Fluoroborondipyrromethene fluorescent probe, preparation method and application thereof

Abstract

The application relates to a kind of fluoroboronic cyanine fluorescent probes and preparation method and application thereof, the fluorescent probe includes hydrophilic group and fluorescent dye connected by amide bond, can be obtained by adjusting the hydrophobicity of fluorescent dye fluoroboronic cyanine fluorescent probe of amphiphilic molecule or hydrophilic molecule, so that the fluorescent probe can be high signal-to-noise ratio, high specificity to cell membrane or mitochondria is fluorescently imaged;The fluoroboronic cyanine fluorescent probe provided by the application enriches the type of existing fluorescent probe, and the synthesis process is simple, the reaction condition is mild, and has the prospect of industrial production.

Description

Technical Field

[0001] This invention relates to the field of fluorescent dyes, specifically to a fluorinated boron cyanine fluorescent probe, its preparation method, and its application. Background Technology

[0002] Fluorescence imaging, as a visualization technique, possesses significant advantages such as high sensitivity, non-destructive nature, real-time detection, and high spatiotemporal resolution, and has been widely applied in bioscience and biomedicine. Small molecule organic fluorophores can help visualize gene expression, post-transcriptional modifications, biomolecule concentrations, organelle transport, disease detection, drug development, and response evaluation. They can also guide tumor surgery through imaging. Therefore, when modern dye chemistry is combined with modern synthetic chemistry, fluorophores have been endowed with considerable functionality in bioimaging.

[0003] Traditional small-molecule fluorescent probes primarily utilize mature, commercially available "core" structures as fluorophores, including coumarin, naphthimide, boron fluoride complexed dipyrrolemethyl ether (BODIPY), fluorescein, rhodamine, and cyanine. These traditional fluorescent dyes have limitations in both chemical and optical properties. In recent years, several emerging fluorophores have been developed and are rapidly progressing.

[0004] However, the variety of fluorescent dyes is still insufficient at present, and the fluorescence imaging effect and specificity of commercial fluorescent dyes on cell membranes and mitochondria need to be further improved. Therefore, the development of functional fluorescent dyes that are easy to promote subsequent functional applications is still of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a fluoroboron cyanine fluorescent probe, which is synthesized based on the borondifluoro indolenine (BFI) electron acceptor. This not only enriches the types of existing fluorescent probes, but also allows for the specific labeling of cell membranes and / or mitochondria, which has important application value for the study of physiological phenomena and disease diagnosis.

[0006] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0007] A fluorescent probe containing fluorinated boron cyanine has the structural formula shown in Formula I:

[0008]

[0009] In formula I, R 1 Selected from hydrogen, halogen, C1-C4 alkyl, phenyl, carboxyl, sulfonic acid, methoxy, formate, and amide; X is selected from C0-C3 saturated alkyl, polyethylene glycol, tertiary amine, and quaternary ammonium salt; R 2Selected from hydrogen, alkyl, sulfonic acid, carboxyl, phosphate, ester, quaternary ammonium salt, triphenylphosphine; R 3 Selected from C1 to C 16 Chain alkyl groups, polyethylene glycol groups, C1-C 16 Alkyl carboxylic acids and their active esters, C1-C5 alkyl sulfonic acids and their salts, C1-C4 alcohols; y = 1-16, z = 1-8, m = 1-3.

[0010] In this technical solution, the fluoroboron cyanine fluorescent probe has the chemical structure shown in Formula I. The left-hand group, linked by an amide bond, is a hydrophilic group, and the right-hand group is a fluoroboron cyanine fluorescent dye, which is based on R... 3 The different functional groups can result in either hydrophilic or hydrophobic fluorescent probes. When the fluoroboron cyanine fluorescent dye is hydrophobic, the fluorescent probe is an amphoteric molecule, making it difficult to enter the hydrophobic environment inside the cell membrane, thus allowing the tetrazine membrane probe to attach to the cell membrane surface for fluorescence imaging. Conversely, when the fluoroboron cyanine fluorescent dye is hydrophilic, the fluorescent probe is a hydrophilic molecule, enabling it to enter the internal environment of the cell membrane for fluorescent labeling of mitochondria. Therefore, the fluorescent probe of Formula I possesses the ability to perform high signal-to-noise ratio and high specificity fluorescence imaging of the cell membrane and mitochondria.

[0011] In this technical solution, R 1 The group is selected from hydrogen, halogen, C1-C4 alkyl, phenyl, carboxyl, sulfonic acid, methoxy, formate, and amide. R 1 The functional group can be an electron-donating group or an electron-withdrawing group, used to adjust the photophysical properties of the fluorescent probe, such as the maximum absorption wavelength, maximum fluorescence wavelength, and Stokes shift. In one or more preferred embodiments, R 1 The group is selected from hydrogen, halogen, and C1-C4 alkyl groups. More preferably, R... 1 The radical is hydrogen, chlorine, bromine or iodine.

[0012] In this technical solution, R 2 The group is selected from hydrogen, alkyl, sulfonic acid, carboxyl, phosphate, ester, quaternary ammonium salt, triphenylphosphine, and X is selected from C0-C3 saturated alkyl, polyethylene glycol, tertiary amine, and quaternary ammonium salt. R 2 At least one of the groups, R and X, is a hydrophilic group, and both work together to enhance the hydrophilicity of the fluoroboron cyanine fluorescent probe. In some preferred embodiments, R 2 Both the R group and the X group are hydrophilic groups, for example, R 2 The group is a sulfonic acid group, a carboxyl group, a phosphate group, or a quaternary ammonium salt, and the X group is a tertiary amine or a quaternary ammonium salt. In one or more embodiments, R 2 The group or X group is a hydrophobic group, but the group to the left of the amide group is generally hydrophilic, for example, R. 2If the group is alkyl or ester, then group X is a tertiary amine or quaternary ammonium salt; or if group X is a C0-C3 saturated alkyl group, then R... 2 Groups include sulfonic acid group, carboxyl group, and phosphate group.

[0013] In this technical solution, R 3 The groups are selected from C1 to C2. 16 Chain alkyl groups, polyethylene glycol groups, C1-C 16 Alkyl carboxylic acids and their reactive esters, C1-C5 alkyl sulfonic acids and their salts, and C1-C4 alcohols. R 3 The group affects the hydrophilicity or hydrophobicity of the fluoroboron cyanine fluorescent dye to the right of the amide group. For example, when R 3 If the functional group is polyethylene glycol, the fluorescent probe is more likely to label mitochondria. 3 When the functional group is a chain alkyl group, the fluorescent probe has a better labeling ability on the cell membrane.

[0014] In this technical solution, the values ​​of y and z determine the length of the alkyl chain, which in turn affects the hydrophilicity and hydrophobicity of the fluorescent probe. Preferably, y = 1–16, z = 1–8. m is the degree of polymerization of the alkenyl group, preferably m = 1–3.

[0015] As a preferred embodiment of the present invention, R 1 Selected from hydrogen, halogens, C1-C4 alkyl groups, X selected from tertiary amines or quaternary ammonium salts, R 2 Selected from hydrogen, alkyl, sulfonic acid, carboxyl, phosphate, ester, and quaternary ammonium salts; R 3 Selected from C1 to C 16 Alkyl chain, C1 to C6 alcohols.

[0016] In this technical solution, X is selected from tertiary amines or quaternary ammonium salts, and R... 2 Selected from hydrogen, alkyl, sulfonic acid, carboxyl, phosphate, ester, and quaternary ammonium salts, while R 3 Selected from C1 to C 16 The use of chain alkyl groups and C1-C4 alcohols makes these fluoroboronic cyanine fluorescent probes amphoteric molecules, meaning one end is hydrophilic and the other is hydrophobic. This makes it difficult for these probes to enter the hydrophobic environment inside the cell membrane, and they are more likely to remain on the cell membrane surface for fluorescent imaging of the cell membrane.

[0017] In some preferred embodiments, R 1 Selected from hydrogen, chlorine, bromine, and iodine; R 3 Selected from C1 to C 16 Alkyl chain.

[0018] Furthermore, the fluorinated boron cyanine fluorescent probe is selected from the following compounds:

[0019]

[0020] As another preferred embodiment of the present invention, R 1 Selected from hydrogen, halogens, C1-C4 alkyl groups, X selected from C0-C3 saturated alkyl groups, polyethylene glycol groups, tertiary amines, quaternary ammonium salts, R 2 Selected from hydrogen, alkyl, sulfonic acid, carboxyl, phosphate, ester, quaternary ammonium salt, triphenylphosphine; R 3 Selected from polyethylene glycol groups, C1-C 16 Alkyl carboxylic acids and their active esters, C1-C5 alkyl sulfonic acids and their salts.

[0021] In this technical solution, R 3 Selected from polyethylene glycol groups, C1-C 16 Alkyl carboxylic acids and their active esters, C1-C5 alkyl sulfonic acids and their salts, make these fluoroboron cyanine fluorescent probes hydrophilic molecules, allowing them to easily cross the cell membrane and enter the cell interior to label organelles. Simultaneously, using R... 2 The structural features of the group enable better binding to mitochondria, thus allowing for fluorescence imaging of mitochondria.

[0022] In some preferred embodiments, R 1 Selected from hydrogen, halogens, C1-C4 alkyl groups; X is selected from tertiary amines or quaternary ammonium salts; R 2 Selected from hydrogen, sulfonic acid group, carboxyl group, phosphate group, quaternary ammonium salt, triphenylphosphine group; R 3 Selected from polyethylene glycol groups;

[0023] Furthermore, this type of fluoroboron cyanine fluorescent probe for labeling mitochondria has the structural formula shown in Formula II:

[0024]

[0025] In Formula II, X is selected from tertiary amines or quaternary ammonium salts, y = 1–16, z = 1–8, m = 1–3, and n = 0–12. Preferably, n = 0–6.

[0026] In this technical solution, R 3 The use of polyethylene glycol groups gives the fluoroboron-based anthocyanin fluorescent probe good hydrophilicity, making it easier to penetrate the cell membrane. Meanwhile, R... 2 The use of triphenylphosphine groups, whose positive charge allows them to bind more effectively to the negative potential of mitochondria, enhances the targeting ability of the fluoroboronic cyanine fluorescent probe to mitochondria.

[0027] Another object of the present invention is to provide a method for preparing any of the aforementioned fluoride-boron cyanine fluorescent probes, specifically, the preparation method includes the following steps:

[0028] The compound of Formula III was reacted with the fluoroboronindolenine Cyanine fluorescent dye of Formula IV in a reaction solvent to obtain the fluoroboronindolenine fluorescent probe.

[0029]

[0030] In Equation III, R 5 Selected from hydroxyl, amino, or ammonium salts; in Formula IV, R 4 Selected from carboxylic acids or active carboxylic acid esters.

[0031] In this technical solution, the compound of formula III is a hydrophilic compound, and its R 5 The group is selected from hydroxyl, amino, or ammonium salts, preferably amino or ammonium salts. The compound of formula IV is a fluoroboron-based cyanine fluorescent dye. This fluorescent dye is prepared by reacting a boron difluoride-bridged difluoroboron pseudoindole electron acceptor with Fisher aldehyde, and its R... 4 The group is a carboxylic acid or a carboxylic acid active ester.

[0032] In this technical solution, the reaction can be completed at room temperature for 0.5–24 hours, under mild conditions. The reaction can be carried out under alkaline or non-alkaline conditions. For R 5 When BCy is a carboxylic acid group, a certain amount of condensing agent needs to be added during the reaction to form a probe derivative of BCy ester or amide. In one or more embodiments, the condensing agent can be 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and its hydrochloride), or DCC (N,N'-dicyclohexylcarbodiimide).

[0033] In one or more embodiments, the reaction is carried out in an alkaline environment, wherein the base is sodium bicarbonate, potassium bicarbonate, triethylamine, diisopropylethylamine, pyridine, 2,6-dimethylpyridine, sodium acetate, potassium acetate, potassium carbonate, or sodium carbonate.

[0034] In some embodiments, the reaction solvent is dichloromethane, dichloroethane, chloroform, acetone, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, toluene, chlorobenzene, xylene, and mixtures thereof. The base is at least one selected from triethylamine, diisopropylaminoethylamine, pyridine, sodium acetate, potassium acetate, potassium carbonate, sodium bicarbonate, sodium carbonate, and potassium tert-butoxide.

[0035] In this technical solution, the tetrazine membrane probe synthesized by water-soluble tetrazine and fluorine boron cyanine fluorescent dye has mild reaction conditions, a short synthesis route, and is easy to scale up for production.

[0036] Furthermore, the fluorescent dye of formula IV is selected from the following compounds:

[0037]

[0038] Furthermore, it also includes the following step: reacting the compound of formula V and the compound of formula VI under the action of a base to obtain R. 2 Compound of formula III, where X is a triphenylphosphine group and X is a tertiary amine;

[0039]

[0040] In formula V, M is a halogen; in formula VI, R... 6 Selected from hydroxyl or tert-butyloxycarbonyl amino.

[0041] In this technical solution, the M group in formula V is a halogen, preferably chlorine, bromine, or iodine. The R group in formula V... 6 The group can be hydroxyl or tert-butoxycarbonylamino. Wherein, when R... 6 When the group is hydroxyl, R in the compound of formula III synthesized from formulas V and VI 5 The group is a hydroxyl group; when R 6 The group is tert-butoxycarbonylamino. The intermediate products after the reactions of formulas V and VI require further deprotection to obtain R. 5 Compounds of formula III with an amino group or that react further to form an ammonium salt.

[0042] In one or more embodiments, the base used in the reaction is sodium bicarbonate, potassium bicarbonate, triethylamine, diisopropylethylamine, pyridine, 2,6-dimethylpyridine, sodium acetate, potassium acetate, potassium carbonate, or sodium carbonate.

[0043] Another object of the present invention is to provide a fluorescence imaging reagent for fluorescence imaging of cell membranes and / or mitochondria, the fluorescence imaging reagent comprising any of the aforementioned fluoroboronic acid cyanine fluorescent probes.

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0045] 1. The fluorinated boron cyanine fluorescent probe provided by the present invention comprises a hydrophilic group and a fluorescent dye connected by an amide bond. By adjusting the hydrophilicity or hydrophobicity of the fluorescent dye, a fluorinated boron cyanine fluorescent probe of amphiphilic or hydrophilic molecule can be obtained, so that the fluorescent probe can perform fluorescence imaging of cell membrane or mitochondria with high signal-to-noise ratio and high specificity.

[0046] 2. The fluorinated boron cyanine fluorescent probe provided by this invention enriches the types of existing fluorescent probes. It has a simple synthesis process, mild reaction conditions, and is promising for industrial production.

[0047] 3. The hydrophilic group of the present invention adopts the structure of triphenylphosphine and tertiary amine, which makes the hydrophilic group have good hydrophilicity. At the same time, the triphenylphosphine can bind well to negatively charged mitochondria, which is beneficial for fluorescent probe labeling mitochondria. Attached Figure Description

[0048] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0049] Figure 1 For the fluorescence no-wash imaging of MemBcy1 to MemBcy4 probes in a specific embodiment of the present invention, Cell MaskGreen is a commercially available staining agent;

[0050] Figure 2 For the specific embodiments of the present invention, the cells treated / untreated with d-TPP are used for fluorescence imaging, and Cell MaskGreen is a commercially available staining agent;

[0051] Figure 3 Time-dependent fluorescence imaging of MemBCy-2 and commercially available DIO staining agent in A549 cells at different time points in a specific embodiment of the present invention;

[0052] Figure 4 The following are fluorescence images of mitochondria and tumors obtained by BCy-TPP in specific embodiments of the present invention: A is a confocal microscopy image of A549 cells treated with MemBCy-6 (500 nM) or Mito-tracker Green (200 nM) for 15 minutes; B is a time-dependent fluorescence image of tumor-bearing mice injected with MemBCy-6; C is the average fluorescence intensity in B; and D is a fluorescence image of major organs incubated with MemBCy-6. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0054] The term "link" as used in this invention can mean either a direct link or an indirect link via other groups, unless otherwise specified.

[0055] The sources of all raw materials used in this invention are not particularly limited; they can be purchased commercially or prepared using conventional methods well-known to those skilled in the art. The purity of all raw materials used in this invention is not particularly limited; however, analytical grade or the purity requirements conventional in the field of fluorescent dyes are preferred. The designations and abbreviations of all raw materials used in this invention are conventional designations and abbreviations in the art, and each designation and abbreviation is clearly defined within its relevant application. Those skilled in the art can obtain these materials from commercially available sources or prepare them using conventional methods based on the designation, abbreviation, and corresponding application.

[0056] The present invention does not impose any particular restrictions on the expression of the substituents, and all expressions are well known to those skilled in the art. Based on common sense, those skilled in the art can correctly understand their meaning according to their expression.

[0057] I. Preparation of the pseudoindole electron acceptor from difluoroboron

[0058]

Example 1

[0059]

[0060] 5.0 g (31.4 mmol) of 2,3,3-trimethyl-3H-pseudoindole was added to 50 mL of acetic anhydride, and 5 mL (41 mmol) of boron trifluoride diethyl ether was added under argon protection. The mixture was then stirred at 120 °C for 6 hours. After removing the solvent under vacuum using a rotary evaporator, the product was purified by silica gel column chromatography to give 2.8 g of the pale yellow product BFI 1, with a yield of 35%.

[0061] 1 H NMR (400MHz, CDCl3) δ7.65 (d, J = 7.9Hz, 1H), 7.39–7.30 (m, 2H), 7.26–7.21 (m, 1H), 5.71 (s, 1H), 2.28 (s, 3H), 1.43 (s, 6H).

[0062] 13 C NMR (101MHz, CDCl3) δ181.44,179.57,143.00,140.66,128.68,125.88,121.84,116.21,116.19,116.17,91.17,91.13,49.64,24.49,23.59.

[0063]

Example 2

[0064]

[0065] 6.1 g (30 mmol) of 2,3,3-trimethyl-3H-indole-5-carboxylic acid was added to 60 mL of acetic anhydride, and 4.5 mL (36 mmol) of boron trifluoride diethyl ether was added under argon protection. The mixture was then stirred at 120 °C for 8 hours. After removing the solvent under vacuum using a rotary evaporator, the product was purified by silica gel column chromatography to give 3.3 g of the pale yellow product BFI 2, with a yield of 37%.

[0066] 1 ¹H NMR (400 MHz, dimethyl sulfoxide-d6) δ 13.05 (s, 1H), 8.19 (d, J = 1.6 Hz, 1H), 8.04 (d, J = 8.3 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 2.31 (s, 3H), 1.49 (s, 6H).

[0067] 13 C NMR (101 MHz, dimethyl sulfoxide) δ 183.98, 181.88, 167.32, 146.41, 141.81, 131.08, 128.65, 124.42, 115.11, 92.83, 49.98, 24.12, 23.81.

[0068]

Example 3

[0069]

[0070] 5.0 g (24 mmol) of 1,1,2-trimethyl-1H-benzo[e]indole was added to 35 mL of acetic anhydride, and 3.8 mL (31 mmol) of boron trifluoride diethyl ether was added under argon protection. The mixture was then stirred at 130 °C for 10 hours. After removing the solvent under vacuum using a rotary evaporator, the product was purified by silica gel column chromatography to give 2.39 g of the pale yellow product BFI 3, with a yield of 33%.

[0071] 1 H NMR (400MHz, CDCl3) δ7.98(d,J=8.2Hz,1H),7.95(d,J=8.1Hz,1H),7.93–7.86(m,2H),7.58(ddd, J=8.4,6.9,1.4Hz,1H),7.48(ddd,J=8.2,6.8,1.2Hz,1H),5.81(s,1H),2.32(s,3H),1.69(s,6H).

[0072] 13C NMR (101MHz, CDCl3) δ182.66,179.10,140.69,133.96,132.36,130.21,129 .93,128.47,127.14,125.13,122.17,115.58,91.06,51.25,24.06,23.66.

[0073]

Example 4

[0074]

[0075] 2.5 g (13 mmol) of 5-chloro-2,3,3-trimethylindole was added to 20 mL of acetic anhydride, followed by the addition of 2 mL (16 mmol) of boron trifluoride diethyl ether under argon protection. The mixture was then stirred at 120 °C for 8 hours. After removing the solvent under vacuum using a rotary evaporator, the product was purified by silica gel column chromatography to give 1.65 g of the pale yellow product BFI 4, with a yield of 45%.

[0076] 1 H NMR (400MHz, Chloroform-d) δ7.56(d,J=8.2Hz,1H),7.40–7.30(m,2H),5.72(d,J=2.4Hz,1H),2.30(s,3H),1.45(s,6H).

[0077] 13 C NMR (101MHz, CDCl3) δ181.37,180.43,142.43,141.58,131.63,128.87,122.54,117.09,117.07,117.05,91.25,49.81,24.45,23.71.

[0078]

Example 5

[0079]

[0080] BFI 1 (750 mg, 3 mmol) and N-chlorosuccinimide (NCS, 479.7 mg, 3.6 mmol) were added sequentially to a reaction flask, followed by 15 mL of chloroform and dimethyl sulfoxide (45 μL, 0.6 mmol). The reaction mixture was stirred overnight at room temperature. After removing the solvent under vacuum using a rotary evaporator, the mixture was purified by silica gel column chromatography to give 245 mg of the white solid product BFI 5, with a yield of 87%.

[0081] 1H NMR (400MHz, CDCl3) δ7.66 (d, J = 7.8Hz, 1H), 7.45–7.23 (m, 3H), 2.43 (s, 3H), 1.71 (s, 6H).

[0082] 13 C NMR (101MHz, CDCl3) δ176.63,176.22,142.03,141.65,128.81,126.72,121.53,116.62,52.58,22.10,21.67.

[0083]

Example 6

[0084]

[0085] N-chlorosuccinimide (400 mg, 3 mmol) and dimethyl sulfoxide (38 μL, 0.5 mmol) were added to a mixture of BFI 2 (596 mg, 2 mmol), CHCl3 (12 mL), and N-methyl-2-pyrrolidone (0.6 mL). The reaction was stirred at room temperature for 8 hours. After removing the solvent under vacuum using a rotary evaporator, the product was purified by silica gel column chromatography to give 536.3 mg of white solid product BFI 6, with a yield of 82%.

[0086] 1 ¹H NMR (400 MHz, dimethyl sulfoxide-d6) δ 13.11 (s, 1H), 8.21 (s, 1H), 8.06 (d, J = 9.9 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 2.47 (s, 3H), 1.73 (s, 6H).

[0087] 13 C NMR (101 MHz, dimethyl sulfoxide) δ 178.95, 178.47, 167.16, 145.30, 142.43, 131.21, 129.56, 124.18, 115.64, 102.10, 52.82, 22.27, 21.77.

[0088]

Example 7

[0089]

[0090] A mixture of BFI 3 (449 mg, 1.5 mmol) and N-chlorosuccinimide (300 mg, 2.25 mmol) was dissolved in dimethyl sulfoxide (32 L, 0.45 mmol) and CHCl3 (8 mL), and stirred at room temperature for 5 hours. After complete conversion, the solvent was removed under vacuum using a rotary evaporator, and the product was purified by silica gel column chromatography to obtain 413.6 mg of a yellow solid, BFI 7, in 83% yield.

[0091] 1 H NMR(400MHz,Chloroform-d)δ8.02(d,J=8.0Hz,1H),7.97(d,J=8.2Hz,1H),7.90(q,J =8.8Hz,2H),7.62(t,J=8.3Hz,1H),7.52(t,J=8.1Hz,1H),2.46(s,3H),1.97(s,6H).

[0092] 13 C NMR (101MHz, CDCl3) δ177.69,175.68,139.50,132.88,130.42,130.05,127.72,127.30,125.48,122.34,115.54,101.46,54.18,21.70,20.72.

[0093] II. Preparation of Fluoroboron Cyanide Fluorescent Probes

[0094]

Example 8

[0095]

[0096] BFI 2 (43.9 mg, 0.15 mmol) and 1,3,3-trimethyl-2-(formylmethylene)indoline (42 mg, 0.2 mmol) were dissolved in a mixed solution of toluene (2 mL), piperidine (40 μL), and acetic acid (20 μL) under argon protection. The mixture was then stirred at 90 °C for 3 hours. After removing the solvent under reduced pressure, most impurities were removed by silica gel column chromatography to obtain BCY1.

[0097] BCy 1 (approximately 0.15 mmol) and N,N'-disuccinimidyl carbonate (76.8 mg, 0.3 mmol) were dissolved in anhydrous dichloromethane (2 mL), followed by the addition of diisopropylethylamine (49 μL, 0.3 mmol). The mixture was stirred at room temperature for 5 hours, and the solvent was removed under vacuum using a rotary evaporator. The solution was then purified by silica gel column chromatography to yield 62.7 mg of the blue product BCy 2, with a yield of 75%.

[0098] 1H NMR(400MHz,Chloroform-d)δ8.26(t,J=13.4Hz,1H),8.15(dd,J=8.4,1.8Hz,1H),8.00(d,J=1.7Hz,1H),7.62(d,J=8.3Hz,1H),7.29–7.22(m,3H),7.0 4(t,J=7.3Hz,1H),6.85–6.80(m,1H),5.89(d,J=13.6Hz,1H),5.62(d,J=13 .0Hz,1H),5.60(s,1H),3.30(s,3H),2.92(s,4H),1.67(s,6H),1.46(s,6H).

[0099] 13 C NMR (101MHz, CDCl3) δ178.95,174.45,169.44,167.82,161.77,150.12,143.66,143.33,140.80,139.87,132.48,128. 04,124.04,122.34,121.96,119.61,114.62,114.15,107.82,96.75,91.36,48.29,47.55,29.70,28.71,25.81,25.72.

[0100]

[0101] Sulfonate (15.6 mg, 0.06 mmol) and diisopropylethylamine (25 μL, 0.15 mmol) were dissolved in 0.6 mL of dimethyl sulfoxide, and then BCY 2 (22.9 mg, 0.04 mmol) was added. The mixture was stirred at 25 °C for 12 h. After the reaction was complete, the product was precipitated overnight at 4 °C in 30 mL of MBTE. The precipitate was purified by reversed-phase silica gel column chromatography using acetonitrile containing 0.1% formic acid and water as eluents, yielding 20.1 mg of product MemBCy-1, in 74% yield.

[0102] 1H NMR(400MHz, Methanol-d4)δ8.24(t,J=13.4Hz,1H),7.92(d,J=1.7Hz,1H),7.86(dd,J=8.3,1.8Hz,1H), 7.45(d,J=8.1Hz,1H),7.31(d,J=6.3Hz,1H),7.25(td,J=7.7,1.2Hz,1H),7.04–6.99(m,1H),6.96(d,J=7 .9Hz,1H),6.01(d,J=13.6Hz,1H),5.85(s,1H),5.78(d,J=13.1Hz,1H),3.59–3.48(m,4H),3.47–3.39(m, 2H),3.33(s,3H),3.12(s,6H),2.87(t,J=6.7Hz,2H),2.26–2.18(m,2H),2.17–2.09(m,2H),1.65(s,6H).

[0103] 13 C NMR(101MHz,MeOD)δ179.33,173.85,168.61,167.53,146.95,143.81,142.49,140.98,139.64,129.30,127.92,127.83,121.86 ,121.48,121.21,113.38,107.81,96.37,90.38,62.39,61.94,50.13,48.37,47.09,36.49,28.58,27.52,24.50,22.71,18.49.

[0104]

Example 9

[0105]

[0106] BFI 6 (65.4 mg, 0.2 mmol) and 1,3,3-trimethyl-2-(formylmethylene)indoline (52.3 mg, 0.26 mmol) were dissolved in a mixed solution of toluene (3 mL), piperidine (60 μL), and acetic acid (30 μL) under argon protection. The mixture was then stirred at 60 °C for 5 hours. After removing the solvent under reduced pressure, most impurities were removed by silica gel column chromatography to obtain BCY3.

[0107] BCY 3 (approximately 0.2 mmol) and N,N'-disuccinimidyl carbonate (76.8 mg, 0.3 mmol) were dissolved in anhydrous dichloromethane (2 mL), followed by the addition of diisopropylethylamine (49 μL, 0.3 mmol). The resulting mixture was stirred at room temperature for 5 hours, and then the solvent was removed. Purification by silica gel column chromatography yielded 103.9 mg of the blue product BCY 4, in 85% yield.

[0108] 1 H NMR (400MHz, CDCl3) δ8.33(t,J=13.4Hz,1H),8.04(dd,J=8.4,1.7Hz,1H),7.89(d,J=1.7Hz,1H),7.50(d,J=8.3Hz,1H),7.27–7.20(m,2H),7.0 3(t,J=7.4Hz,1H),6.83(d,J=8.1Hz,1H),6.41(d,J=13.4Hz,1H),5.76(d,J=13.3Hz,1H),3.31(s,3H),2.84(s,4H),1.67(s,6H),1.61(s,6H).

[0109] 13 C NMR (101MHz, CDCl3) δ172.61,170.20,170.13,169.41,161.67,149.43,146.74,143.26,141.37,140.21,132.41,128.2 0,123.67,123.25,122.07,119.78,114.51,109.62,108.51,100.11,98.59,51.08,48.14,30.01,28.71,25.71,22.94.

[0110]

[0111] Sulfonate (15.6 mg, 0.06 mmol) and diisopropylethylamine (25 μL, 0.15 mmol) were dissolved in 0.6 mL of dimethyl sulfoxide, and then BCY 4 (24.2 mg, 0.04 mmol) was added. The mixture was stirred at 25 °C for 12 h. After the reaction was complete, the product was precipitated overnight at 4 °C in 30 mL of methyl tert-butyl ether. The precipitate was purified by reversed-phase silica gel column chromatography using acetonitrile containing 0.1% formic acid and water as eluents, yielding 23.6 mg of product MemBCy-2, in 82% yield.

[0112] 1H NMR(400MHz, Methanol-d4)δ8.43(t,J=13.4Hz,1H),7.87(d,J=1.7Hz,1H),7.84(dd,J=8.4,1.9Hz,1H), 7.42(d,J=8.2Hz,1H),7.38(d,J=7.4Hz,1H),7.32(t,J=7.2Hz,1H),7.12(t,J=7.2Hz,1H),7.08(d,J=8. 0Hz,1H),6.50(d,J=13.3Hz,1H),6.02(d,J=13.4Hz,1H),3.60–3.50(m,4H),3.44(s,3H),3.43–3.38(m, 2H),3.12(s,6H),2.88(t,J=6.6Hz,2H),2.25–2.16(m,2H),2.16–2.08(m,2H),1.76(s,6H),1.69(s,6H).

[0113] 13 C NMR (101MHz, CDCl3) δ173.53,168.94,168.70,167.57,145.76,145.02,143.47,141.36,140.03,130.20,128.47,128.01,122.69,12 2.06,121.65,113.91,110.09,108.07,99.66,98.19,63.04,62.54,51.55,50.87,47.86,47.71,37.05,29.80,28.65,22.70,19.07.

[0114]

Example 10

[0115]

[0116] BFI 6 (65.4 mg, 0.2 mmol) and Fischer aldehyde (59.0 mg, 0.26 mmol) were dissolved in a mixed solution of toluene (3 mL), piperidine (60 μL), and acetic acid (30 μL) under argon protection. The mixture was then stirred at 50 °C for 2 hours. After removing the solvent under reduced pressure, most impurities were removed by silica gel column chromatography to obtain BCY 5.

[0117] BCY 5 (approximately 0.2 mmol) and N,N'-disuccinimidyl carbonate (76.8 mg, 0.3 mmol) were dissolved in anhydrous dichloromethane (2 mL), followed by the addition of diisopropylethylamine (49 μL, 0.3 mmol). The mixture was then stirred at room temperature for 5 hours. After solvent removal, the product was purified by silica gel column chromatography to yield 98.7 mg of the blue product BCY 6, in 78% yield.

[0118] 1 H NMR(400MHz, CDCl3)δ8.12(dd,J=8.4,1.7Hz,1H),8.01–7.89(m,2H),7.61( d,J=8.4Hz,1H),7.55(t,J=13.1Hz,2H),7.30–7.21(m,3H),7.03(t,J=7.5Hz ,1H),6.81(d,J=7.8Hz,1H),6.54(d,J=13.9Hz,1H),6.34(t,J=12.0Hz,1H), 5.63(d,J=12.8Hz,1H),3.29(s,3H),2.92(s,4H),1.75(s,7H),1.64(s,6H).

[0119] 13 C NMR (101 MHz, dimethyl sulfoxide) δ 168.86, 165.28, 164.61, 161.10, 156.84, 146.57, 144.31, 141.61, 138.99, 136.86, 134.99, 127.63, 123.35, 118.97, 118.63, 117.49, 117.16, 115.61, 110.18, 107.32, 103.02, 96.25, 93.78, 46.67, 42.54, 24.94, 23.50, 20.96, 18.05.

[0120]

[0121] Sulfonate (15.6 mg, 0.06 mmol) and diisopropylethylamine (25 μL, 0.15 mmol) were dissolved in 0.6 mL of dimethyl sulfoxide solution, and then BCY 6 (25.3 mg, 0.04 mmol) was added. The mixture was stirred at 25 °C for 12 h. After the reaction was complete, the product was precipitated overnight at 4 °C in 30 mL of MBTE. The precipitate was purified by reversed-phase silica gel column chromatography using acetonitrile containing 0.1% formic acid and water as eluents, to give 25.5 mg of product MemBCy-3, in 86% yield.

[0122] 1¹H NMR (400 MHz, dimethyl sulfoxide-d6) δ 8.60 (t, J = 5.7 Hz, 1H), 8.06–7.97 (m, 2H), 7.96–7.84 (m, 2H), 7.45 (d, J = 6.8 Hz, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.32–7.27 (m, 1H), 7.14 (d, J = 7.9 Hz, 1H), 7.07 (t, J = 5.7 Hz, 1H) 7.4Hz,1H),6.50–6.37(m,2H),5.90(d,J=13.1Hz,1H),3.46–3.44(m,4H),3.40(s,3H),3.37 –3.35(m,2H),3.04(s,6H),2.54–2.52(m,2H),2.08–1.95(m,4H),1.72(s,6H),1.61(s,6H).

[0123] 13 C NMR (101 MHz, dimethyl sulfoxide) δ 174.24, 172.07, 167.45, 167.35, 165.93, 150.73, 148.40, 144.97, 143.52, 140.83, 130.26, 128.33, 128.02, 123.57, 122.43, 122.00, 121.42, 113.02, 109.49, 108.96, 99.79, 98.87, 62.32, 61.20, 50.99, 50.08, 47.64, 47.27, 36.43, 31.56, 29.43, 27.41, 24.85, 22.50, 18.90.

[0124]

Example 11

[0125]

[0126] BFI 6 (49.2 mg, 0.15 mmol) and hemicyanine intermediate (84.4 mg, 0.18 mmol) were dissolved in 2 mL of dichloromethane, followed by the addition of 59 μL of diisopropylethylamine (0.36 mmol). The reaction mixture was stirred at 50 °C for 90 min. After cooling, N,N'-disuccinimidyl carbonate (76 mg, 0.3 mmol) and diisopropylethylamine (49 μL, 0.3 mmol) were added, and the mixture was column-sected at Rt 12 h. TLC showed a P:E ratio of 3:2, and 72 mg of BCY 7 was obtained, representing a yield of 72%.

[0127] BCY 7 (172.8 mg, 0.4 mmol) was dissolved in 5 mL of methylene chloride, and then 4 M HCl (2 mmol, 0.5 mL) dissolved in 1,4-dioxane was added at 0 °C. The solution was reacted at room temperature for 60 min. After evaporating the solvent, the solid fraction was recrystallized in MeOH / CHCl3 / MTBE to give 128 mg of red solid product BCY 8, with a yield of 87%.

[0128] 1 H NMR(400MHz,Chloroform-d)δ8.42(t,J=13.4Hz,1H),8.15–8.05(m,1H),7.95(d,J =1.7Hz,1H),7.55(d,J=8.3Hz,1H),7.53–7.43(m,4H),7.34–7.28(m,4H),7.14–7.0 7(m,2H),6.92–6.86(m,1H),6.45(d,J=13.3Hz,1H),5.84(d,J=13.4Hz,1H),3.80(t ,J=7.7Hz,2H),2.91(s,4H),2.16(s,4H),1.73(s,10H),0.90(q,J=5.8,4.6Hz,3H).

[0129]

[0130] Sulfonate (13.0 mg, 0.05 mmol) and diisopropylethylamine (16 μL, 0.1 mmol) were dissolved in 0.5 mL of dimethyl sulfoxide solution, and then BCY 7 (25.3 mg, 0.04 mmol) was added. The mixture was stirred at 25 °C for 12 h. After the reaction was complete, the product was purified by reversed-phase silica gel column chromatography using acetonitrile containing 0.1% formic acid and water as eluents, yielding 26.8 mg of MemBCy-4, with a yield of 86%.

[0131] 1H NMR(400MHz, DMSO-d6)δ8.49(s,1H),8.28(t,J=13.3Hz,1H),8.05–7.85(m,2H),7.43(d, J=7.3Hz,1H),7.22(d,J=8.2Hz,3H),7.03(t,J=7.4Hz,1H),6.81(d,J=7.9Hz,1H),6.41( d,J=13.3Hz,1H),5.76(d,J=13.1Hz,1H),3.81–3.41(m,8H),3.12(s,6H),2.98–2.80(m, 2H),2.33–2.02(m,4H),1.68(s,8H),1.62(s,6H),1.43–1.31(m,6H),0.92–0.81(m,3H).

[0132] 13 C NMR (101MHz, CDCl3) δ173.39,169.16,168.46,167.66,145.96,145.54,143 .12,141.41,140.35,130.31,128.56,128.13,122.86,122.26,121.90,113 .90,109.76,108.47,99.69,98.14,63.42,62.78,51.63,50.92,48.06,47.85,43.33,37.14,31.60,29.83,28.85,26.85,22.92,22.65,19.28,14.12.

[0133]

Example 12

[0134]

[0135] Sulfonate (234.7 mg, 0.9 mmol) and diisopropylaminoethylamine (180 μL, 1.1 mmol) were dissolved in 8 mL of dimethyl sulfoxide, followed by the addition of 418.4 mg of Boc-NH-PEG3-NHS ester (1 mmol). The reaction mixture was stirred at 25 °C for 12 h. After the reaction was complete, 5 mL of dichloromethane was added for dilution, followed by precipitation with 80 mL of methyl tert-butyl ether. The precipitate was filtered, washed with methyl tert-butyl ether, and the product was obtained, yielding approximately 451 mg of the initial product, which was directly used in the next reaction step.

[0136]

[0137] The primordial product (316.2 mg, 0.6 mmol) was dissolved in 9 mL of dichloromethane. Then, at 0 °C, 4 M HCl dissolved in 1 mL of dioxane was added, and the mixture was reacted at room temperature for 3 h. After evaporating the solvent, 3 mL of isopropanol and approximately 0.3 mL of methanol were added. Under heating, chloroform / methyl tert-butyl ether (20 mL) was added. After precipitation, the supernatant was removed to give 264 mg of intermediate 1, with a yield of 95%.

[0138] 1 H NMR(400MHz, Methanol-d4)δ3.79–3.70(m,4H),3.70–3.60(m,8H),3.57–3.50(m,2H),3.40–3.31(m,4H),3.14(t ,J=5.2Hz,2H),3.11(s,6H),2.88(t,J=6.6Hz,2H),2.50(t,J=6.1Hz,2H),2.24–2.14(m,2H),2.04–1.95(m,2H).

[0139] 13 C NMR (101MHz, CD3OD) δ172.99,70.05,69.94,69.75,69.71,66.76,66.43,62.17,61.52,50.31,39.23,36.09,35.73,22.47,18.47.

[0140]

[0141] Under argon protection, diisopropylethylamine (59 μL, 0.33 mmol) was added to a mixture of BFI 6 (49 mg, 0.15 mmol) and hemicyanine (99 mg, 0.2 mmol) in dichloromethane (2 mL). The mixture was then stirred at 50 °C for 90 min. After cooling to room temperature, N,N'-disuccinimidyl carbonate (76 mg, 0.3 mmol) and diisopropylethylamine (33 μL, 0.2 mmol) were added to the solution. The mixture was stirred at room temperature for 12 h, and then the solvent was removed. Purification by silica gel column chromatography yielded 45.5 mg of product BCY 9, in a yield of 46%.

[0142] 1H NMR(400MHz,Chloroform-d)δ8.14(dd,J=8.4,1.7Hz,1H),8.00(d,J=1.7Hz,1H),7.83(dd,J=14 .1,12.1Hz,1H),7.65(d,J=8.3Hz,1H),7.25–7.16(m,3H),7.01(dd,J=13.9,11.6Hz,1H),6.95(t ,J=7.4Hz,1H),6.73(d,J=7.8Hz,1H),6.65(d,J=14.1Hz,1H),6.41(t,J=13.0Hz,1H),6.22(dd,J =13.7,11.7Hz,1H),5.53(d,J=12.5Hz,1H),3.22(s,3H),2.92(s,4H),1.75(s,6H),1.62(s,6H).

[0143] 13 C NMR (101MHz, CDCl3) δ174.71,169.85,169.34,162.54,161.53,149.78,149.36,148.75,144.23,141.85,140.10,139.35,132.37,1 27.98,126.13,124.63,123.76,121.76,121.23,120.93,115.38,115.12,101.63,98.04,51.73,46.62,29.43,28.32,25.72,22.65.

[0144]

[0145] Intermediate 1 (13.9 mg, 0.03 mmol) and diisopropylethylamine (8 μL, 0.05 mmol) were dissolved in 0.3 mL of dimethyl sulfoxide, followed by the addition of BCY 9 (13.1 mg, 0.02 mmol). The mixture was stirred at 25 °C for 12 h. After the reaction was complete, the product was directly purified by column chromatography (D:M = 2:3) to remove small polar impurities. Further purification by reversed-phase column chromatography yielded MemBCy-5 in 84% yield.

[0146] 11H NMR (400 MHz, Methanol-d4) δ 7.91 (d, J = 1.7 Hz, 1H), 7.89–7.86 (m, 1H), 7.73 (s, 1H), 7.50 (d, J = 8.1 Hz, 1H), 7.39–7.32 (m, 1H), 7.23–7.18 (m, 2H), 7.15–7.07 (m, 1H), 6.92 (t, J = 7.4 Hz, 1H), 6.83 (d, J = 7.9 Hz, 1H), 6.63 (d, J = 14.0 Hz, 1H), 6.42 (t, J = 13.1 Hz, 1H), 6.26 (t, J = 12.7 Hz, 1H), 5.62 (d, J = 12.5 Hz, 1H), 3.72–3.67 (m, 4H), 3.65 (s, 4H), 3.64–3.57 (m, 8H), 3.54–3.50 (m, 2H), 3.33 (d, J = 5.0 Hz, 1H), 3.30–3.26 (m, 4H), 3.08 (s, 6H), 2.88 (t, J = 6.6 Hz, 2H), 2.43 (t, J = 6.0 Hz, 2H), 2.22–2.15 (m, 2H), 2.00–1.94 (m, 2H), 1.76 (s, 6H), 1.59 (s, 6H).

[0147] 13 13C NMR (101 MHz, MeOD) δ 175.93, 174.17, 169.85, 169.29, 163.87, 150.67, 149.91, 146.78, 145.52, 143.43, 143.05, 141.76, 140.53, 132.26, 129.31, 128.97, 126.89, 125.87, 122.63, 122.30, 122.16, 115.56, 108.28, 71.40, 71.32, 71.20, 71.12, 70.44, 68.04, 63.63, 63.05, 53.06, 51.51, 47.59, 40.95, 37.52, 36.98, 28.59, 23.80, 23.01, 19.75.

[0148]

Example 13

[0149]

[0150] tert-butyl(3-(methylamino)propyl)carbamate tert-butyl ester (338 mg, 1.8 mmol), (3-bromopropyl)-triphenylphosphine bromide (696 mg, 1.5 mmol), and potassium carbonate (207 mg, 1.5 mmol) were dissolved in acetonitrile (8 mL). The solution was stirred at 80 °C for 10 h. After the reaction mixture was cooled to room temperature, the solvent was removed under vacuum. The resulting solid was washed, neutralized, and purified by silica gel column chromatography to give intermediate 2 (625 mg, 73%).

[0151]

[0152] 1,4-dioxane in 1 mL of 4 M HCl (4 mmol) was added to 8 mL of dichloromethane solution containing intermediate 2 (571 mg, 1 mmol). The solution was heated to room temperature at 0 °C. After stirring for 60 minutes, the solvent was evaporated to obtain intermediate 3.

[0153]

[0154] Compound 16 (18 mmol, 6.5 g) and 2,3,3-trimethylindonin (18 mmol, 2.8 g) were heated overnight at 110 °C. The red mixture was then cooled, dissolved in a minimal amount of CH₂Cl₂, and poured into diethyl ether with stirring. After removing the diethyl ether, the red oily substance was washed three times with diethyl ether to obtain a dark red oily substance (9.2 g), which was compound 17.

[0155] 1 H NMR (400MHz, CDCl3) δ7.82–7.77(m,1H),7.67(d,J=8.1Hz,2H),7.58–7.44(m,3H),7.05(d,J=7.9Hz,2H),4.96 (t,J=5.0Hz,2H),4.04–3.92(m,2H),3.62–3.43(m,12H),3.36(s,3H),2.91(s,3H),2.29(s,3H),1.54(s,6H).

[0156]

[0157] Compound 17 (781.5 mg, 1.5 mmol) and malondialdehyde bis(phenylimine) hydrochloride (510 mg, 2.3 mmol) were added to a solution of Ac₂O (8 mL) and AcOH (1 mL). The reaction mixture was stirred at 100 °C for 2 h. After removing the solvent under vacuum, the resulting solid was passed through a silica gel column to remove most of the impurities, yielding intermediate 4. Intermediate 4 was unstable and was used directly in the next coupling reaction.

[0158] Dichloromethane (2 mL) and DIEPA (49 μL, 0.3 mmol) were added to a mixture of BFI 6 (49.2 mg, 0.15 mmol) and intermediate 4 (138 mg, 0.2 mmol). The reaction mixture was stirred at 50 °C for 60 min. After cooling to room temperature, N,N'-disuccinimidyl carbonate (76 mg, 0.3 mmol) and diisopropylethylamine (49 μL, 0.3 mmol) were added. The mixture was stirred at room temperature for 12 h and purified by rapid chromatography to give the product as a deep blue solid BCY 10 (62 mg, 51%).

[0159] 1 H NMR(400MHz,Chloroform-d)δ8.12(dd,J=8.4,1.7Hz,1H),7.99(d,J=1.7Hz,1H),7.93(d,J=13.6Hz,1H),7 .61(d,J=8.3Hz,1H),7.56(t,J=13.1Hz,1H),7.22(d,J=7.5Hz,2H),7.06–6.99(m,1H),6.89(d,J=7.9Hz,1 H),6.54(d,J=13.9Hz,1H),6.34(t,J=12.8Hz,1H),5.74(d,J=12.8Hz,1H),3.95(t,J=6.1Hz,2H),3.75(t, J=6.0Hz,2H),3.64–3.57(m,10H),3.56–3.52(m,2H),3.36(s,3H),2.92(s,4H),1.75(s,6H),1.63(s,6H).

[0160] 13 C NMR (101MHz, CDCl3) δ173.55,169.96,169.43,165.46,161.58,151.23,149.0 4,146.47,143.49,141.62,139.56,132.35,128.04,123.73,123.49,122.31,1 21.80,120.37,114.88,112.07,108.49,101.00,98.81,71.91,71.13,70.66,70.62,70.59,70.50,67.46,59.03,51.41,47.37,43.22,28.32,25.71,22.79.

[0161]

[0162] BCY 10 (12.2 mg, 0.015 mmol) was added to a solution of intermediate 3 (11.5 mg, 0.025 mmol) and diisopropylethylamine (13 μL, 0.08 mmol) in 0.2 mL of LMF. The mixture was stirred at 25 °C for 3 hours and purified by reverse-phase column chromatography to obtain the product BCy-TPP.

[0163] 1 H NMR(400MHz,Chloroform-d)δ8.50(t,J=5.6Hz,1H),8.43(s,1H),8.35(s,1H),8.19(s,1H),7.99(d,J=8.4Hz,1H),7.89–7.75(m,1 0H),7.67(dt,J=8.1,4.6Hz,6H),7.52–7.42(m,2H),7.21(d,J=7.7Hz,2H),6.99(t,J=7.4Hz,1H),6.86(d,J=7.9Hz,1H),6.55(d,J =14.0Hz,1H),6.32(t,J=12.8Hz,1H),5.69(d,J=12.6Hz,1H),3.93(t,J=6.1Hz,2H),3.89–3.80(m,2H),3.74(t,J=6.1Hz,2H),3.6 6–3.58(m,10H),3.57–3.49(m,6H),3.37(s,3H),3.11(t,J=7.3Hz,2H),2.67(s,3H),2.27–2.12(m,4H),1.73(s,6H),1.62(s,6H).

[0164] 13 C NMR (101MHz, CDCl3) δ174.50,168.62,167.05,166.62,163.90,149.38,145.38,144.42,143.69,141.48, 139.31,135.36,135.33,133.70,133.60,130.67,130.54,128.56,127.96,123.41,121.79,121.71,117. 87,117.01,114.49,112.77,108.11,100.66,98.19,71.87,71.08,70.62,70.57,70.55,70.45,67.39,59.00,55.54,55.33,53.79,51.94,47.00,43.03,40.50,36.99,28.32,23.91,22.62,20.70,20.17,18.64.

[0165]

Example 14

[0166]

[0167] DIPEA (9.8 μL, 0.06 mmol) was added to a mixture of PPh3-NH2 (12 mg, 0.03 mmol) and BCY4 (12.2 mg, 0.02 mmol) in anhydrous DCM (0.5 mL). The mixture was stirred at room temperature for 6 h under an argon atmosphere. The substrate was purified by TLC (DCM:MeOH = 15:1) to give the desired (11.5 mg, 71%) blue solid, MemBCy-6.

[0168] 1 H NMR(400MHz,Chloroform-d)δ9.58(t,J=6.3Hz,1H),8.57(s,1H),8.30(t,J=13.4Hz,1H),8. 15(d,J=8.3Hz,1H),7.78–7.72(m,10H),7.60(td,J=7.7,3.3Hz,7H),7.25(dd,J=4.2,3.0Hz, 1H),7.05(t,J=7.5Hz,1H),6.85(s,1H),6.49(d,J=13.6Hz,1H),5.75(d,J=13.1Hz,1H),4.0 0–3.87(m,3H),3.77–3.70(m,3H),3.33(s,3H),2.05–1.90(m,5H),1.81(s,6H),1.67(s,6H).

[0169] III. Fluorescent probes are used to label cell membranes.

[0170]

Example 15

[0171] Fluoroboron cyanine fluorescent probes were used for wash-free fluorescence imaging. A549 cells were treated with Hoechst (1 μg / mL), followed by treatment with MemBCy-1, MemBCy-2, MemBCy-3, or MemBCy-4 for 15 minutes, and then treated with Cell Mask Green cell membrane staining agent for 20 minutes. The dosage of MemBCy-1, MemBCy-2, and MemBCy-3 was 200 nM, and the dosage of MemBCy-4 was 500 nM.

[0172] Imaging results as follows Figure 1 As shown, the excitation wavelength of MemBCy-1 is λ. ex =580nm, emission wavelength λ em=590-660nm; the excitation wavelength of MemBCy-2 is λ ex =613nm, emission wavelength λ em =620-690nm; the excitation wavelength of MemBCy-3 is λ ex =650nm, emission wavelength λ em =660-750nm; the excitation wavelength of MemBCy-4 is λ ex =725nm, emission wavelength λ em =740-850nm, scale bar 20μm. This indicates that the far-infrared to near-infrared fluorinated boron cyanine fluorescent probe can achieve multicolor imaging of cell membranes.

[0173]

Example 16

[0174] In this embodiment, the fluorescence of MemBCy-2 in A549 cells was treated with d-TPP. dTPP is a cytotoxic inhibitor that induces oxidative stress damage in cells.

[0175] Cells were treated with d-TPP (5 μM) for 30 minutes (d-TPP(+) Cell Mask group), or cells were not treated with d-TPP (d-TPP(-) Cell Mask group), followed by treatment with Hoechst (1 μg / mL), Cell mask Green cell membrane staining agent (0.5 mg / mL) and MemBCy-2 (200 nM) for 5 minutes.

[0176] from Figure 2 It can be seen that the commercially available Cell Mask Green cell membrane staining agent is difficult to detect cell membrane permeability, while MemBCy-2 can detect cell membrane permeability after cell damage and vesicles formed within the cell.

[0177]

Example 17

[0178] In this embodiment, A549 cells were labeled with MemBCy-2 and commercially available DIO staining agent, wherein the excitation wavelength of MemBCy-2 was λ. ex =613nm, emission wavelength λ em =620-700nm, the excitation wavelength of DIO is λ ex =488nm, emission wavelength λ em =495-560nm, scale bar is 10μm.

[0179] from Figure 3 It can be seen that MemBCy-2 has a longer labeling time on the cell membrane than commercially available DIO.

[0180] IV. Fluorescent probes are used to label mitochondria.

[0181]

Example 18

[0182] This embodiment utilizes a fluorinated boron cyanine fluorescent probe to detect mitochondria and tumors. Because the mitochondrial membrane potential of cancer cells is higher than that of normal cells, delocalized lipophilic cations such as BCy-TPP provided by this invention can be selectively detected and accumulated in cancer cells and tissues.

[0183] Specifically, A549 cells were subcutaneously injected into 4- to 6-week-old nude mice to obtain tumor-bearing mice for approximately 21 days. In this embodiment, all mice were bred and housed at the Sichuan University Animal Experiment Center, and the protocol was approved by the Sichuan University Institutional Animal Care and Use Committee.

[0184] Both tumor-bearing and normal mice were injected with BCy-TPP and imaged at different time points. For tissue imaging, tumors and normal organs (including lungs, liver, kidneys, spleen, and heart) were isolated for in vivo organ imaging. After washing with PBS, tumors and organs were incubated with BCy-TPP (25 μM) for 30 minutes before imaging.

[0185] Experimental results are as follows Figure 4 As shown, the NIR fluorescence signal of BCy-TPP completely overlaps with the green channel of the commercially available Mito-Tracker Green staining agent. When BCy-TPP was injected subcutaneously into the tumor sites of a549 tumor-bearing mice and the corresponding sites in normal nude mice, strong fluorescence signals were observed in the transplanted tumor areas of nude mice, while weak fluorescence was observed in the transplanted tumor areas of normal mice. This demonstrates the selective detection of tumor cells within the tissue. Figure 4 C is Figure 4 Quantitative statistical analysis of B fluorescence intensity. Figure 4 D represents fluorescent staining of ex vivo tissue, indicating that BCy-TPP can selectively stain tumors.

[0186] Therefore, the fluorescent probe of the present invention can transfer uniquely labeled particles and, due to its near-infrared characteristics, can be used for active tumor imaging.

[0187] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fluorescent probe for fluorinated boron cyanine flowers, characterized in that, The fluorinated boron cyanine fluorescent probe is selected from the following compounds:

2. A method for preparing a fluorinated boron cyanine fluorescent probe, characterized in that, The preparation method includes the following steps: The fluoroboron cyanine fluorescent probe was obtained by mixing the compound of formula III with the fluorescent dye of formula IV in a reaction solvent. In Equation III, R 5 Selected from amino or ammonium salts, R 2 The selection of , y, z, and X matches the fluorescent probe of the fluorine-boron cyanine in claim 1; The fluorescent dye of formula IV is selected from the following compounds:

3. The method for preparing a fluorinated boron cyanine fluorescent probe according to claim 2, characterized in that, The reaction solvent is dichloromethane, dichloroethane, chloroform, acetone, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N-methylpyrrolidone, toluene, chlorobenzene, xylene, and mixtures thereof, and the base is at least one selected from triethylamine, diisopropylaminoethylamine, pyridine, sodium acetate, potassium acetate, potassium carbonate, sodium bicarbonate, sodium carbonate, and potassium tert-butoxide.

4. A fluorescence imaging reagent, characterized in that, The fluorescent imaging reagent is used for fluorescent imaging of cell membranes and / or mitochondria, and the fluorescent imaging reagent includes the fluoroboronic acid cyanine fluorescent probe of claim 1.

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