Meso amide substituted 1,3,5,7-tetramethyl-bodipy compounds, methods of making and uses thereof

Abstract

The present application belongs to the technical field of fluorescence analysis, and relates to a fluorescent dye used for fluorescence analysis, in particular to a fluorescent dye with a meso amide substituted BODIPY structure, a preparation method and application. Especially, it relates to a meso amide substituted 1,3,5,7-tetramethyl-BODIPY compound, and anti-quenching characteristics and application in biological imaging. The compound has excellent amphiphilicity, certain anti-water quenching ability, high fluorescence quantum yield in organic solvents and water, and can be used for fluorescence labeling, biological imaging and development of new fluorescent probes. The fluorescent dye with the meso amide substituted BODIPY structure has the following skeleton structure (I):

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fluorescence analysis, and relates to a fluorescent dye used for fluorescence analysis, in particular to a fluorescent dye with a meso amide-substituted BODIPY structure, a preparation method and application. In particular, it relates to a meso amide-substituted 1,3,5,7-tetramethyl-fluorobodipy compound, and its anti-quenching characteristics and application in biological imaging. BACKGROUND

[0002] The prior art discloses that fluorescence imaging has the advantages of non-destructive microenvironment, high resolution, high sensitivity, in-situ online, visualization of various complex physiological processes, etc., so that the fluorescence imaging technology gradually plays an important role in biomedical research. In recent years, small organic molecule fluorescent dyes with small molecular weight and high fluorescence quantum yield have unique advantages such as ultra-sensitivity, rapidness, safety and low cost, so small molecule fluorescent dyes have broad application prospects, including: (1) high-definition imaging: for example, clear visualization of the presence of various proteins and muscle tissues, which is a powerful tool for visualizing living cells and tissues; (2) biomolecular labeling: for example, the biomolecular labeling function of fluorescent dyes on neurons can be used to study the pathogenesis of various neurodegenerative diseases; (3) biological detection and environmental monitoring: for example, small molecule fluorescent probes can be used for gas signaling molecules, bioactive oxides, bioactive sulfides, etc.; (4) clinical diagnosis and treatment: for example, the fluorescence molecular imaging technology is developing rapidly, and with the help of fluorescent molecules and fluorescence endoscopy equipment, tumors invisible to the naked eye can be "precisely lit", which is widely used for early diagnosis, improving the detection rate in the early stage of cancer and guiding doctors to perform tumor cutting surgery, etc.

[0003] Fluorescent dyes gradually play a crucial role in fluorescence imaging and sensing technology. The photophysical and physicochemical properties of small molecule fluorescent dyes are very crucial to their application effect. The current fluorescent dyes are mostly aromatic conjugated structures, which have strong liposolubility and usually lack sufficient water solubility, which is very unfavorable for biological applications. The introduction of water-soluble functional groups such as sulfonate groups into the dye structure is a classic method to improve the water solubility of dyes (such as CF, Dy, ATTO and Alexa dyes). However, such structural modification often produces polar dyes with poor membrane permeability. Therefore, there is still a lack of fluorescent dyes with strong fluorescence emission in organic solvents and aqueous environments so far.

[0004] BODIPY is a kind of small molecule fluorescent dye which has been widely studied and applied in recent years. It has excellent biological and chemical properties such as neutral core, low biological toxicity, fluorescence signal insensitive to environmental polarity and pH, easy to be chemically modified, and many other excellent optical properties such as high fluorescence quantum yield, high molar extinction coefficient, adjustable fluorescence characteristics, strong photo-thermal stability, high detection sensitivity, etc. However, BODIPY dyes are also prone to aggregation-caused quenching (ACQ) in polar solvents, resulting in insufficient fluorescence brightness and ineffective excitation in aqueous solution. The existing strategies to increase water solubility mainly introduce ionic hydrophilic groups (quaternary ammonium salt, sulfonate, carboxylate or zwitterionic moiety) or oligomers (ethylene glycol) into the BODIPY dye; these modifications often lead to complex preparation, difficult purification, greatly increased molecular weight and reduced penetration through the cell membrane, etc.

[0005] 1,3,5,7-tetramethyl-BODIPY is commonly used as a fluorophore, and the spatial repulsion of the two methyl groups at 1,7 positions drives the meso position functional group to twist out of the BODIPY plane, thereby preventing the hydrophobic π-π stacking of BODIPY. Functionalization of BODIPY dyes is usually carried out at the 2,6-, 3,5- or 8-(meso) positions, and modification of the meso position substituent has attracted much attention due to the electronic effect of the meso position substituent, in which photoinduced electron transfer (PET) plays a crucial role in adjusting the fluorescence properties of BODIPYs. For example, compound Ref-1 (λ abs = 511 nm, λ em = 526 nm, Φ f = 0.019, PBS) shows poor fluorescence emission in aqueous solution, while compound Ref-1 (λ abs = 496 nm, λ em = 511 nm, Φ f = 0.58) has significantly increased fluorescence in aqueous solution, mainly due to the change in electronic effect from ester group to carboxyl group, but the polarity of carboxyl group limits its further application.

[0006] Based on the current status of the prior art, the inventors of the present application propose a class of BODIPY fluorescent dyes with amide structure at the meso position. This class of compounds has strong fluorescence emission in organic solvents and aqueous solution environments, and the double-substituted compound on the amide N atom has good anti-quenching ability in aqueous solution system, and the amide site can provide a connection site to allow various derivatization modifications, which has important significance for the biological application of fluorescent dyes. SUMMARY

[0007] The present application aims at providing a novel BODIPY compound with good anti-quenching ability and its application based on the current situation of the prior art, and particularly relates to a BODIPY fluorescent dye with amide structure at the meso position, and more particularly to a 1,3,5,7-tetramethyl-fluorobodipy compound with amide substitution at the meso position.

[0008] Specifically, the present application provides a 1,3,5,7-tetramethyl-BODIPY compound with amide substitution at the meso position with the structure of general formula (I):

[0009]

[0010] wherein R1 and R2 are independently selected from H, unsubstituted and substituted C1-C6 alkyl, C1-C6 unsaturated alkyl, C1-C6 alkylamino, C1-C6 alkoxy, aryl; in addition, R1 and R2 can be connected to form a ring with a size of 4 to 6-membered ring, which can contain heteroatoms such as oxygen and nitrogen,

[0011] Compared with the analogues with amide substitution at the meso position, the BODIPY dye with amide substitution at the meso position has good amphiphilicity, good fluorescence quantum yield in organic solvents and water, good anti-quenching ability, and is beneficial to the application in molecular imaging.

[0012] In the present application, the compound has the structure of the following compounds 1-23:

[0013]

[0014] For example, the compound 1 and 17, the compound of the present application is prepared as follows:

[0015]

[0016] The present application further aims at providing the application of the 1,3,5,7-tetramethyl-BODIPY dye compound with amide substitution at the meso position in different fields such as biological imaging, fluorescent probe, fluorescent sensor, and particularly in the preparation of cell fluorescent imaging agent.

[0017] The compounds of the present invention can be applied in various fields such as bioimaging, fluorescent probes, and fluorescent sensors by linking functional structures to amide substituents at the 2, 6, and meso positions of 1,3,5,7-tetramethyl-BODIPY. Functional structures include, but are not limited to, amino acids, proteins, glycoproteins, lipoproteins, receptors, antibodies or fragments thereof, antigens, aptamers, polysaccharides, oligosaccharides, nucleosides, nucleotides, oligonucleotides, nucleic acids, drugs, inhibitors, hormones, nutrients, metabolites, growth factors, lipids, polymers, polymeric microparticles, cells, viruses, enzyme substrates, etc.; they can link mitochondrial targeting groups (such as triphenylphosphine groups), lysosomal targeting groups (such as morpholino groups, dimethylamino groups, etc.), endoplasmic reticulum targeting groups (such as pentafluorobenzoyl groups, p-toluenesulfonyl groups, etc.) for fluorescent probes targeting organelles (such as imaging of mitochondria, lysosomes, endoplasmic reticulum, and Golgi apparatus); they can link protein labeling structures (such as SNAP-tags, Halo-tags, etc.), antibody-linked structures, and tumor-targeting structures (such as biotin, folic acid, etc.) for high-resolution cell microscopy and tumor-targeting imaging; they can link triplet quenching structures to enhance the photostability of fluorophores; and they can link click chemistry reaction structures (such as azide groups, alkynyl groups, etc.) for click chemistry, etc.

[0018] For example, compound 13 can be used for single-cell imaging of HeLa cells, compounds 11 (Lyso-01), 12 (Lyso-02), and 14 (Lyso-03) can be used for targeted imaging of lysosomes in HeLa cells, compound 21 (ER-1) can be used for targeted imaging of lysosomes in HeLa cells, and compound 22 can be used for imaging of exogenous hypochlorous acid in HeLa cells and imaging of endogenous hypochlorous acid in Raw 264.7 cells.

[0019] In this invention, the HeLa cells and Raw 264.7 cells used are commercially available to those skilled in the art.

[0020] The compounds of this invention exhibit strong fluorescence emission in organic solvents and aqueous solutions. In particular, compounds with double substitution on the N atom of the amide exhibit good resistance to quenching in aqueous systems, and the amide site can provide a linking site to allow for various derivatization modifications, which is of great significance for the application of fluorescent dyes in biology. Attached Figure Description

[0021] Figure 1 Cell imaging experiments of compounds Ref-2, 1, 2, and 13: (A) Ref-2; (B) 1; (C) 2; (D) 13, scale bar: 20 μm.

[0022] Figure 2Lysosomal localization experiments of compounds 11 (Lyso-01), 12 (Lyso-02), and 14 (Lyso-03): (A1, B1, C1) Green fluorescence images of compounds Lyso-01–Lyso-03; (A2, B2, C2) Red fluorescence images after incubation with Lyso-tracker Red (50 nM); (A3, B3, C3) Superimposed images of the green fluorescence and red fluorescence of compounds Lyso-01–Lyso-03 and Lyso-tracker Red; (A4, B4, C4) Corresponding fluorescence intensity superimposed curves. Scale bar: 10 μm.

[0023] Figure 3 Cell survival rate-concentration bar chart of compounds in HeLa cells. (A) Compound 11 (Lyso-01); (B) Compound 12 (Lyso-02); (C) Compound 14 (Lyso-03).

[0024] Figure 4 (A) Green fluorescence of compound 21 (ER-1); (B) Red fluorescence of ER-tracker Red; (C) Overlay of A and B; (D) Corresponding fluorescence intensity overlay curves, scale bar: 10 μm.

[0025] Figure 5 Probe 22 was detected in HeLa cells and Raw 264.7. – The confocal fluorescence images show that (A) HeLa cells were incubated with probe 22 (10 μM) for 30 minutes; and (B) HeLa cells were pre-incubated with probe (10 μM) for 30 minutes, followed by incubation with ClO2. – (C) Incubate raw 264.7 cells with probe (10 μM) for 30 minutes; (D) Stimulate raw 264.7 cells with 100 μg / mL LPS for 24 h, then with 1 μg / mL PMA for 1 h, and then incubate with probe (10 μM) at 37 °C for 30 minutes; (E) During PMA stimulation, co-incubate with the MPO inhibitor 4-ABAH (100 μM); other steps are the same. (Top: bright field image, middle: fluorescence image, bottom: merged image, Ex = 514 nm), scale bar: 20 μm.

[0026] Figure 6 The cell survival rate-concentration bar chart of compound 22 in HeLa cells. Detailed Implementation

[0027] The present application can be described in detail by the following examples, but it does not mean any unfavorable limitation to the present application. The described examples are part of the examples of the present application, but not all of them. Based on the examples in the present application, all the examples obtained by the ordinary skilled in the art without making creative labor are within the scope of protection of the present application.

[0028] For all the following examples, standard procedures and purification methods known to those skilled in the art can be used. Unless otherwise stated, all temperatures are in °C (degrees Celsius). The structural formula of the compounds is determined by 1 H NMR, 13 C NMR, MS (ESI), HRMS (ESI).

[0029] Example 1: Synthesis of compound 1

[0030] (1) Synthesis of compound Ref-1

[0031]

[0032] To a solution of 2,4-dimethyl-lH-pyrrole (924 mg, 9.71 mmol) in dry CH2Cl2(30 mL) at 0 °C under nitrogen, a solution of oxalyl chloride (573 mg) in dry CH2Cl2(5 mL) was added dropwise; stirred at room temperature for 2 h, then added successively trimethylamine (Et3N, 3.64 g, 39 mmol) and boron trifluoride diethyl etherate (BF3·OEt2, 5 mL, 60 mmol) under ice-bath conditions, stirred at room temperature for 2 h, rotary evaporation, column chromatography PE / CH2Cl2(4 / 1, v / v) to give Ref-1 as a brown powder (890 mg, yield: 30.7%).

[0033] (2) Synthesis of compound Ref-2

[0034]

[0035] Compound Ref-1 (0.15 g, 0.5 mmol) was dissolved in anhydrous EtOAc (10 mL), then lithium iodide (0.35 g, 2.0 mmol) was added, and stirred at reflux overnight. After cooling to room temperature, EtOAc (50 mL) was added. The mixture was washed with 0.1 N HC1 (10 mL), dried, and rotary evaporated. Ref-2 was obtained as a brown powder (1.11 g, yield: 75.0%) using EtOAc as eluent.

[0036] (3) Synthesis of compound 1

[0037]

[0038] Ref-2 (60 mg, 0.21 mmol) was dissolved in dry dichloromethane (10 mL), 1 drop of DMF was added, and oxalyl chloride (80 mg, 0.63 mmol) was added dropwise slowly. The reaction was allowed to proceed for 30 min at room temperature, and then was evaporated to dryness. The residue was redissolved in 5 mL of dry tetrahydrofuran and used directly in the next step. The above oxalyl chloride solution was added dropwise to an ammonia solution (1 mL) in dry tetrahydrofuran under ice-bath conditions. The reaction was completed in a few minutes, and the mixture was washed with water, extracted with dichloromethane, dried over anhydrous Na2S04, and evaporated to dryness. The residue was chromatographed on silica gel, and column chromatography (hexane / DCM = 4:1) gave 1 as an orange-red solid (40 mg, 68.9%). 1 H NMR (400 MHz, Chloroform-d) δ 6.08 (s, 4H), 2.53 (s, 6H), 2.32 (s, 6H) ppm; 13 C NMR (151 MHz, Chloroform-d) δ 166.12, 157.46, 141.79, 131.90, 128.49, 121.37, 14.75, 13.03 ppm; MS (ESI): Calcd for C 14 H 16 BF2N3O [M+H] + 292.1, found 292.0; HRMS (ESI): Calcd for [M+H] + 292.1427, found 292.1427.

[0039] Example 2: Synthesis of compound 2

[0040]

[0041] Compound Ref-2 (60 mg, 0.21 mmol) was dissolved in dry dichloromethane (10 mL), 1 drop of DMF was added, and oxalyl chloride (80 mg, 0.63 mmol) was added dropwise slowly. The reaction was allowed to proceed for 30 min at room temperature, and then was evaporated to dryness. The residue was redissolved in 5 mL of dry tetrahydrofuran and used directly in the next step. The above oxalyl chloride solution was added dropwise to an ammonia solution (1 mL) in dry tetrahydrofuran under ice-bath conditions. The reaction was completed in a few minutes, and the mixture was washed with water, extracted with dichloromethane, dried over anhydrous Na2S04, and evaporated to dryness. The residue was chromatographed on silica gel, and column chromatography (hexane / DCM = 4:1) gave 1 as an orange-red solid (40 mg, 68.9%). 1H NMR (400 MHz, Chloroform-d) δ 6.06 (s, 2H), 3.50 (td, J = 7.2, 5.4 Hz, 2H), 2.52 (s, 6H), 2.22 (s, 6H), 1.29 - 1.24 (m, 3H); 13 C NMR (151 MHz, Chloroform-d) δ 164.97, 157.32, 141.67, 132.73, 129.02, 121.20, 26.73, 14.74, 12.81; MS (ESI): Calculated for C 15 H 18 BF2N3O [M+H] + 306.2, found 305.9; HRMS (ESI): Calculated for [M+H] + 306.1584, found 306.1586.

[0042] Example 3: Synthesis of compound 3

[0043]

[0044] The synthesis method of compound 3 is the same as the synthesis route of compound 2, and the equivalents are as follows: compound Ref-2 (60 mg, 0.21 mmol), drop 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), 70% aqueous solution of ethylamine 0.2 mL, triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 3:1) to obtain orange red solid 3 (40 mg, 62.5%). 1 H NMR (400 MHz, Chloroform-d) δ 6.06 (s, 2H), 3.50 (td, J = 7.2, 5.4 Hz, 2H), 2.52 (s, 6H), 2.22 (s, 6H), 1.29 - 1.24 (m, 3H); 13 C NMR (151 MHz, Chloroform-d) δ 164.20, 157.29, 141.71, 133.00, 129.01, 121.17, 35.07, 14.74, 14.12, 13.02; MS (ESI): Calculated for C 16 H 20 BF2N3O [M+H] + 320.2, found 319.9; HRMS (ESI): Calculated for [M+H] + 320.1740, found 320.1743.

[0045] Example 4: Synthesis of compound 4

[0046]

[0047] The synthesis of compound 4 was carried out as described for compound 2, with the following equivalents: compound Ref-2 (60 mg, 0.21 mmol), dropwise addition of 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), n-butylamine (45 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 1 :2) to give orange red solid 4 (25 mg, 35.7%). 1 H NMR (400 MHz, Chloroform-d) δ 6.06 (s, 3H), 3.44 (q, J = 6.8 Hz, 2H), 2.52 (s, 6H), 2.22 (s, 6H), 1.63 (d, J = 8.1 Hz, 1H), 1.41 (h, J = 7.4 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H); 13 C NMR (151 MHz, Chloroform-d) δ 164.29, 157.30, 141.73, 133.11, 129.06, 121.18, 40.11, 30.80, 20.26, 14.73, 13.63, 12.99; MS (ESI): Calcd for C 18 H 24 BF2N3O [M+H] + 348.2, found 347.9; HRMS (ESI): Calcd for [M+H] + 348.2053, found 348.2057.

[0048] Example 5: Synthesis of compound 5

[0049]

[0050] The synthesis of compound 5 was carried out as described for compound 2, with the following equivalents: compound Ref-2 (60 mg, 0.21 mmol), dropwise addition of 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), tert-butylamine (45 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 1 :2) to give orange red solid 5 (40 mg, 57.1%). 1H NMR (400 MHz, Chloroform-d) δ 6.05 (s, 2H), 5.80 (s, 1H), 2.52 (s, 6H), 2.32 (s, 6H), 1.49 - 1.44 (m, 9H); 13 CNMR (151 MHz, Chloroform-d) δ 163.34, 157.20, 141.99, 134.10, 129.23, 121.13, 53.07, 28.84, 14.75, 13.93; MS (ESI): Calculated for C 18 H 24 BF2N3O [M+H] + 348.2, found 347.9; HRMS (ESI): Calculated for [M+H] + 348.2053, found 348.2053.

[0051] Example 6: Synthesis of compound 6

[0052]

[0053] The synthesis method of compound 6 is the same as the synthesis route of compound 2, and the equivalents are as follows: compound Ref-2 (60 mg, 0.21 mmol), drop 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), n-hexylamine (63 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 1:2) to obtain orange red solid 6 (30 mg, 42.9%). 1 H NMR (400 MHz, Chloroform-d) δ 6.05 (s, 2H), 5.80 (s, 1H), 2.52 (s, 6H), 2.32 (s, 6H), 1.49 - 1.44 (m, 9H); 13 CNMR (151 MHz, Chloroform-d) δ 163.34, 157.20, 141.99, 134.10, 129.23, 121.13, 53.07, 28.84, 14.75, 13.93; MS (ESI): Calculated for C 20 H 28 BF2N3O [M+H] +376.2, found 376.0; HRMS (ESI): Calculated for [M+H] + 376.2366, found 376.2369..

[0054] Example 7: Synthesis of compound 8

[0055]

[0056] The synthesis method of compound 7 is the same as the synthesis route of compound 2, and the equivalents are as follows: compound Ref-2 (60 mg, 0.21 mmol), drop 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), allylamine hydrochloride (59 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 1:2) to obtain orange-red solid 7 (28 mg, 42.4%). 1 H NMR (400 MHz, Chloroform-d) δ 6.09 (d, J = 19.2 Hz, 3H), 5.99 - 5.82 (m, 1H), 5.39 - 5.18 (m, 2H), 4.15 - 4.03 (m, 2H), 2.53 (s, 6H), 2.22 (s, 6H); 13 C NMR (151 MHz, Chloroform-d) δ 164.05, 157.42, 141.71, 132.65, 132.13, 128.98, 121.25, 118.91, 42.63, 14.75, 13.08; MS (ESI): Calculated for C 17 H 20 BF2N3O [M+H] + 332.2, found 331.8; HRMS (ESI): Calculated for [M+H] + 332.1740, found 332.1743..

[0057] Example 8: Synthesis of compound 8

[0058]

[0059] Synthesis of compound 8 following the synthetic route of compound 2, with the following equivalents: compound Ref-2 (60 mg, 0.21 mmol), drop 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), propargylamine (35 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 1 :2) to give orange red solid 8 (37 mg, 56.0%). 1 H NMR (400 MHz, Chloroform-d) δ 6.29 (s, 1H), 6.07 (s, 2H), 4.26 (dd, J = 5.4, 2.6 Hz, 2H), 2.53 (s, 6H), 2.32 (t, J = 2.6 Hz, 1H), 2.22 (s, 6H); 13 C NMR (151 MHz, Chloroform-d) δ 163.88, 157.64, 141.82, 131.80, 128.93, 121.33, 77.57, 73.02, 29.53, 14.76, 13.05; MS (ESI): Calcd for C 17 H 18 BF2N3O [M+H] + 330.2, found 329.8; HRMS (ESI): Calcd for [M+H] + 330.1584, found 330.1587.

[0060] Example 9: Synthesis of compound 9

[0061]

[0062] Synthesis of compound 9 following the synthetic route of compound 2, with the following equivalents: compound Ref-2 (60 mg, 0.21 mmol), drop 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), aniline (59 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 1 :2) to give orange red solid 9 (16 mg, 21.9%). 1 H NMR (400 MHz, Chloroform-d) δ 6.29 (s, 1H), 6.07 (s, 2H), 4.26 (dd, J = 5.4, 2.6 Hz, 2H), 2.53 (s, 6H), 2.32 (t, J = 2.6 Hz, 1H), 2.22 (s, 6H); 13C NMR (151 MHz, Chloroform-d) δ 163.19, 157.30, 142.22, 134.19, 129.33, 121.26, 54.09, 41.94, 36.31, 29.53, 14.96, 14.25; MS (ESI): Calcd for C 20 H 20 BF2N3O [M+H] + 368.2, found 368.0; HRMS (ESI): Calcd for [M+H] + 368.1740, found 368.1744.

[0063] Example 10: Synthesis of compound 10

[0064]

[0065] The synthesis of compound 10 was carried out as for compound 2, with the following equivalents: compound Ref-2 (60 mg, 0.21 mmol), 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), adamantylamine (95 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 3: 1) to give 10 (53 mg, 62.4%) as an orange red solid. 1 H NMR (400 MHz, Chloroform-d) δ 6.06 (s, 2H), 5.69 (s, 1H), 2.52 (s, 6H), 2.35 (s, 6H), 2.13 (s, 9H), 1.72 (d, J = 2.8 Hz, 6H); 13 C NMR (151 MHz, Chloroform-d) δ 163.19, 157.30, 142.22, 134.19, 129.33, 121.26, 54.09, 41.94, 36.31, 29.53, 14.96, 14.25; MS (ESI): Calcd for C 24 H 30 BF2N3O [M+H] + 426.2, found 425.9; HRMS (ESI): Calcd for [M+H] + 448.2342, found 448.2343.

[0066] Example 11: Synthesis of compound 11

[0067]

[0068] Compound Ref-2 (60 mg, 0.21 mmol) was dissolved in dry DCM (15 mL), 1 drop of DMF was added, oxalyl chloride (80 mg, 0.63 mmol) was added dropwise slowly, stirred at room temperature for 15 min, dried by rotary evaporation, redissolved in 5 mL of dry DCM, added dropwise to a solution of N-aminopropyl morpholine (90 mg, 0.63 mmol) and triethylamine (63 mg, 0.63 mmol) in dry DCM (5 mL) under ice bath condition, stirred at room temperature for 5 min. Washed with water, extracted with DCM, washed with saturated NH4Cl, washed with saturated NaCl, dried over anhydrous Na2SO4, dried by rotary evaporation, column chromatography (DCM / CH3OH = 50:1). Product: orange red solid 11 (Lyso-01, 15 mg, 17.5%). 1 H NMR (400 MHz, Chloroform-d) δ 8.13 (s, 1H), 6.07 (d, J = 8.6 Hz, 2H), 3.53 (d, J = 27.0 Hz, 8H), 2.55 (s, 6H), 2.43 (s, 4H), 2.24 (d, J = 8.0 Hz, 6H), 1.81 (s, 2H) ppm; 13 C NMR (151 MHz, Chloroform-d) δ 164.06, 157.21, 141.43, 133.52, 128.98, 121.05, 66.62, 57.64, 53.53, 40.22, 23.56, 14.76, 13.08 ppm; MS (ESI): Calcd for C 21 H 29 BF2N4O2[M+H] + 419.2, found 419.0; HRMS (ESI): Calcd for [M+Na] + 441.2244, found 441.2248.

[0069] Example 12: Synthesis of compound 12

[0070]

[0071] Compound Ref-2 (60 mg, 0.21 mmol) was dissolved in 15 mL of dry dichloromethane, 1 drop of DMF was added, oxalyl chloride (80 mg, 0.63 mmol) was added dropwise, stirred at room temperature for 15 minutes, rotary evaporation, 5 mL of dry dichloromethane was added, and the solution was added dropwise to a solution of N,N-dimethylaminoethylamine (55 mg, 0.63 mmol) and triethylamine (63 mg, 0.63 mmol) in dichloromethane (5 mL) under ice bath conditions, stirred at room temperature for 5 minutes, and the reaction was completed. Washed with water, extracted with DCM, washed with saturated NH4Cl, washed with saturated NaCl, dried with anhydrous Na2SO4, rotary evaporation, and column chromatography (DCM / CH3OH = 50:1) to give the product: orange solid 12 (Lyso-01, 22 mg, 30%). 1 H NMR (400 MHz, Chloroform-d) δ 6.77 (s, 1H), 6.07 (d, J = 10.2 Hz, 2H), 3.50 (d, J = 6.4 Hz, 3H), 2.53 (s, 8H), 2.22 (d, J = 3.0 Hz, 12H) ppm; 13 C NMR (151 MHz, Chloroform-d) δ 164.18, 157.15, 141.66, 133.12, 129.06, 121.11, 56.54, 44.87, 37.13, 14.73, 12.91 ppm; MS (ESI): Calcd for C 18 H 25 BF2N4O [M+H]+363.2, found 363.0; HRMS (ESI): Calcd for C 18 H 25 BF2N4O [M+Na]+385.1982, found 385.1979.

[0072] Example 13: Synthesis of compound 13

[0073]

[0074] The synthesis method of compound 3c is the same as that of compound 3b, and the equivalents are as follows: compound Ref-2 (60 mg, 0.21 mmol), 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), 0.2 mL of dimethylamine in methanol (2 mol / L), triethylamine (63 mg, 0.63 mmol), and DCM column chromatography to give orange solid 13 (30 mg, 48.4%). 1H NMR (400 MHz, Chloroform-d) δ 6.08 (s, 2H), 3.13 (s, 3H), 3.03 (s, 3H), 2.55 (s, 6H), 2.13 (s, 6H); 13 C NMR (151 MHz, Chloroform-d) δ 164.79, 157.03, 141.15, 132.84, 128.39, 121.10, 37.96, 34.43, 14.72, 12.58; MS (ESI): Calculated for C 16 H 20 BF2N3O [M+H] + 320.2, found 319.9; HRMS (ESI): Calculated for [M+H] + 320.1740, found 320.1743.

[0075] Example 14: Synthesis of compound 14

[0076]

[0077] Compound Ref-2 (60 mg, 0.21 mmol) was dissolved in 15 mL of dry dichloromethane, 1 drop of DMF was added, and excess oxalyl chloride (80 mg, 0.63 mmol) was added dropwise. After stirring at room temperature for 15 minutes, it was rotary evaporated, redissolved in 5 mL of dry dichloromethane, and added dropwise to a solution of N-methyl (N,N-dimethylaminoethyl)amine (64 mg, 0.63 mmol) and triethylamine (63 mg, 0.63 mmol) in dichloromethane (5 mL) under ice bath condition. After stirring at room temperature for 5 minutes, the reaction was completed. It was washed with water, extracted with DCM, washed with saturated NH4Cl, washed with saturated NaCl, dried over anhydrous Na2SO4, rotary evaporated, and column chromatographed (DCM / CH3OH = 50:1). Product: orange red solid 14 (Lyso-03, 21 mg, 26.6%). 1 H NMR (400 MHz, Chloroform-d) δ 6.05 (s, 2H), 3.64 (t, J = 6.3 Hz, 2H), 3.05 (s, 3H), 2.64 (s, 2H), 2.53 (s, 6H), 2.31 (s, 6H), 2.17 (s, 6H); 13 C NMR (151 MHz, Chloroform-d) δ 164.91, 156.98, 141.29, 133.13, 128.37, 121.04, 55.99, 45.31, 44.89, 36.96, 14.73, 12.83; MS (ESI): Calculated for C19 H 27 BF2N4O[M+H] + 377.2, found 377.2; HRMS (ESI): Calculated for C 19 H 27 BF2N4O[M+H] + 377.2319, found 377.2315.

[0078] Example 15: Synthesis of compound 15

[0079]

[0080] The synthesis method of compound 15 was the same as the synthesis route of compound 2, and the equivalent was as follows: compound Ref-2 (60 mg, 0.21 mmol), drop 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), diethylamine (46 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 3:1) to obtain orange red solid 15 (24 mg, 34.8%). 1 H NMR (400 MHz, Chloroform-d) δ 6.07 (s, 2H), 3.60 (q, J = 7.2 Hz, 2H), 3.40 (q, J = 7.2 Hz, 2H), 2.54 (s, 6H), 2.18 (s, 6H), 1.30 (d, J = 7.3 Hz, 3H), 1.10 (t, J = 7.2 Hz, 3H); 13 C NMR (151 MHz, Chloroform-d) δ 164.32, 156.87, 141.31, 133.37, 128.63, 121.12, 43.07, 39.26, 14.73, 13.20, 12.85, 12.03; MS (ESI): Calculated for C 18 H 24 BF2N3O[M+H] + 348.2, found 347.8; HRMS (ESI): Calculated for [M+H] + 348.2053, found 348.2054.

[0081] Example 16: Synthesis of compound 16

[0082]

[0083] The synthesis of compound 16 was carried out according to the synthetic route of compound 2 with the following equivalents: compound Ref-2 (60 mg, 0.21 mmol), dropwise addition of 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), azetidine (36 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 3:1) to give 16 (20 mg, 30.3%) as an orange red solid. 1 H NMR (400 MHz, Chloroform-d) δ 6.05 (s, 2H), 3.63 (t, J = 6.7 Hz, 2H), 3.35 (t, J = 6.4 Hz, 2H), 2.53 (s, 6H), 2.16 - 2.13 (m, 6H), 2.02 - 1.95 (m, 4H); 13 C NMR (151 MHz, Chloroform-d) δ 164.00, 157.23, 141.20, 130.66, 128.55, 121.14, 50.84, 48.44, 15.79, 14.74, 12.80; MS (ESI): Calcd for C 17 H 20 BF2N3O [M+H] + 332.2, found 331.9; HRMS (ESI): Calcd for [M+H] + 332.1740, found 332.1744.

[0084] Example 17: Synthesis of compound 17

[0085]

[0086] The synthesis of compound 16 was carried out according to the synthetic route of compound 2 with the following equivalents: compound Ref-2 (60 mg, 0.21 mmol), dropwise addition of 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), azetidine (36 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 3:1) to give 16 (20 mg, 30.3%) as an orange red solid. 1 H NMR (400 MHz, Chloroform-d) δ 6.05 (s, 2H), 3.63 (t, J = 6.7 Hz, 2H), 3.35 (t, J = 6.4 Hz, 2H), 2.53 (s, 6H), 2.16 - 2.13 (m, 6H), 2.02 - 1.95 (m, 4H); 13C NMR (151 MHz, Chloroform-d) δ 163.19, 156.84, 141.26, 133.37, 128.46, 121.15, 47.63, 41.90, 25.52, 24.60, 24.26, 14.71, 13.06; MS (ESI): Calculated for C 18 H 22 BF2N3O [M+H] + 360.2, found 359.9; HRMS (ESI): Calculated for [M+H] + 360.2053, found 360.2059.

[0087] Example 18: Synthesis of compound 18

[0088]

[0089] The synthesis method of compound 18 is the same as the synthesis route of compound 2, and the equivalents are as follows: compound Ref-2 (60 mg, 0.21 mmol), drop 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), hexahydropyridine (54 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 4:1) to obtain orange red solid 18 (45 mg, 69.2%). 1 H NMR (600 MHz, Chloroform-d) δ 6.06 (s, 2H), 3.71 (d, J = 5.5 Hz, 2H), 3.39 - 3.31 (m, 2H), 2.53 (s, 6H), 2.19 (s, 6H), 1.69 (p, J = 2.8 Hz, 4H), 1.53 (d, J = 7.2 Hz, 2H); 13 C NMR (151 MHz, Chloroform-d) δ 163.19, 156.84, 141.26, 133.37, 128.46, 121.15, 47.63, 41.90, 25.52, 24.60, 24.26, 14.71, 13.06; MS (ESI): Calculated for C 19 H 24 BF2N3O [M+H] + 360.2, found 359.9; HRMS (ESI): Calculated for [M+H] + 360.2053, found 360.2059.

[0090] Example 19: Synthesis of compound 19

[0091]

[0092] The synthesis of compound 19 was carried out according to the synthetic route of compound 2, with the following equivalents: compound Ref-2 (60 mg, 0.21 mmol), dropwise addition of 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), morpholine (55 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 1 :2) to give 19 as an orange red solid (51 mg, 80.9%). 1 H NMR (400 MHz, Chloroform-d) δ 6.08 (s, 2H), 3.79 (hept, J = 3.6, 3.2 Hz, 4H), 3.66 - 3.60 (m, 2H), 3.42 (dq, J = 4.7, 1.6 Hz, 2H), 2.54 (s, 6H), 2.20 (s, 6H); 13 C NMR (151 MHz, Chloroform-d) δ 163.93, 157.60, 141.30, 132.19, 128.60, 121.62, 66.27, 66.11, 47.01, 41.59, 14.94, 13.28; MS (ESI): Calcd for C 18 H 22 BF2N3O [M+H] + 362.2, found 361.8; HRMS (ESI): Calcd for [M+H] + 362.1846, found 362.1850.

[0093] Example 20: Synthesis of compound 20

[0094]

[0095] The synthesis of compound 19 was carried out according to the synthetic route of compound 2, with the following equivalents: compound Ref-2 (60 mg, 0.21 mmol), dropwise addition of 1 drop of DMF, oxalyl chloride (80 mg, 0.63 mmol), morpholine (55 mg, 0.63 mmol), triethylamine (63 mg, 0.63 mmol), column chromatography (hexane / DCM = 1 :2) to give 19 as an orange red solid (51 mg, 80.9%). 1H NMR (400 MHz, Chloroform-d) δ 6.07 (s, 2H), 3.76 (t, J = 5.1 Hz, 2H), 3.56 (t, J = 5.2 Hz, 2H), 3.40 (s, 4H), 2.53 (s, 6H), 2.17 (s, 6H), 1.46 (s, 9H) ppm; 13 C NMR (151 MHz, Chloroform-d) δ 163.72, 157.41, 154.30, 141.03, 132.06, 128.36, 121.43, 80.73, 46.38, 41.00, 28.32, 14.75, 13.11; MS (ESI): Calcd for C 23 H 31 BF2N4O3[M+H] + 461.2, found 461.2; HRMS (ESI): Calcd for [M+Na] + 483.2349, found 483.2347.

[0096] Example 21: Synthesis of compound 21 (ER-1)

[0097]

[0098] (1) Synthesis of N-3-bromopropylamino-2,3,4,5,6-pentafluorobenzamide (A)

[0099]

[0100] Compound 3-bromopropylamine hydrobromide (876 mg, 4.0 mmol) and DIPEA (4.0 mmol) were dissolved in DCM (10 mL) solution, slowly added pentafluorobenzoyl chloride (916 mg, 4.0 mmol) dropwise under ice bath condition, and stirred for 30 min. After reaction, washed with water (20 mL), 1 N HC1 (20 mL), extracted with DCM, washed with NaCl, dried with Na2S04, rotary evaporated, and the residue was subjected to silica gel column chromatography (hexane / DCM = 1:4) to obtain compound A, yield: 93.9%; 1 H NMR (600 MHz, Chloroform-d) δ 3.65 (q, J = 6.4 Hz, 2H), 3.52 - 3.45 (m, 2H), 2.27 - 2.16 (m, 2H) ppm; 13C NMR (151 MHz, Chloroform-d) δ 157.60, 144.96, 143.25, 141.44, 138.41, 136.91, 111.16, 38.93, 31.48, 30.39 ppm.

[0101] (2) Synthesis of N-3-(4-Boc-l-piperazinyl)propylamino-2,3,4,5,6-pentafluorobenzamide (B)

[0102]

[0103] Compound A (600 mg, 1.82 mmol) was dissolved in acetonitrile (10 mL) solution, K2CO3 and N-Boc-piperazine (406 mg, 2.18 mmol) were added, stirred at room temperature for 12 h. After the reaction was completed, filtered, concentrated, extracted with DCM, washed with water, dried, rotary evaporated, and column chromatography on silica gel (DCM / CH3OH = 20: 1) to obtain intermediate B, yield: 69.0%, directly to the next step.

[0104] (3) Synthesis of 3-(l-piperazinyl)propyl-2,3,4,5,6-pentafluorobenzamide trifluoroacetate (C)

[0105]

[0106] Compound 6 (177 mg, 0.40 mmol) was dissolved in TFA / DCM (1 / 5, v / v) (10 mL) solution, after stirring for 1 h, LC-MS showed [M+H] + : 337.9, rotary evaporated, directly used in the next step.

[0107] (4) Synthesis of compound 21 (ER-1)

[0108]

[0109] Compound 21 (ER-1) was prepared from N-piperazine propyl pentafluorobenzamide trifluoroacetate (C). ER-1 was prepared in the same way as compound 2, column chromatography (DCM / CH3OH = 30: 1) to obtain orange red solid ER-1, yield: 20.0%; 1 H NMR (400 MHz, Chloroform-d) δ 6.07 (s, 2H), 3.64 (d, J = 51.9 Hz, 6H), 3.35 (s, 2H), 2.55 (d, J = 15.4 Hz, 10H), 2.12 (s, 6H), 1.80 (s, 2H) ppm; 13C NMR (151 MHz, Chloroform-d) δ 163.47, 157.34, 156.99, 145.00, 143.20, 142.93, 141.02, 138.47, 136.89, 132.14, 128.36, 121.37, 111.75, 56.89, 52.60, 51.96, 46.44, 41.07, 39.96, 24.76, 14.74, 12.98 ppm. MS (ESI): Calcd for [M+H] + 612.2, found 612.2; HRMS (ESI): Calcd for [M+Na] + 634.2195, found 634.2188.

[0110] Example 22: Synthesis of compound 22

[0111]

[0112] (1) Synthesis of compound (D)

[0113]

[0114] Compound D was prepared by Vilsmeier Haack reaction. Phosphorus oxychloride (408 mg, 2.66 mmol) was added dropwise into DMF (237 mg, 3.24 mmol) under ice bath condition, stirred at room temperature for 30 min, after dissolved in DCE, slowly added into compound 13 (85 mg, 0.27 mmol) in DCE (10 mL), stirred at 60 °C for 1 h. After the reaction was completed, cooled to room temperature, added saturated NaOAc until no more bubbles, the reaction mixture was extracted with EtOAc, washed with saturated NaCl, dried over anhydrous Na2SO4, rotary evaporated. Column chromatography (DCM / CH3OH = 50:1), product: 50 mg (red solid), yield: 53.4%. 1 H NMR (600 MHz, Chloroform-d) δ 10.13 - 9.97 (m, 1H), 6.24 (s, 1H), 3.14 (d, J = 2.5 Hz, 3H), 3.03 - 2.97 (m, 3H), 2.79 (s, 3H), 2.60 (s, 3H), 2.41 (s, 3H), 2.18 (s, 3H); 13C NMR (151 MHz, Chloroform-d) δ 185.72, 163.95, 163.54, 157.53, 145.27, 140.73, 134.60, 131.43, 126.53, 126.14, 124.09, 37.92, 34.53, 15.29, 12.99, 10.08. MS (ESI): Calculated for [M+H] + 348.2, found 348.2; HRMS (ESI): Calculated for [M+H] + 348.1689, found 348.1688.

[0115] (2) Synthesis of compound 22

[0116]

[0117] Compound D (40 mg, 0.11 mmol) was dissolved in 5 ml of absolute ethanol, NH2OH HCI (11 mg, 0.16 mmol) was added, refluxed for 30 min, rotary evaporated, column chromatography, DCM / CH3OH (100:1) to obtain 22 (25 mg, yield: 65.9%); 1 HNMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 10.6 Hz, 1H), 6.16 (s, 1H), 3.15 (d, J = 10.7 Hz, 3H), 3.03 (d, J = 10.4 Hz, 3H), 2.68 (d, J = 10.6 Hz, 3H), 2.62 - 2.52 (m, 3H), 2.28 - 2.19 (m, 3H), 2.16 (d, J = 10.5 Hz, 3H) ppm; 13 C NMR (151 MHz, Chloroform-d) δ 164.54, 159.66, 155.54, 144.48, 142.91, 137.85, 133.42, 129.57, 127.38, 122.38, 121.60, 37.97, 34.49, 14.94, 13.98, 12.80, 10.78 ppm. MS (ESI): Calculated for [M+H] + 363.2, found 363.2; HRMS (ESI): Calculated for [M+H] + 363.1798, found 363.1796.

[0118] Example 21: Test of optical parameters of compounds

[0119] The synthesized compounds were tested for maximum absorption wavelength (λ abs ), molar extinction coefficient (ε), fluorescence emission wavelength (λ em ), fluorescence half-peak width (Fwhm), Stocks shift and fluorescence quantum yield (Φ f ) in water, PBS, ethanol, acetonitrile, tetrahydrofuran, chloroform, and the corresponding data are shown in Table 1.

[0120] Table 1 Spectral properties of BODIPY dyes

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] [a]1×10 4 M –1 cm –1 , [b] Φ f , the determination of fluorescence quantum yield used 1,3,5,7,8-pentamethyl-BODIPY as reference (Φ F = 0.82, MeCN).

[0127] After introducing amide groups at the meso position of BODIPY, the maximum absorption wavelength (λ abs ), molar extinction coefficient (ε), fluorescence emission wavelength (λ em ), half-peak width, Stocks shift of the resulting compounds did not change much, but the fluorescence quantum yield (Φ f ) had a large difference; among which the amide mono-substituted compounds 1-12 (except 6 and 10), the fluorescence quantum yield (Φ f ) in polar solvents of deionized water, PBS, ethanol was significantly higher than that in acetonitrile, tetrahydrofuran, chloroform, and the substituent side chain of compounds 6 and 10 was too lipophilic to aggregate in water, resulting in fluorescence quenching; while the amide di-substituted compounds 13-20 (except 16) maintained a high fluorescence quantum yield (Φ f ) in the tested solvents of deionized water, PBS, ethanol, acetonitrile, tetrahydrofuran, chloroform, and did not quench fluorescence in polar environment, so this class of compounds has excellent fluorescence quantum yield (Φ f), which has anti-quenching ability in water, making up the water quenching defect of traditional liposoluble fluorescent dyes.

[0128] Example 22: Cell imaging test

[0129] (1) Cell culture: HeLa cells (human cervical cancer cells) were selected for the experiment, and the culture medium was DMEM (HyClone), which contained 10% fetal bovine serum (FBS) and 1% penicillin (10,000 units / mL)-streptomycin (10,000 μg / mL) (HyClone). After the cells were resuscitated, they were cultured in a 37°C incubator containing 5% CO2.

[0130] (2) Cell imaging: HeLa cells were seeded into a confocal imaging culture dish and incubated at 37°C for 24 h with DMEM medium containing 10% FBS. Dye 11 (Lyso-01), 12 (Lyso-02), and 14 (Lyso-03) were dissolved in DMSO to 1 mM, and 10 μL was diluted to 10 μM with medium. The culture dish was removed, the medium was removed, and the cells were washed three times with PBS; the medium containing the dye (10 μM) was added and incubated with the HeLa cells for 30 min; the cells were washed three times with PBS, and confocal microscopy was used for imaging, and the results are shown in Figure 1

[0131] As shown in Figure 1 , the green fluorescence images of compounds Ref-2, 1, 2, and 13 (A, B, C, and D) show that compounds 1, 2, and 13 have improved membrane permeability compared with compound Ref-2 and can be applied to cell imaging, and the imaging of compound 13 is clearer.

[0132] (3) Lysosome targeting imaging: In order to study the lysosome organelle positioning ability, lysosome colocalization experiments were carried out. The laser confocal live cell workstation was used, and HeLa cells were incubated with compound 11 (Lyso-01), 12 (Lyso-02), and 14 (Lyso-03) and commercially available organelle dyes, and the positioning effect was judged according to the positioning coincidence of the two. HeLa cells were seeded into a confocal imaging culture dish and incubated at 37°C for 24 h with DMEM medium containing 10% fetal bovine serum. The culture dish was removed, the medium was removed, and the cells were washed three times with PBS; the medium containing Lyso-tracker Red (50 nM) was added and incubated with the HeLa cells for 30 min; the cells were washed with PBS; medium containing compound 11 (Lyso-01), compound 12 (Lyso-02), and compound 14 (Lyso-03) (10 μM) was added and incubated with the HeLa cells for 30 min; the cells were washed three times with PBS; 1 mL of D++ was added, and confocal microscopy was used for imaging, and the results are shown in Figure 2 ​Shown,

[0133] As Figure 2 shown, (A1, B1, C1) green fluorescence images of compounds Lyso-01~Lyso-03; (A2, B2, C2) red fluorescence images after incubation with Lyso-tracker Red (50 nM); (A3, B3, C3) overlay images of green fluorescence of BODIPY compounds and red fluorescence of Lyso-tracker Red, scale bar: 10 μm. (A4, B4, C4) overlay graphs of corresponding fluorescence intensity in figures A, B, C,

[0134] From the shown localization effect, and the corresponding fluorescence colocalization curve shows that: compounds L11 (Lyso-01), 12 (Lyso-02), 14 (Lyso-03) and Lyso-tracker Red show good coincidence, and the Pearson correlation coefficient is 0.97, 0.95, 0.96, respectively.

[0135] (4) Endoplasmic reticulum targeting imaging: In order to study the endoplasmic reticulum organelle localization ability, endoplasmic reticulum colocalization experiment was carried out. The HeLa cells were incubated with compound 21 (ER-1) and commercially available organelle dye, respectively, and the localization effect was judged according to the localization coincidence of the two. The HeLa cells were seeded into a confocal imaging culture dish and incubated at 37℃ with DMEM medium containing 10% fetal bovine serum for 24h. The culture dish was taken out, the culture medium was removed, and the cells were washed with PBS three times; add dye (10 μM) medium to incubate HeLa cells for 30 minutes; add medium containing ER-tracker Red (1 μM) to incubate HeLa cells for 30 minutes; wash with PBS three times; add 1 mL of D++ and use confocal microscope to image, and the results are shown in Figure 4

[0136] As Figure 4 shown, (A) green fluorescence image of compound 21 (ER-1); (B) red fluorescence image after incubation with ER-tracker Red (1 μM); (C) overlay image of green fluorescence of compound ER-1 and red fluorescence of ER-tracker Red, scale bar: 10 μm. (D) overlay graph of corresponding fluorescence intensity in figures A, B,

[0137] From the shown localization effect, and the corresponding fluorescence colocalization curve shows that: compound 21 (ER-1) and ER-tracker Red show good coincidence, and the Pearson correlation coefficient is 0.90.

[0138] (5) Fluorescence imaging of compound 22 for detecting exogenous and endogenous hypochlorous acid: as​Figure 5 Figure 22 shows confocal fluorescence images of compound 22 in HeLa cells and Raw 264.7 cells. (A) HeLa cells were incubated with compound 22 (10 μΜ) for 30 min; (B) HeLa cells were pre-incubated with compound 22 (10 μΜ) for 30 min, and then treated with CIO4for 30 min (Ex = 514 nm); (C) Raw 264.7 cells were incubated with compound 22 (10 μΜ) for 30 min; (D) Raw 264.7 cells were stimulated with 100 μg / mL LPS for 24 h, and then stimulated with 1 μg / mL PMA for 1 h, and then incubated with compound 22 (10 μΜ) for 30 min at 37 °C; (E) MPO inhibitor 4-ABAH (100 μΜ) was co-incubated during PMA stimulation; other steps were the same. (Top: bright field, middle: fluorescence, bottom: merged, E = 514 nm), scale bar: 20 μm. – x Figure 23 shows confocal fluorescence images of compound 22 in HeLa cells and Raw 264.7 cells. (A) HeLa cells were incubated with compound 22 (10 μΜ) for 30 min; (B) HeLa cells were pre-incubated with compound 22 (10 μΜ) for 30 min, and then treated with CIO4for 30 min (Ex = 514 nm); (C) Raw 264.7 cells were incubated with compound 22 (10 μΜ) for 30 min; (D) Raw 264.7 cells were stimulated with 100 μg / mL LPS for 24 h, and then stimulated with 1 μg / mL PMA for 1 h, and then incubated with compound 22 (10 μΜ) for 30 min at 37 °C; (E) MPO inhibitor 4-ABAH (100 μΜ) was co-incubated during PMA stimulation; other steps were the same. (Top: bright field, middle: fluorescence, bottom: merged, E = 514 nm), scale bar: 20 μm. –

[0139] Figure 24 shows confocal fluorescence images of compound 22 in HeLa cells and Raw 264.7 cells. (A) HeLa cells were incubated with compound 22 (10 μΜ) for 30 min; (B) HeLa cells were pre-incubated with compound 22 (10 μΜ) for 30 min, and then treated with CIO4for 30 min (Ex = 514 nm); (C) Raw 264.7 cells were incubated with compound 22 (10 μΜ) for 30 min; (D) Raw 264.7 cells were stimulated with 100 μg / mL LPS for 24 h, and then stimulated with 1 μg / mL PMA for 1 h, and then incubated with compound 22 (10 μΜ) for 30 min at 37 °C; (E) MPO inhibitor 4-ABAH (100 μΜ) was co-incubated during PMA stimulation; other steps were the same. (Top: bright field, middle: fluorescence, bottom: merged, E = 514 nm), scale bar: 20 μm.

[0140] Figure 25 shows the results of dye cytotoxicity (CCK-8) of compound 22 in HeLa cells. Cells were incubated in 96-well plates at a concentration of 14 x 10 5 cells / 0.1 ml for 24 h, and then the cells were added with compound at a concentration of 0, 1.25, 2.5, 5, 10, 20 μΜ, with 3 replicates for each concentration, and incubated for 48 h. 100 μl of 10% CCK8 solution was added to each well to measure the ability of viable cells in each well. The cells were incubated for 30 min, and then removed and measured for OD value at 450 nm using a microplate reader. The results are shown in Figure 25. Figure 3 6

[0141] The results show that the survival rate of cells was still greater than 85% at a concentration of 20 μΜ for compounds 11, 12, 14 and 22, indicating that the dyes have small cytotoxicity and can be used in biological systems.​​​​

Claims

1. A kind meso 1,3,5,7-Tetramethyl-BODIPY compounds substituted with an amide, wherein the compounds are selected from the following structures: 。 2. The method of claim 1 meso Use of 1,3,5,7-tetramethyl-BODIPY compounds substituted with amides in the preparation of fluorescent imaging agents.

Images