, preparation method is 1 mM of DP1 and 1 mM of B(C6F5)3 reacted in dichloromethane for 3 min and then the solvent was spin-dried) in glycerol and ethanol mixed solution with increasing glycerol content: (A) UV-Vis absorption spectra; (B) fluorescence emission spectra, excitation wavelength is 580 nm; (C) DP1 ·+ Plot of the change of the emission intensity at 657 nm in different content of glycerol and ethanol mixed solution with the emission intensity at 657 nm in ethanol solution (I / I0);

[0066] Figure 12 Electron spin resonance (EPR) spectra of compound DP1 (100 mM) with different oxidants (100 mM) in acetonitrile solvent: (A) tris(pentafluorophenyl)borane B(C6F5)3; (B) nitrosyl hexafluoroantimonate NOSbF6; (C) copper perchlorate Cu(ClO4)2; (D) triethyloxonium hexachloroantimonate Et3OSbCl6;

[0067] Figure 13 Magnetization of solid powder of compound DP1 reacted with oxidant tris(pentafluorophenyl)borane B(C6F5)3 in dichloromethane for 5 min;

[0068] Figure 14 Oxidation cyclic voltammograms of compound I: (A) oxidation cyclic voltammogram of compound DP1; (B) oxidation cyclic voltammogram of compound DP2; (C) oxidation cyclic voltammogram of compound DP3; (D) oxidation cyclic voltammogram of compound DP4; (E) oxidation cyclic voltammogram of compound DP5; (F) oxidation cyclic voltammogram of compound DP6; (G) oxidation cyclic voltammogram of compound DP7; (H) oxidation cyclic voltammogram of compound DP1-OCH3;

[0069] Figure 15 Reduction cyclic voltammograms of compound I: (A) reduction cyclic voltammogram of compound DP1; (B) reduction cyclic voltammogram of compound DP2; (C) reduction cyclic voltammogram of compound DP3; (D) reduction cyclic voltammogram of compound DP4; (E) reduction cyclic voltammogram of compound DP5; (F) reduction cyclic voltammogram of compound DP6; (G) reduction cyclic voltammogram of compound DP7; (H) reduction cyclic voltammogram of compound DP1-OCH3;

[0070] Figure 16 Fluorescence emission spectra of compound DP1 ·+ Pictures of fluorescence staining of MCF-7 living cells by FeCl3;

[0071] Figure 17 For compound DP1 ·+ @FeCl3 fluorescence imaging and fluorescence spectrum in Lambda mode in MCF-7 cells: (A) fluorescence staining photo in Lambda mode; (B) fluorescence spectrum of the selected fluorescence staining area in (A);

[0072] Figure 18 For compound DP1 ·+ @FeCl3 fluorescence staining photo of MCF-7 fixed cells;

[0073] Figure 19 For compound DP1 ·+ @FeCl3 cytotoxicity results of MCF-7 cells;

[0074] Figure 20 For compound DP1 ·+ @Dynamic light scattering and transmission electron microscopy characterization of B(C6F5)3 nanoparticles: (A) dynamic light scattering characterization; (B) transmission electron microscopy characterization;

[0075] Figure 21 For compound DP1 ·+ @UV-Vis absorption spectrum, fluorescence emission spectrum and relative fluorescence intensity change chart of B(C6F5)3 nanoparticles stored at 4℃ for different time: (A) UV-Vis absorption spectrum; (B) fluorescence emission spectrum, excitation wavelength is 560 nm; (C) DP1 ·+ @B(C6F5)3 nanoparticles at different time points, the ratio change chart of the absorption or emission intensity at 627 nm or 664 nm to that at 0 h (A / A0 or I / I0);

[0076] Figure 22 For compound DP1 ·+ @B(C6F5)3 nanoparticles fluorescence staining photo of L929 cells;

[0077] Figure 23 For compound DP1 ·+ @B(C6F5)3 nanoparticles cytotoxicity results of L929 cells;

[0078] Figure 24 For compound DP1 ·+ @B(C6F5)3 nanoparticles fluorescence staining photo of zebrafish vascular imaging. DETAILED DESCRIPTION

[0079] The application will be described in further detail below with reference to the examples, but the embodiments of the application are not limited thereto.

[0080] Example 1: synthesis of compound DP1

[0081]

[0082] Compound 1: 4-methoxyaniline (1.00 g, 8.1 mmol) and 2,5-hexanedione (1.43 mL, 12.2 mmol) were dissolved in THF, 20 μL trifluoroacetic acid (TFA) was added dropwise, and the reaction was allowed to proceed at room temperature for 4 h. The product was purified by column chromatography to give white solid 1 (1.43 g, 87.7%).

[0083] Compound 1': N,N-dimethylformamide DMF (1.55 mL, 20.0 mmol) was first added to a flask, followed by dropwise addition of oxalyl chloride (COCl)2(1.70 mL, 20.0 mmol) under ice bath conditions. A large amount of white smoke was produced, and the mixture was stirred for 10 min. A solution of compound 1 (1.00 g, 5.0 mmol) in dichloromethane (30 mL) was added dropwise, the ice bath was removed, and the reaction was allowed to proceed at room temperature for 30 min. After confirming that the reaction was complete, a saturated aqueous sodium acetate (6.80 g, 50.0 mmol) solution (50 mL) was added dropwise, followed by stirring at room temperature for 3 h. The reaction was stopped, and the reaction mixture was extracted with dichloromethane (30 mL) and saturated brine (50 mL x 3 times). The organic phase was dried with anhydrous sodium sulfate for 30 min, then distilled under reduced pressure, and purified by column chromatography to give white solid compound 1' (986.3 mg, 86.0%).

[0084] Compound 1": Compound 1' (900.0 mg) was dissolved in 20 mL acetonitrile MeCN, 20 mL 39% aqueous formaldehyde was added with stirring, then 50 μL concentrated hydrochloric acid was added dropwise, and the reaction was allowed to proceed at room temperature for 5 h to give a white solid precipitate. The precipitate was collected by centrifugation and washed with water 3 times, and dried to give compound 1" (453.2 mg, 49.1%).

[0085] Compound DP1: Compound 1" (471.0 mg, 1.0 mmol) was dissolved in 10 mL methanol MeOH under ice bath conditions, followed by slow addition of sodium borohydride (378.3 mg, 10.0 mmol) under argon protection, and stirring for 30 min under ice bath conditions. After the reaction was completed, methanol was added dropwise to ensure that the sodium borohydride was completely reacted (no gas bubbles were produced), then the solvent was removed by distillation under reduced pressure, and water was added to ultrasonicate to give a white solid. The precipitate was collected by centrifugation and washed with water 3 times, and dried to give compound DP1 (427.6 mg, 90.1%). The structure was characterized as follows: 1H NMR (400 MHz, CD3OD) δ (ppm): 7.05 (d, J = 8.8 Hz, 4H), 7.00 (d, J = 8.8 Hz, 4H), 4.37 (s, 4H), 3.83 (s, 6H), 3.74 (s, 2H), 1.95 (s, 6H), 1.88 (s, 6H); 13 C NMR (100 MHz, CD3OD) δ (ppm): 160.6, 133.2, 130.7, 127.9, 125.8, 118.9, 118.8, 115.3, 56.2, 56.0, 21.3, 10.8, 10.5.

[0086] Example 2: Synthesis of compound DP1-OCH3

[0087]

[0088] Compound DP1-OCH3: Compound DP1 (474.0 mg, 1.0 mmol) and sodium hydride (60% in mineral oil, 400.0 mg, 10.0 mmol) were dissolved in 10 mL dry tetrahydrofuran under ice bath condition, which was stirred for 30 min, then methyl iodide (622.0 μL, 10.0 mmol) was added, and the reaction was carried out at room temperature for 8 h. After the reaction was completed, the residual sodium hydride was quenched with ice water, and the reaction mixture was extracted with ethyl acetate (20 mL) and saturated brine (30 mL*3 times). The organic phase was dried over anhydrous sodium sulfate for 1 h, distilled under reduced pressure, and purified by column chromatography to obtain compound DP1-OCH3 (302.0 mg, 60.0%) as a yellow solid. The structure was characterized as follows: 1 H NMR (400 MHz, CD3OD) δ (ppm): 7.08-7.04 (m, 4H), 7.04 -6.99 (m, 4H), 4.21 (s, 4H), 3.84 (s, 6H), 3.68 (s, 2H), 3.25 (s, 6H), 1.94 (s, 6H), 1.85 (s, 6H); 13 C NMR (100 MHz, CD3OD) δ (ppm): 158.8, 132.4, 129.7, 127.2, 124.9, 117.6, 115.4, 114.2, 66.0, 57.5, 55.6, 10.8, 10.8, 10.7.

[0089] Example 3: Synthesis of compound DP2

[0090]

[0091] The preparation method of this example is similar to that of Example 1.

[0092] Compound DP2 was obtained in 92.0% yield. The structural characterization is as follows: 1 H NMR (400 MHz, CD2CI2) δ (ppm): 7.50-7.40 (m, 6H), 7.18 (d, J = 6.8 Hz, 4H), 4.33 (s, 4H), 3.72 (s, 2H), 2.01 (s, 6H), 1.97 (s, 6H), 1.41 (s, 2H); 13 C NMR (100 MHz, CD2CI2) δ (ppm): 139.0, 129.1, 128.6, 127.8, 127.1, 124.5, 118.6, 118.4, 55.8, 20.3, 10.4, 10.1.

[0093] Example 4: Synthesis of compound DP3

[0094]

[0095] The preparation method of this example is similar to Example 1. In the preparation of compound 3, 4-methylaniline is replaced with 4-methylthioaniline in Example 1.

[0096] Compound DP3 was obtained in 95.0% yield. The structural characterization is as follows: 1 H NMR (400 MHz, d6-DMSO) δ (ppm): 7.35 (d, J = 8.4 Hz, 4H), 7.11 (d, J = 8.4 Hz, 4H), 4.21 (s, 6H), 3.63 (s, 2H), 2.52 (s, 6H), 1.91 (s, 6H), 1.85 (s, 6H); 13 C NMR (100 MHz, d6-DMSO) δ (ppm): 138.0, 135.9, 129.1, 126.7, 125.2, 123.8, 119.5, 117.5, 54.7, 20.5, 15.0, 10.9, 10.8.

[0097] Example 5: Synthesis of compound DP4

[0098]

[0099] The preparation method of this example is similar to Example 1. In the preparation of compound 4, 4-methoxyaniline is replaced with 4-cyanianiline in Example 1.

[0100] Compound DP4 was obtained in 90.0% yield. The structural characterization is as follows: 1H NMR (400 MHz, CD3OD) δ (ppm): 7.76 (d, J = 12.8 Hz, 4H), 7.27 (d, J = 8.4 Hz, 4H), 4.28 (s, 4H), 3.67 (s, 2H), 1.91 (s, 6H), 1.82 (s, 6H); 13 C NMR (100 MHz, CD3OD) δ (ppm): 143.3, 133.0, 129.3, 126.1, 124.1, 119.1, 118.5, 117.8, 111.0, 54.4, 19.8, 9.5, 9.2.

[0101] Synthesis of compound DP5 of Example 6

[0102]

[0103] The preparation method of this example is similar to Example 1. In the preparation of compound 5, 3,6-octanedione is used to replace 2,5-hexanedione in Example 1.

[0104] The yield of compound DP5 is 91.0%. The structural characterization is as follows: 1 H NMR (400 MHz, CD3OD) δ (ppm): 7.76 (d, J = 12.8 Hz, 4H), 7.27 (d, J = 8.4 Hz, 4H), 4.28 (s, 4H), 3.67 (s, 2H), 1.91 (s, 6H), 1.82 (s, 6H); 13 CNMR (100 MHz, CDCl3) δ (ppm): 159.4, 133.3, 131.5, 131.0, 129.9, 117.1, 116.9, 113.8, 54.7, 54.6, 19.6, 17.5, 17.2, 15.0, 13.8.

[0105] Synthesis of compound DP6 of Example 7

[0106]

[0107] The preparation method of this example is similar to Example 1. In the preparation of compound 6, isopropylamine is used to replace 4-methoxyaniline in Example 1. In the preparation of compound DP6, ethyl acetate extraction is used to replace water washing in Example 1.

[0108] The yield of compound DP6 is 79.0%. The structural characterization is as follows: 1H NMR (400 MHz, CD3OD) δ (ppm): 4.53-4.46 (m, 2H), 4.24 (s, 4H), 3.62 (s, 2H), 2.24 (s, 6H), 2.19 (s, 6H), 1.46 (s, 6H), 1.44 (s, 6H). 13 C NMR (100 MHz, CD3OD) δ (ppm): 125.1, 122.6, 117.9, 117.4, 54.6, 46.7, 21.7, 19.7, 10.0, 9.7.

[0109] Synthesis of compound DP7 of Example 8

[0110]

[0111] The preparation method of this example is similar to Example 1. In the preparation of compound 7, n-butylamine is used to replace 4-methoxyaniline in Example 1. In the preparation of compound DP7, ethyl acetate extraction is used to replace water washing in Example 1.

[0112] The yield of compound DP7 is 94.0%. The structural characterization is as follows: 1 H NMR (400 MHz, CD3OD) δ (ppm): 4.53-4.46 (m, 2H), 4.24 (s, 4H), 3.62 (s, 2H), 2.24 (s, 6H), 2.19 (s, 6H), 1.46 (s, 6H), 1.44 (s, 6H). 13 C NMR (100 MHz, CD3OD) δ (ppm): 125.1, 122.6, 117.9, 117.4, 54.6, 46.7, 21.7, 19.7, 10.0, 9.7.

[0113] In-situ generation of compound DP1 in oxidant tris(pentafluorophenyl)borane B(C6F5)3 ·+

[0114]

[0115] 40 μL of a dichloromethane solution of compound DP1 (10 mM) and 40 μL of a dichloromethane solution of different molar concentrations of oxidant tris(pentafluorophenyl)borane B(C6F5)3 (2, 4, 6, 8, 10, 12, 14, 16 and 20 mM) were added to 3920 μL of dichloromethane, and after mixing, compound DP1 could be converted into the corresponding cationic radical compound DP1 ·+ in 1 minute, which could be proved by UV-visible absorption spectrum and fluorescence emission spectrum DP1​·+ The results are shown in Figure 6. The characteristic absorption of the cationic radical compound DP1 Figure 1 at 630 nm and the near-infrared emission at 710 nm were observed.

[0116] Example 10 In-situ generation of compound DP1 ·+ from oxidant FeCl3

[0117]

[0118] To 3920 μL acetonitrile, 40 μL of compound DP1 in acetonitrile (10 mM) and 40 μL of different molar concentration of oxidant anhydrous FeCl3 in acetonitrile (2, 4, 6, 8, 10, 12, 14, 16 and 20 mM) were added. After mixing, compound DP1 ·+ was converted to the corresponding cationic radical compound DP1 ·+ in 1 minute. The results are shown in Figure 6. The characteristic absorption of the cationic radical compound DP1 Figure 2 at 630 nm and the near-infrared emission at 710 nm were observed.

[0119] Example 11 In-situ generation of compound DP1 ·+ from different oxidants

[0120]

[0121] 40 μL of a 10 mM dichloromethane solution of compound DP1 was added to 3960 μL of a dichloromethane solution containing trifluoroacetic acid (TFA) (62.5 nL / mL, serially diluted) and reacted for 1 min; 40 μL of a 10 mM acetonitrile solution of compound DP1 and 40 μL of a 10 mM acetonitrile solution containing nitrosamine hexafluoroantimony (NOSbF6) were added to 3920 μL of a acetonitrile solution and reacted for 1 min; 40 μL of a 10 mM acetonitrile solution of compound DP1 was added to 3960 μL of a 10 mM acetonitrile solution containing sulfuric acid (H2SO4) (5.0 nL / mL, serially diluted) and reacted for 1 min; 40 μL of a 10 mM acetonitrile solution of compound DP1 and 40 μL of oxygen... An acetonitrile solution (10 mM) of the oxidizing agent [bis(trifluoroacetoxy)iodide]benzene PIFA was added to a mixed solvent of 1920 μL acetonitrile and 2 mL water and reacted for 1 min; 40 μL of a dichloromethane solution (10 mM) of compound DP1 and 40 μL of a dichloromethane solution (10 mM) of the oxidizing agent triethyloxonium hexachloroantimonyate Et3OSbCl6 were added to a 3920 μL acetonitrile solution and reacted for 5 min; 40 μL of an acetonitrile solution (10 mM) of compound DP1 and 40 μL of an acetonitrile solution (10 mM) of the oxidizing agent copper perchlorate Cu(ClO4)2 were added to a 3920 μL acetonitrile solution and reacted for 1 min; after thorough mixing, all reactions were converted into the corresponding cationic free radical compound DP1. ·+ DP1 can be verified by ultraviolet-visible absorption spectroscopy. ·+ Generate. Result as follows: Figure 3 As shown, all exhibit characteristic absorption of cation radicals in the 600-700 nm range.

[0122] Example 12 Compound DP1 under Lewis acid solid-state milling ·+ In-situ generation

[0123]

[0124] Compound DP1 (10.0 mg) and Lewis acid tris(pentafluorophenyl)borane B(C6F5)3 (10.8 mg) were added to a mortar and mixed thoroughly. The mixture was then ground with a pestle for 4 minutes to convert it into the corresponding cationic free radical compound DP1. ·+ DP1 can be demonstrated through bright-field images, excitation spectra, and fluorescence emission spectra. ·+ Generate. Result as follows: Figure 4 As shown, after grinding, the powder changes from colorless to blue-green, generating excitation and emission wavelengths of cationic free radicals at approximately 680 nm and 720 nm, respectively. Compared to the solution state, a red shift occurs, which is attributed to molecular aggregation.

[0125] Example 13 Compound DP1 under solid-state milling of proton acid ·+ In-situ generation

[0126]

[0127] Compound DP1 (16.0 mg) and protonic acid 3-bromopyruvic acid (5.6 mg) were added into a mortar and ground for 4 min with a pestle after mixing well, to transform into the corresponding cation radical compound DP1 ·+ DP1 was proved to be generated by bright field image, excitation spectrum and fluorescence emission spectrum. The results are shown in ·+ Figure 5 After grinding, the powder was transformed from colorless to blue-green, and the excitation wavelength and emission wavelength of the cation radical were generated at about 680 nm and 720 nm, respectively.

[0128] Example 14 In-situ generation of compound DP1 under light ·+

[0129]

[0130] 20 μL of dichloromethane solution of compound DP1 with different concentrations was dropped onto a silica gel plate, and irradiated with 365 nm ultraviolet light for 1 min to transform into the corresponding cation radical compound DP1 ·+ DP1 was proved to be generated by excitation spectrum and fluorescence emission spectrum. The results are shown in ·+ Figure 6 The fluorescence emission was blue-shifted compared with the solution state and the solid state, which was considered to be caused by the adsorption of the cation radical by the porous silica gel, resulting in a decrease in the degree of intermolecular stacking.

[0131] Example 15 In-situ generation of compound II in organic solvent

[0132] 40 μL of dichloromethane solution (10 mM) of compound II (DP2-DP7) and 40 μL of dichloromethane solution (10 mM) of oxidant tris(pentafluorophenyl)borane B(C6F5)3 were added into 3920 μL of dichloromethane to react for different time (DP2, DP6, DP7 reacted for 1 min; DP3, DP4 reacted for 2 min; DP5 reacted for 30 min), to transform into the corresponding cation radical compound II, which was proved to be generated by ultraviolet-visible absorption spectrum and fluorescence emission spectrum. The results are shown in Figure 7 It was shown that the pyrrole dimers with different substituents could all generate the corresponding cation radicals.

[0133] ​​​The 200 μL compound DP1-OCH3 solution (10 mM) in dichloromethane and 200 μL oxidant tris(pentafluorophenyl)borane B(C6F5)3 solution (10 mM) in dichloromethane were added to 3600 μL dichloromethane and reacted for 30 min to transform into the corresponding cationic radical compound II, and the generation of the cationic radical was proved by UV-visible absorption spectrum and fluorescence emission spectrum. The results are shown in Figure 8 .

[0134] Example 16 In-situ generation of compound II under solid grinding

[0135] The equal molar equivalent ratio of compound II (DP2-DP7, DP1-OCH3) and oxidant tris(pentafluorophenyl)borane B(C6F5)3 was weighed, mixed uniformly in a mortar, and ground with a pestle for 4 min to transform into the corresponding cationic radical compound II, and the generation of the cationic radical was proved by bright field pictures, excitation spectrum and fluorescence emission spectrum. The results are shown in Figure 9 and Figure 10 .

[0136] Example 17 Compound DP1 ·+ Spectral changes in different viscosities (glycerol / ethanol)

[0137] The compound DP1 and oxidant tris(pentafluorophenyl)borane were mixed in equal molar equivalent in dichloromethane solution for 3 min, and the final concentration of the molecules was 1 mM. After the solvent was spin-dried, an equal volume of ethanol was added for dissolution, and diluted into glycerol / ethanol mixed according to different proportions (glycerol / ethanol = 80 / 20, 60 / 40, 40 / 60, 20 / 80, 10 / 90, 0 / 100), and the final concentration of compound DP1 was 100 μM. Then, UV-visible absorption spectrum and fluorescence emission spectrum (excitation wavelength was 580 nm) tests were carried out, as shown in Figure 11 ; and quantum yield and fluorescence lifetime tests, as shown in Table 2. With the increase of glycerol content, the relative fluorescence intensity ratio and quantum yield of compound DP1 ·+ both gradually increased, which indicated that the fluorescence intensity and photophysical properties of the compound would be further enhanced under the condition of limited molecular motion. At the same time, with the increase of glycerol content, the emission wavelength was blue-shifted, indicating that with the increase of viscosity, the molecular aggregation was destroyed, and the state was transformed from aggregation to monodisperse state.

[0138] Example 18 Verification of the generation of cationic radicals

[0139] (1) Electron paramagnetic resonance spectrum test

[0140] Take 10 mg of compound DP1 and equimolar amount of oxidant tris (pentafluorophenyl) borane B (C6F5) 3, nitrous oxide antimony hexafluoride NOSbF6, copper perchlorate Cu (ClO4) 2, triethyl oxonium antimony chloride Et3OSbCl6, dissolved in acetonitrile to prepare 100 mM solution, react for 5 min, and then suck it into the glass tube for testing. After sealing, the EPR spectrum was recorded on the Bruker E500-10 / 12 electron paramagnetic resonance spectrometer, and the results are shown in Figure 12 Different oxidants were used to oxidize, and obvious free radical signal peaks were collected, proving the generation of cationic free radicals.

[0141] (2) Magnetic susceptibility test

[0142] Take 10 mg of compound DP1 and 10.8 mg of oxidant tris (pentafluorophenyl) borane B (C6F5) 3, dissolve in dichloromethane and react for 5 min, spin dry the solvent to collect the solid powder, and take 4.5 mg of the powder to test the magnetic susceptibility on the Quantum Design MPMS 3 superconducting quantum interference magnetic measurement system. The test conditions are: direct current, external magnetic field 0.1 T, 2-300 K temperature drop test of magnetic moment change. The results are shown in Figure 13 It has paramagnetic properties, proving the existence of cationic free radicals.

[0143] Quantum yield and fluorescence lifetime of compound II in example 19

[0144] The quantum yield and fluorescence lifetime of compound II generated by in situ oxidation of oxidant tris (pentafluorophenyl) borane B (C6F5) 3 were detected. Compound I dichloromethane solution (10 mM, 40 μL) and oxidant tris (pentafluorophenyl) borane dichloromethane solution (10 mM, 40 μL) were added to 3920 μL of dichloromethane solution to react for different times to prepare compound II (DP1, DP2, DP6 and DP7 react for 1 min, DP3 and DP4 react for 2 min, and DP5 react for 30 min), and then the quantum yield and fluorescence lifetime were tested. The results are shown in Table 1. Different substituents can produce high luminous efficiency.

[0145] Table 1 Maximum absorption wavelength, maximum emission wavelength, quantum yield and fluorescence lifetime of compound II

[0146] Compound II Absorption wavelength (nm) Emission wavelength (nm) Quantum yield (%) Fluorescence lifetime (ns) DP1 ·+ ]]> 630 711 34.7 5.01 DP2 ·+ ]]> 630 710 32.1 5.23 DP3 ·+ ]]> 638 719 37.8 4.75 DP4 ·+ ]]> 676 714 43.5 4.25 DP5 ·+ ]]> 675 717 43.4 5.45 DP6 ·+ ]]> 630 650 37.4 6.86 DP7 ·+ ]]> 637 666 44.1 6.76

[0147] Example 20 Compound DP1 ·+ Quantum yield and fluorescence lifetime in different glycerol / ethanol systems

[0148] Detecting compound DP1 generated by in situ oxidation of oxidant tris (pentafluorophenyl) borane ·+Quantum yield and fluorescence lifetime in different glycerol / ethanol systems. The dichloromethane solution of compound DP1 (10 mM, 500 μL) and the dichloromethane solution of oxidant tris(pentafluorophenyl)borane (10 mM, 500 μL) were added to 4 mL dichloromethane solution for 3 min, after the solvent was spun dry, 5 mL ethanol was added to dissolve, diluted into glycerol and water mixed system, so that the final volume ratio of glycerol / ethanol was 80 / 20, 60 / 40, 40 / 60, 20 / 80, 10 / 90, 0 / 100, the final concentration of compound DP1 was 100 μM, then the quantum yield and fluorescence lifetime test was carried out. The results are shown in Table 2.

[0149] Table 2 Compound DP1 ·+ Maximum absorption wavelength, maximum emission wavelength, quantum yield and fluorescence lifetime in glycerol system

[0150] Glycerol ratio (%) Absorption wavelength (nm) Emission wavelength (nm) Quantum yield (%) Fluorescence lifetime (ns) 0 625 716 8.9 3.03 10 625 716 9.9 2.84 20 625 716 10.7 2.94 40 625 715 12.4 2.76 60 625 658 15.1 1.74 80 625 657 18.2 2.55

[0151] Example 21

[0152] The cyclic voltammogram of compound I was detected. The glassy carbon electrode was used as the working electrode, the platinum wire was used as the counter electrode, Ag / Ag + The ferrocene cation / ferrocene was used as the internal standard, and the scanning rate was 100 mV s -1 Anhydrous dimethylformamide and anhydrous dichloromethane (0.1 M) containing tetrabutylammonium hexafluorophosphate were used as the supporting electrolyte for negative scanning and positive scanning, respectively. The oxidation cyclic voltammogram is shown in Figure 14 The reduction cyclic voltammogram is shown in Figure 15 The redox potential is shown in Table 3, and the pyrrole dimer has a low oxidation potential.

[0153] Table 3 Redox potential of compound I

[0154] Compound I Oxidation potential (V) Reduction potential (V) DP1 0.35 -1.10 DP1 -OCH3 0.37 -1.13 DP2 0.39 -1.13 DP3 0.41 -1.11 DP4 0.43 -1.13 DP5 0.41 -1.14 DP6 0.31 -1.13 DP7 0.33 -1.14

[0155] Example 22

[0156] DP1 ·+ Application of @FeCl3 in cell imaging:

[0157] DP1 ·+ Live cell imaging of @FeCl3: After counting the MCF-7 cells (human breast cancer cells) by hemocytometer, they were inoculated on laser confocal dishes at a density of about 1 x 10 5 cells / dish, after 24 h of culture, an equal volume of acetonitrile of compound DP1 (10 mM) and FeCl3 (10 mM) was mixed to generate DP1 ·+FeCl3solution, then 4.0 μL of the mixed solution was added to 1 mL of PBS to make a 20 μM staining solution. After incubation for 1 h, the cells were co-stained with commercial dyes Hoechst 33342 and Mito-Tracker Green. The results showed that DP1 ·+ FeCl3can be used for specific fluorescence imaging of mitochondria in living MCF-7 cells. Figure 16 DP1 ·+ Lambda imaging mode of FeCl3uses a 639 nm laser, and collects fluorescence signals of 640-750 nm. Figure 17 The maximum emission wavelength was found to be around 670 nm, indicating that it came from the luminescence of cation radical DP1 ·+ .

[0158] DP1 ·+ Imaging of fixed cells with FeCl3: After counting MCF-7 cells (human breast cancer cells) by a hemocytometer, they were seeded on laser confocal dishes at a density of about 1 x 10 5 cells / dish. After incubation for 24 h, the cells were washed and fixed with 1 mL of 2.5% glutaraldehyde for 6 h. After washing with PBS, 1 mL of PBS solution containing DP1 ·+ FeCl3(the solution was prepared by mixing equal volumes of DP1(10 mM) and FeCl3(10 mM) in acetonitrile in situ) was added. After incubation for 1 h, the cells were co-stained with commercial dyes Hoechst 33342 and Mito-Tracker Green. The results showed that DP1 ·+ FeCl3solution (5 mM), then 4.0 μL of the mixed solution was added to 1 mL of PBS to make a 20 μM staining solution. After incubation for 1 h, the cells were co-stained with commercial dyes Hoechst 33342 and Mito-Tracker Green. The results showed that DP1 ·+ FeCl3can be used for specific fluorescence imaging of mitochondria in fixed MCF-7 cells. Figure 18 DP1 ·+ Mitochondrial staining with FeCl3does not depend on mitochondrial membrane potential.

[0159] The products of DP2-DP7 by oxidation with oxidants also have similar mitochondrial staining imaging.

[0160] Example 23

[0161] DP1 ·+ Cytotoxicity of FeCl3( Figure 19 ):

[0162] MCF-7 cells (human breast cancer cells) were seeded on laser confocal dishes at a density of about 1 x 10 4Cells were seeded at a density of 1 cell / well in 96-well plates, with 5 replicates per group, and a blank control group (culture medium only) was added. After 24 hours, the cell culture medium was aspirated, and cells containing different concentrations of DP1 were added. ·+ Cell culture medium containing FeCl3 (DP1 dissolved in acetonitrile to prepare a 10mM stock solution, FeCl3 dissolved in acetonitrile to prepare a 10mM stock solution, and equal volumes of both were mixed for 30 seconds and then diluted into complete culture medium) was prepared. The wells were then placed in a cell culture incubator and cultured at 37°C for 24 hours. Afterward, the culture medium was aspirated, and the cells were washed once with 100 μL of PBS. 100 μL of medium containing MTT (0.5 mg / mL) was added to each well, and the cells were incubated at 37°C for 4 hours. The medium was then aspirated, and 100 μL of biological-grade dimethyl sulfoxide was added to each well. The plates were then shaken on a shaker at room temperature for 10 minutes to fully dissolve the precipitate. Finally, the absorbance of each well at 570 nm was measured using a microplate reader. The results are shown below. Figure 19 As shown. The results indicate that DP1 ·+ @FeCl3 retains good biocompatibility even at a concentration of 100 μM.

[0163] Example 24

[0164] DP1 ·+ @B(C6F5)3 nanoparticle imaging

[0165] Preparation of nanoparticles: Prepare 40 mM acetonitrile solution of DP1 and 40 mM acetonitrile solution of B(C6F5)3. F127(M w =12600) aqueous solution 20 mg / mL. Take 100 μL each of DP1 and B(C6F5)3 solution and react for 1 min, then add dropwise to F127 aqueous solution under high-speed stirring (1000 rpm, 5 min). Remove unencapsulated DP1 through 0.45 μm filter. ·+ The nanoparticles were then concentrated by ultrafiltration and centrifugation at 4°C (molecular weight cutoff 30 kDa, centrifugation speed 6000 rpm, centrifugation time 30 min, repeated three times) to further remove acetonitrile and unencapsulated molecules, thus obtaining purified nanoparticles.

[0166] Nanoparticle size and morphology characterization: The purified nanoparticles were diluted in ultrapure water and measured by dynamic light scattering. The particle size was 104 nm, the polydispersity index was 0.173, and the distribution was uniform. Figure 20 A). An aqueous solution of nanoparticles was dropped onto a copper mesh, and the morphology was characterized using transmission electron microscopy. Figure 20 B) The size is smaller than that of the dynamic light scattering results because the dynamic light scattering test is for hydrated particle size, while the transmission characterization will be performed by air drying and laser irradiation may cause the particles to shrink.

[0167] Determination of nanoparticle concentration: The concentrated nanoparticle aqueous solution (800 μL) was lyophilized to get 45 mg powder. Since the nanoparticle aqueous solution is stable, the density of the nanoparticle suspension can be estimated as ~1 g / cm3. 3 The average size of the nanoparticle was determined by TEM as ~50 nm, so the concentration of the nanoparticle can be calculated by the following equation: total number of particles in 800 μL nanoparticle suspension = total volume of nanoparticle / average volume of nanoparticle = (45 x 10 -3 / 1) / (4 x π x (25 x 10 -7 ) 3 / 3) = 6.9 x 10 14 , concentration of particles in 800 μL nanoparticle suspension = (6.9 x 10 14 / (6.02 x 10 23 )) / (0.8 x 10 -3 ) = 1.43 μM.

[0168] Characterization of cation radical stability in the nanoparticle and quantum yield and fluorescence lifetime characterization: The purified nanoparticle was diluted in ultrapure water, and the ultraviolet-visible absorption spectrum and fluorescence emission spectrum were tested at 4°C for different time (test results are shown in Figure 21 The cation radical in the nanoparticle can be stably maintained for more than 4 days at low temperature, indicating that the nanoparticle can be stored for a long time. At the same time, the quantum yield and fluorescence lifetime of the nanoparticle were tested (Table 4), and the nanoparticle still has high luminescent efficiency.

[0169] Table 4 Maximum absorption wavelength, maximum emission wavelength, quantum yield and fluorescence lifetime of the nanoparticle aqueous solution

[0170] Absorption wavelength (nm) Emission wavelength (nm) Quantum yield (%) Fluorescence lifetime (ns) Nanoparticle 625 662 14.9 3.69

[0171] Cell imaging of the nanoparticle: After counting the L929 cells (mouse fibroblasts) by a hemocytometer, the cells were inoculated on a laser confocal dish at a density of about 1 x 10 5 cells / dish, and after 24 h of culture, 1.0 mL of serum-free medium containing the nanoparticle was added, and confocal imaging was performed after 30 min of incubation (test results are shown in Figure 22 The nanoparticle has excellent cell entry efficiency.

[0172] Cytotoxicity of the nanoparticle: L929 cells were inoculated on a laser confocal dish at a density of about 1 x 10 4The density of 1 cell / hole is seeded in 96-well plates, 5 wells are set in each group, and a blank control group (only medium is added) is set. After 24 h, the cell culture solution is removed, and cell culture solution containing different concentrations of nanoparticles is added. Then, the well plate is placed in a cell culture box, and incubated at 37°C for 24 h. Then, the culture solution is removed, and washed once with 100 μL of PBS. 100 μL of medium containing MTT (0.5 mg / mL) is added to each well, and incubated at 37°C for 4 h. The culture medium is removed, and then 100 μL of biological grade dimethyl sulfoxide is added to each well, and placed on a shaker at room temperature for 10 min to dissolve the precipitate. Finally, the absorbance of each well at 570 nm is measured by an enzyme-labeled instrument (the test results are shown in Table 1). Figure 23 The results show that the nanoparticles have good biocompatibility.

[0173] Zebrafish vascular imaging of nanoparticles: First, the vascular transfection flk1: GFP (expressing green fluorescence) zebrafish larvae are cultured to 3 dpf, and the zebrafish is anesthetized using tricaine. Then, the concentrated nanoparticle solution is injected intravenously into the zebrafish blood vessels using microinjection technology (injection volume 1.0 nL). The agarose is fixed to the zebrafish, and after 1 h of circulation, the vascular imaging effect is photographed using confocal (the test results are shown in Table 2). Figure 24 The results show that the nanoparticles can effectively image the zebrafish blood vessels.

[0174] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A luminescent, stable cationic radical compound based on methylene-bridged pyrrole dimers, characterized in that: Its cationic free radical has the structure of formula II and is stabilized by anion or stabilizer; Where R 1 For substituted or unsubstituted alkyl groups, substituted or unsubstituted aryl groups; R 2 It is hydrogen, substituted or unsubstituted alkyl, alkyloxy, alkylamino; R 3 Alkyl groups, substituted alkyl groups; R 1 In this context, the alkyl group is a straight-chain or branched alkyl group; the substituted alkyl group refers to a group that substitutes hydrogen in the alkyl group formed by a cyclic compound, and the cyclic compound is a cyclic compound formed by carbon, hydrogen and one or more heteroatoms of N, S, and O. R 1 In this context, the aryl group refers to a monocyclic or polycyclic aromatic group having 6-20 carbon atoms; R 1 In this context, the substituted aryl group refers to the hydrogen atom on the aryl ring being replaced by one or more of alkoxy, alkylthio, amino, carboxyl, and cyano groups; the substitution is monosubstituted, disubstituted, or trisubstituted. R 2 The substituted alkyl group mentioned above refers to the alkyl group in which hydrogen atoms are replaced by hydroxyl, methoxy, carboxyl, or halogen groups; R 3 In this context, the substituted alkyl group refers to an alkyl group in which hydrogen atoms are replaced by hydroxyl, methoxy, carboxyl, or halogen groups. The anion is an anion provided by an oxidizing agent, a Lewis acid, or a protic acid.

2. The luminescent stable cationic radical compound based on methylene-bridged pyrrole dimers according to claim 1, characterized in that: R 1 In the above, the cyclic compound is A group formed by the loss of a hydrogen atom from a cyclic compound replaces a hydrogen atom in an alkyl group; R 1 In this context, the alkyl group is C10. 1-10 Alkyl group; the aryl group is phenyl, naphthyl, anthraceneyl, or pyreneyl. R 1 In the substituted aryl group, the aryl group is phenyl, naphthyl, anthraceneyl, or pyrene; R 2 In this context, the alkyl group is C10. 1-10 Alkyl, alkyloxy group is C 1-10 Alkyloxy and alkylamino are C 1-10 Alkylamino; R 3 In this context, the alkyl group is C10. 1-10 Alkyl; the alkyl group in the substituted alkyl group is C10. 1-10 alkyl; The oxidant is one or more of the following: trifluoroacetic acid, sulfuric acid, p-bromotriphenylamine radical cation hexachloroantimonate, tris(2,4-dibromotriphenyl)amine hexachloroantimonate, tris(pentafluorophenyl)borane, sodium tetra(pentafluorophenyl)borate, nitrosyl hexafluoroantimonate, triethyloxonium hexachloroantimonate, [bis(trifluoroacetoxy)iodide]benzene, thallium trifluoroacetate, m-chloroperoxybenzoic acid, thallium trifluoroacetate, copper perchlorate, ferric chloride, and silver hexafluoroantimonate. Lewis acid or protic acid is one or more of the following: tris(pentafluorophenyl)borane, citric acid monohydrate, phosphomolybdic acid, phosphotungstic acid, p-toluenesulfonic acid, pyruvic acid, 3-bromopyruvic acid, oxalic acid, 4-sulfonylcalix[4] aromatic hydrocarbon, and glycolic acid. The stabilizer is silica gel.

3. The luminescent stable cationic radical compound based on methylene-bridged pyrrole dimers according to claim 2, characterized in that: R 1 It is an alkyl, phenyl, naphthyl, or substituted aryl group; the substituent in the substituted aryl group is one or more of alkoxy, alkylthio, carboxyl, and cyano groups, and the aryl group in the substituted aryl group is phenyl or naphthyl. R 2 It is an alkyl group; R 3 It is an alkyl group substituted with a hydroxyl group.

4. The method for preparing the luminescent stable cationic radical compound based on methylene-bridged pyrrole dimers according to any one of claims 1 to 3, characterized in that: Includes the following steps: Yes The compound of formula I is oxidized in a solvent by an oxidizing agent, or oxidized in the solid state under the action of Lewis acid or protic acid, or oxidized under ultraviolet light in an oxygen atmosphere to obtain a luminescent stable cationic radical compound based on methylene-bridged pyrrole dimer. Compound of Formula I: R 1 ~R 3 R of formula II in luminescent stable cationic radical compounds based on methylene-bridged pyrrole dimers 1 ~R 3 .

5. The method for preparing the luminescent stable cationic radical compound based on methylene-bridged pyrrole dimers according to claim 4, characterized in that: The oxidant is one or more of the following: trifluoroacetic acid, sulfuric acid, p-bromotriphenylamine radical cation hexachloroantimonate, tris(2,4-dibromotriphenyl)amine hexachloroantimonate, tris(pentafluorophenyl)borane, sodium tetra(pentafluorophenyl)borate, nitrosyl hexafluoroantimonate, triethyloxonium hexachloroantimonate, [bis(trifluoroacetoxy)iodide]benzene, thallium trifluoroacetate, m-chloroperoxybenzoic acid, thallium trifluoroacetate, copper perchlorate, ferric chloride, and silver hexafluoroantimonate. The solvent is an organic solvent, water, or PBS solution; The organic solvent is one or more of acetonitrile, dichloromethane, trichloromethane, carbon tetrachloride, tetrahydrofuran, or 1,4-dioxane; Lewis acid or protic acid is one or more of the following: tris(pentafluorophenyl)borane, citric acid monohydrate, phosphomolybdic acid, phosphotungstic acid, p-toluenesulfonic acid, pyruvic acid, 3-bromopyruvic acid, oxalic acid, 4-sulfonylcalix[4] aromatic hydrocarbon, and glycolic acid. The solid state refers to mixing and grinding a compound of formula I with a Lewis acid or a protic acid. In the solvent, the concentration of the compound of formula I is 1 μM to 1000 mM; the molar ratio of the oxidant to the compound of formula I is (0.001 to 10):

1. In the solvent, the reaction is carried out at room temperature; When exposed to ultraviolet light, the compound of formula I comes into contact with silica gel.

6. A complex containing a luminescent stable cationic radical compound, characterized in that: It is mainly prepared by encapsulating a luminescent stable cationic radical compound with a polymer; specifically, the compound of formula I is oxidized to obtain the luminescent stable cationic radical compound; then the luminescent stable cationic radical compound and the polymer are stirred at high speed in an aqueous phase to remove the solvent and obtain the complex. The luminescent stable cationic radical compound is defined as in any one of claims 1 to 3; Compound of Formula I: R 1 ~R 3 R of formula II in symluminescent stable cationic radical compounds 1 ~R 3 ; The polymer is one or more of the following: poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) copolymer, polyoxyethylene castor oil, distearate phosphatidylethanolamine-polyethylene glycol, hyaluronic acid, or polylactic acid-glycolic acid copolymer.

7. The complex containing a luminescent stable cationic radical compound according to claim 6, characterized in that: The oxidation of compound I specifically refers to oxidizing compound I in a solvent using an oxidizing agent or mixing compound I with a Lewis acid or a protic acid, grinding it, and dissolving the ground product in an organic solvent; then, the oxidized product or the ground product dissolved in the organic solvent is stirred with the polymer in an aqueous phase at high speed to remove the solvent and obtain the complex; the organic solvent is one or more of dimethyl sulfoxide, acetonitrile, and ethanol. In the preparation of the complex, the amount of the polymer and the compound of formula I is 0.01 mg: (10-30) mmol; the concentration of the polymer in the aqueous phase is 10-30 mg / mL; and the speed of the high-speed stirring is 500-2000 rpm.

8. A fluorescent imaging probe, characterized in that: Including luminescent stable cationic radical compounds based on methylene-bridged pyrrole dimers or complexes containing luminescent stable cationic radical compounds; The luminescent stable cationic radical compound based on methylene-bridged pyrrole dimer is defined as in any one of claims 1 to 3; The complex containing the luminescent stable cationic radical compound is as defined in any one of claims 6 to 7.

9. The application of the fluorescence imaging probe according to claim 8, characterized in that: The fluorescent probe is used for organelle-specific fluorescence imaging.

10. The application according to claim 9, characterized in that: The fluorescent probe is used for mitochondrial-specific fluorescence imaging.

11. The application of the fluorescence imaging probe according to claim 8, characterized in that: The fluorescent probe is used for vascular imaging; The fluorescent probe comprises a complex containing a cationic radical compound that emits near-infrared light.

12. A luminescent material, characterized in that: Including luminescent stable cationic radical compounds based on methylene-bridged pyrrole dimers or complexes containing luminescent stable cationic radical compounds; The luminescent stable cationic radical compound based on methylene-bridged pyrrole dimer is defined as in any one of claims 1 to 3; The complex containing the luminescent stable cationic radical compound is as defined in any one of claims 6 to 7.

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