Boron-coordinated salicylhydrazone-based dual-emission fluorophore, preparation method and application thereof

Abstract

The application relates to the technical field of organic synthesis and the technical field of fluorescent materials, in particular to a boron-coordinated salicylhydrazone dual-state emission fluorophore as well as a preparation method and application thereof. The structure of the boron-coordinated salicylhydrazone dual-state emission fluorophore is shown in I, II or III; wherein R1 is selected from one of H, a halogen atom, diethylamino and a thiophenyl group, R2 is selected from one of H, a halogen atom, diethylamino and a thiophenyl group, and R3 is selected from one of an aryl group, a 9,9'-spirobifluorenyl group, an alkoxy group and a halogen atom. The boron-coordinated salicylhydrazone dual-state emission fluorophore has various structures, good solubility, excellent photophysical properties, extremely high quantum yield, excellent biocompatibility and significant liposome targeting. Moreover, the preparation method of the boron-coordinated salicylhydrazone dual-state emission fluorophore adopts a one-pot two-step method, is simple and convenient, has high yield, is green and environment-friendly, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to the fields of organic synthesis technology and fluorescent materials technology, specifically to a boron-coordinated salicylhydrazone-like dual-state fluorophore, its preparation method, and its application. Background Technology

[0002] Fluorescent small molecules, as key functional materials, have irreplaceable application value in the fields of materials science and biomedicine. Among them, boron-containing fluorophores have attracted much attention due to their unique structural characteristics. Typical examples include boron-dipyrrolemethylene (BODIPY) and its derivatives. Their four-coordinate structure can effectively enhance the rigidity of the π-conjugated system and suppress nonradiative transitions through steric hindrance, thus significantly improving luminescence performance.

[0003] However, traditional BODIPY fluorophores suffer from significant solid-state π-π stacking, leading to severe aggregation-induced fluorescence quenching (ACQ). While materials with aggregation-induced emission (AIE) properties can overcome the ACQ effect, their flexible molecular structures often result in insufficient solution-state fluorescence intensity, and there are few reports of achieving near-100% solid-state quantum yield simultaneously. This technical contradiction makes the development of dual-state emission (DSE) fluorescent molecules with both efficient solid-state and solution-state luminescence properties a significant challenge in the field of materials chemistry. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a boron-coordinated salicylhydrazone-based dual-state fluorophore (BOSHY), its preparation method, and its applications. This boron-coordinated salicylhydrazone exhibits diverse structures, good solubility, excellent photophysical properties, and extremely high quantum yield. It also demonstrates excellent biocompatibility and significant liposome targeting. Furthermore, the preparation method for this boron-coordinated salicylhydrazone employs a one-pot, two-step process, which is simple, convenient, yield-efficient, and environmentally friendly, possessing broad application prospects.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a boron-coordinated salicylhydrazone-like dual-state fluorophore, the structure of which is shown in I, II, or III: ; R1 is selected from H, a halogen atom, diethylamino and thiophene group; R2 is selected from H, a halogen atom, diethylamino and thiophene group; and R3 is selected from aryl, 9,9'-spirodifluorenyl, alkoxy and halogen atom.

[0006] Furthermore, R1 is selected from one of H, F, Cl, Br, I, diethylamino, 2-thienyl, and 3-thienyl; R2 is selected from one of H, F, Cl, Br, I, diethylamino, 2-thienyl, and 3-thienyl; and R3 is selected from one of monocyclic aryl, polycyclic aryl, substituted aryl, 9,9'-spirodifluorenyl, methoxy, F, Cl, Br, and I.

[0007] Furthermore, R1 is selected from one of H, Br, diethylamino and 2-thienyl, R2 is selected from one of H, Br, diethylamino and 2-thienyl, and R3 is selected from one of p-phenyl, 4-trifluoromethylphenyl, 4-(bromomethyl)phenyl, 4-methoxyphenyl, 4-(diphenylamino)phenyl, 9,9'-spirodifluorenyl, F and methoxy.

[0008] Furthermore, the structural formulas of the boron-coordinated salicylhydrazone-like dual-state fluorophores are shown in 1a-j, 2a-b, and 3a-b.

[0009]

[0010] .

[0011] Secondly, the present invention provides a method for preparing boron-coordinated salicylhydrazone-like dual-state fluorophores, the preparation method comprising: 1) Under acidic conditions, the precursor is condensed with hydrazine hydrate in a solvent to generate hydrazone ligands; 2) The hydrazone ligands are coordinated with organoboron compounds in a solvent to generate boron-coordinated salicylhydrazone dual-state fluorophores; In step 1), the precursor is a salicylaldehyde derivative and / or a julonidine derivative; In step 2), the organoboron compound is a boric acid derivative and / or a boron trifluoride diethyl ether complex.

[0012] Furthermore, the preparation method further includes: subjecting the boron-coordinated salicylhydrazone-like dual-state fluorophore to a Stille coupling reaction.

[0013] Furthermore, in step 1), the acidic environment is provided by a Lewis acid.

[0014] Furthermore, the Lewis acid is selected from one or more of acetic acid, p-toluenesulfonic acid, hydrochloric acid, nitric acid, and sulfuric acid.

[0015] Furthermore, the salicylaldehyde derivative is selected from one or more of 2-hydroxybenzaldehyde, 4-bromo-2-hydroxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, 4-(diethylamino)-2-hydroxybenzaldehyde, 2,4-dihydroxybenzaldehyde, and 5-bromo-2-hydroxybenzaldehyde.

[0016] Furthermore, the julonidine derivative is 8-hydroxyjulonidine-9-carboxaldehyde.

[0017] Furthermore, in step 1), the solvent is selected from one or more of acetonitrile, chloroform, 1,2-dichloromethane, toluene, chlorobenzene, o-dichlorobenzene, p-dichlorobenzene and m-dichlorobenzene.

[0018] Furthermore, in step 1), the conditions for the condensation reaction include: being carried out in a metal bath at a temperature of 80~140℃ for 10~15h, and with a stirring rate of 200~2000 rpm.

[0019] Furthermore, in step 1), the molar ratio of the precursor substance to hydrazine hydrate is 2 to 4:1.

[0020] Furthermore, the boric acid derivative is selected from phenylboronic acid, 4-trifluoromethylphenylboronic acid, 4-(bromomethyl)phenylboronic acid, 4-methoxyphenylboronic acid, 4-(diphenylamino)phenylboronic acid, 9,9'-spirodifluorene-2-boronic acid, 3-thiopheneboronic acid, 1,4-phenyldiboronic acid, 2-biphenylboronic acid, and trimethyl borate.

[0021] Furthermore, in step 2), the conditions for the coordination reaction include: a temperature of 105~145℃ and a reflux time of 10~15h.

[0022] Furthermore, in step 2), the molar ratio of the organoboron compound to the hydrazone ligand is 2 to 10:1.

[0023] Furthermore, the conditions for the Stille coupling reaction include: coupling the palladium catalyst, organotin reagent, and the boron-coordinated salicylhydrazone-like dual-state emitting fluorophore in toluene under an argon atmosphere at a temperature of 80-140°C and an oil bath time of 20-30 h.

[0024] Thirdly, the present invention provides a boron-coordinated salicylhydrazone-like dual-state fluorophore prepared by the preparation method described in the second aspect.

[0025] Fourthly, the present invention provides a fluorescent probe, which is prepared from a boron-coordinated salicylhydrazone-like dual-state fluorophore as described in the first or third aspect; The fluorescent probe is specifically designed to target liposomes.

[0026] In the above technical solution, this invention innovatively designs a class of borate salicylhydrazone fluorescent molecules (BOSHY). This molecular system is constructed by one-pot condensation of commercially available salicylaldehyde, salicylaldehyde derivatives, or julonidine derivatives with hydrazine and arylboronic acid. By introducing a stereobonded boron center into the 6,7-fused ring backbone, tetracoordinated boron substitution simultaneously achieves the following technical effects: (1) enhancing molecular rigidity; (2) providing necessary steric hindrance to suppress solid-state aggregation quenching; and (3) maintaining the intramolecular charge transfer (ICT) effect between the amino donor and the boron-coordinated salicylhydrazone nucleus, ensuring the fluorescence intensity in solution. This strategy successfully overcomes the performance limitations of traditional boron dyes and provides a new approach for the development of dual-state emission fluorescent materials.

[0027] The boron-coordinated salicylhydrazone-like two-state fluorophores of this invention exhibit significant DSE characteristics, high quantum yield (up to 95% in solution and up to 74% in the solid state), and a large Stokes shift of up to 7200 cm⁻¹. -1 Furthermore, the introduction of axial aryl groups not only improves solubility but also effectively prevents aggregation, while the distorted molar geometry significantly reduces π-π stacking interactions. Importantly, these BOSHYs exhibit exceptional lipid droplet specificity, extremely high targeting precision, and excellent biocompatibility. These superior properties make the BOSHY platform a promising candidate for fluorescent probes in metabolic disorder research and diagnostic applications.

[0028] Meanwhile, the preparation method of the boron-coordinated salicylhydrazone-type dual-state fluorophores of the present invention is simple to operate, has few steps, and uses readily available raw materials, and has broad application prospects.

[0029] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0030] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 The X-ray crystal structures of the boron-coordinated salicylhydrazone-like dual-state fluorophores of the present invention are shown in (a) as the X-ray crystal structure of 1c, (b) as the X-ray crystal structure of 1f, (c) as the X-ray crystal structure of 1i, and (d) as the X-ray crystal structure of 2b, wherein C atoms are light gray; H atoms are white; N atoms are blue; O atoms are red; B atoms are pink; Br atoms are brown; and F atoms are green. Figure 2Normalized absorption spectra (a) and emission spectra (b, c) of the boron-coordinated salicylhydrazone-type dual-state fluorophores of the present invention in dichloromethane and solid state; (d) is the emission intensity of 1a in a buffer system with pH 1-8 (excitation wavelengths of 440 nm). Figure 3 The transient absorption curve (a) and decay kinetic curve (b) of boron-coordinated salicylhydrazone-like dual-state emitting fluorophore 1b and the transient absorption curve (c) and decay kinetic curve (d) of 1i are shown for the present invention. Figure 4 Cell viability of HeLa cells after 24 hours of treatment with different concentrations of 1i(a) and 1j(b); Figure 5 The images show confocal fluorescence images of HeLa cells stained with DAPI and containing the boron-coordinated salicylhydrazone-like dual-state fluorophore 1i (1.0 μM) of the present invention. (a) Bright field image; (b) Fluorescence image after DAPI staining; (c) Fluorescence image of 1i after 2.5 hours of incubation; (d) Combined image of Figures b and c. Scale bar: 50 μm. Lipid droplet colocalization study of 1i (1.0 µM) in HeLa cells: (e) Bright field plot; (f) Lipi-blue staining fluorescence image; (g) Fluorescence image of 1i; (h) Combined image of f and g; (i) Intensity profile of 1i and Lipi-Blue in the stippled region of HeLa cells, Pearson correlation coefficient Rr = 0.96 ± 0.02; (j) Scatter plot of correlation between Lipi-Blue and 1i intensities, Pearson correlation coefficient Rr = 0.98 ± 0.01, overlap coefficient R = 0.99 ± 0.01; Scale bar: 50 µm; (k) Cell viability after co-incubating HeLa cells with 1i at different concentrations for 24 hours; Figure 6 The images show confocal fluorescence images of HeLa cells stained with DAPI and containing the boron-coordinated salicylhydrazone-like dual-state fluorophore 1j (1.0 μM) of the present invention. (a) Bright field image; (b) Fluorescence image after DAPI staining; (c) Fluorescence image of 1j after incubation for 2.5 hours; (d) Combined image of Figures b and c. Scale bar: 50 μm. Study on lipid droplet colocalization of 1j (1.0 µM) in HeLa cells: (e) bright field plot; (f) Lipi-blue staining fluorescence image; (g) 1j fluorescence image; (h) merged image of (f) and (g); (i) intensity profile of 1j and Lipi-Blue in the underlined region of HeLa cells, Pearson correlation coefficient Rr = 0.88 ± 0.05; (j) scatter plot of correlation between Lipi-Blue and 1j intensities, Pearson correlation coefficient Rr = 0.93 ± 0.02, overlap coefficient R = 0.95 ± 0.04, scale bar: 50 µm. Detailed Implementation

[0031] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0032] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0033] In a first aspect, the present invention provides a boron-coordinated salicylic hydrazone-like dual-state fluorophore, the structure of which is shown in I, II, or III. ; R1 is selected from H, a halogen atom, diethylamino and thiophene group; R2 is selected from H, a halogen atom, diethylamino and thiophene group; and R3 is selected from aryl, 9,9'-spirodifluorenyl, alkoxy and halogen atom.

[0034] This invention utilizes the NO2 tridentate coordination mode of salicylaldehyde hydrazone ligands in boron-coordinated salicylaldehyde hydrazone fluorophores. Through asymmetric coordination engineering between the ligand molecule and boric acid, an asymmetric topological dye containing a six- or seven-membered ring at the boron center is constructed, and ring strain is used to modulate the excited-state structural relaxation energy. The substituents at the boron sites protrude vertically outward from the π-surface, and due to the structural strain caused by the tetrahedral geometry of the ring fusion, the ligand exhibits a slight bending state. This structure effectively solves the solubility problem of large conjugated planar structures. Furthermore, through the synergistic effect of the ligand's electronic push-pull effect and steric hindrance, the mutual repulsion between solubility and solid-state luminescence efficiency is overcome. Compared with traditional bidentate systems, NO2-type tridentate ligands substituted with strong electron-donating groups such as diethylamino can generate a significant ring strain effect by forming a six- or seven-membered chelate ring, which not only enhances radiative transitions but also suppresses non-radiative decay. The structures of this type of complex are diverse and can be achieved by modifying the substituents of salicylaldehyde or boric acid derivatives or by conducting post-modification derivatization reactions.

[0035] In a preferred embodiment of the present invention, R1 is selected from one of H, F, Cl, Br, I, diethylamino, 2-thienyl and 3-thienyl, R2 is selected from one of H, F, Cl, Br, I, diethylamino, 2-thienyl and 3-thienyl, and R3 is selected from one of monocyclic aryl, polycyclic aryl, substituted aryl, 9,9'-spirodifluorenyl, methoxy, F, Cl, Br and I.

[0036] In a preferred embodiment of the present invention, R1 is selected from H, Br, diethylamino and 2-thienyl, R2 is selected from H, Br, diethylamino and 2-thienyl, and R3 is selected from p-phenyl, 4-trifluoromethylphenyl, 4-(bromomethyl)phenyl, 4-methoxyphenyl, 4-(diphenylamino)phenyl, 9,9'-spirodifluorenyl, F and methoxy.

[0037] In a preferred embodiment of the present invention, the structure of the boron-coordinated salicylhydrazone-like dual-state fluorophore is shown in 1a-j, 2a-b, and 3a-b.

[0038]

[0039] .

[0040] Secondly, the present invention provides a method for preparing boron-coordinated salicylhydrazone-like dual-state fluorophores, the preparation method comprising: 1) Under acidic conditions, the precursor is condensed with hydrazine hydrate in a solvent to generate hydrazone ligands; 2) The hydrazone ligands are coordinated with organoboron compounds in a solvent to generate boron-coordinated salicylhydrazone dual-state fluorophores; In step 1), the precursor is a salicylaldehyde derivative and / or a julonidine derivative; In step 2), the organoboron compound is a boric acid derivative and / or a boron trifluoride diethyl ether complex.

[0041] This invention utilizes a one-pot, two-step method to synthesize a series of highly soluble BOSHY fluorophores by condensing salicylaldehyde, salicylaldehyde derivatives, or julonidine derivatives with hydrazine hydrate under acidic conditions to obtain ligands, followed by coordination with boron trifluoride, various boric acids, or boric acid derivatives. These fluorophores exhibit excellent photophysical properties. For example, introducing strong electron-donating groups or increasing the conjugation system on the framework results in a significant red shift in the absorption spectrum of the solution, particularly with a liquid-state fluorescence quantum yield as high as 95%. The solid-state quantum yield can also reach as high as 74%. Cell experiments show that this series of dyes exhibits excellent biocompatibility (cell viability >90%) and significant liposome targeting (co-localization coefficient with commercial dyes reaches 0.96). The molecular design paradigm established by this invention and the structure-activity relationship it demonstrates provide a theoretical basis and practical reference for the development of novel BOPPY-based biological probes, with broad application prospects.

[0042] In a preferred embodiment of the present invention, the preparation method further includes: subjecting the boron-coordinated salicylhydrazone-like dual-state fluorophore to a Stille coupling reaction.

[0043] In a preferred embodiment of the present invention, in step 1), the acidic environment is provided by a Lewis acid.

[0044] In a preferred embodiment of the present invention, the Lewis acid is selected from one or more of acetic acid, p-toluenesulfonic acid, hydrochloric acid, nitric acid, and sulfuric acid.

[0045] In a preferred embodiment of the present invention, the salicylaldehyde derivative is selected from one or more of 2-hydroxybenzaldehyde, 4-bromo-2-hydroxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, 4-(diethylamino)-2-hydroxybenzaldehyde, 2,4-dihydroxybenzaldehyde, and 5-bromo-2-hydroxybenzaldehyde.

[0046] In a preferred embodiment of the present invention, the julonidine derivative is 8-hydroxyjulonidine-9-carboxaldehyde.

[0047] In a preferred embodiment of the present invention, in step 1), the solvent is selected from one or more of acetonitrile, chloroform, 1,2-dichloromethane, toluene, chlorobenzene, o-dichlorobenzene, p-dichlorobenzene and m-dichlorobenzene; In a preferred embodiment of the present invention, in step 1), the conditions for the condensation reaction include: being carried out in a metal bath at a temperature of 80~140℃ for 10~15h and a stirring rate of 200~2000 rpm.

[0048] In a preferred embodiment of the present invention, in step 1), the molar ratio of the precursor substance to hydrazine hydrate is 2 to 4:1.

[0049] In a preferred embodiment of the present invention, the boric acid derivative is selected from phenylboronic acid, 4-trifluoromethylphenylboronic acid, 4-(bromomethyl)phenylboronic acid, 4-methoxyphenylboronic acid, 4-(diphenylamino)phenylboronic acid, 9,9'-spirodifluorene-2-boronic acid, 3-thiopheneboronic acid, 1,4-phenyldiboronic acid, 2-biphenylboronic acid, and trimethyl borate.

[0050] In a preferred embodiment of the present invention, in step 2), the conditions for the coordination reaction include: a temperature of 105~145℃ and a reflux time of 10~15h.

[0051] In a preferred embodiment of the present invention, in step 2), the molar ratio of the organoboron compound to the hydrazone ligand is 2 to 10:1.

[0052] In a preferred embodiment of the present invention, the conditions for the Stille coupling reaction include: coupling palladium catalyst, organotin reagent and boron-coordinated salicylhydrazone-like dual-state emitting fluorophore in toluene under an argon atmosphere at a temperature of 80-140°C and an oil bath time of 20-30 h.

[0053] Thirdly, the present invention provides a boron-coordinated salicylhydrazone-like dual-state fluorophore prepared by the preparation method described in the second aspect.

[0054] Fourthly, the present invention provides a fluorescent probe, which is prepared from a boron-coordinated salicylhydrazone-like dual-state fluorophore as described in the first or third aspect; The fluorescent probe is specifically designed to target liposomes.

[0055] In a preferred embodiment of the present invention, the room temperature is 15~30°C.

[0056] The present invention will be described in detail below through examples. In the following examples, the pharmaceuticals and agents are all conventional commercially available products.

[0057] Example 1 The specific synthetic route for 1a is as follows:

[0058] In a 100 mL pressure-resistant reaction flask, 4-(diethylamino)-2-hydroxybenzaldehyde (435 mg, 2 mmol) was dissolved in 20 mL of anhydrous chlorobenzene. Hydrazine hydrate (60 μL, 1 mmol) and glacial acetic acid (5 drops) were added sequentially, and the system was heated and stirred in a metal bath at 105 °C for 12 hours. After confirming complete conversion of the starting material by TLC, phenylboronic acid (1.1 g, 9 mmol, 9 equiv.) was added, and the reaction system was heated to 130 °C and refluxed for 12 hours. After the reaction was complete, the mixture was cooled to room temperature and extracted with dichloromethane (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was purified by column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v) to give an orange-yellow solid product 1a (239 mg, 51%).

[0059] 1 H NMR (400 MHz, CDCl3) δ 8.30 (s, 1H), 7.32 – 7.29 (m, 3H), 7.15 –7.09 (m, 4H), 6.92 (d, J = 8.8 Hz, 1H), 6.55 (d, J = 2.6 Hz, 1H), 6.28 – 6.24 (m,2H), 6.17 (dd, J = 8.8, 2.6 Hz, 1H), 3.42 – 3.35 (m, 8H), 1.19 (q, J = 6.8 Hz, 12H). 13 C NMR (101 MHz, CDCl3) δ 161.5, 160.7, 156.5, 155.3, 153.6, 152.4,137.3, 132.9, 131.9, 128.1, 127.2, 126.8, 111.0, 105.5, 104.7, 104.3, 102.2,98.9, 45.0, 44.7, 12.9. 11 B NMR (128 MHz, CDCl3) δ 4.49 (brs). HRMS (ESI) calcdfor C 28 H 34 BN4O2[M + H] + : 469.4160, found 469.4139. Example 2 Synthesis of 1b: In a 100 mL high-pressure reaction flask, 4-(diethylamino)-2-hydroxybenzaldehyde (386 mg, 2 mmol) was dissolved in 20 mL of chlorobenzene. Hydrazine hydrate (60 μL, 1 mmol) and glacial acetic acid (5 drops) were added sequentially, and the reaction was heated in a metal bath at 105 °C for 12 h. After TLC monitoring showed complete conversion of the starting material, 4-trifluoromethylphenylboronic acid (760 mg, 4 mmol, 4 equiv.) was added, and the reaction mixture was heated to 130 °C and stirred under reflux for 12 h. After the reaction was complete, the mixture was cooled to room temperature and extracted with dichloromethane (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was purified by column chromatography (eluent: dichloromethane / petroleum ether = 1:2, v / v) to give an orange-yellow solid product 1b (280 mg, 52%).

[0060] 1 H NMR (400 MHz, CDCl3) δ 8.33 (d, J = 4.9 Hz, 1H), 7.44 – 7.39 (m, 2H), 7.36 (d, J = 7.9 Hz, 2H), 7.29 (s, 1H), 7.16 (dd, J = 8.9, 1.3 Hz, 1H), 6.95 –6.91 (m, 1H), 6.55 (d, J = 2.5 Hz, 1H), 6.29 (dd, J = 9.0, 2.1 Hz, 1H), 6.23 (d, J = 2.4 Hz, 2H), 3.42 – 3.36 (m, 8H), 1.19 (q, J = 7.1 Hz, 12H). 13 C NMR (101 MHz, CDCl3) δ 161.2, 160.5, 156.3, 155.5, 153.6, 152.5, 137.5, 133.1 (q, J = 6.3 Hz,FC), 132.1, 125.2 (q, J = 30.4 Hz, FC), 124.0 (q, J = 3.8 Hz, FC), 123.5,105.9, 105.7, 104.7, 104.5, 102.3, 102.2, 100.9, 45.1, 44.8, 12.9, 12.8. 11BNMR (128 MHz, CDCl3) δ 3.82 (brs). 19 F NMR (376 MHz, CDCl3) δ -62.34 (s). HRMS(ESI) calcd for C 29 H 33 BF3N4O2[M + H] + : 537.2649, found 537.2661. Example 3 Synthesis of 1c: In a 100 mL pressure-resistant reaction flask, 4-(diethylamino)-2-hydroxybenzaldehyde (435 mg, 2 mmol) was dissolved in 20 mL of anhydrous chlorobenzene. Hydrazine hydrate (60 μL, 1 mmol) and glacial acetic acid (5 drops) were added sequentially, and the system was heated and stirred in a metal bath at 105 °C for 12 h. After confirming complete conversion of the starting material by TLC, 4-(bromomethyl)phenylboronic acid (430 mg, 2 mmol, 1 equiv.) was added, and the reaction system was heated to 130 °C and refluxed for 12 h. After the reaction was complete, the mixture was cooled to room temperature and extracted with dichloromethane (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was purified by column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v) to give an orange-yellow solid product 1c (250 mg, 45%).

[0061] 1 H NMR (400 MHz, CDCl3) δ 8.33 (s, 1H), 7.32 – 7.28 (m, 3H), 7.19 –7.13 (m, 3H), 6.93 (d, J = 8.9 Hz, 1H), 6.56 (s, 1H), 6.31 – 6.21 (m, 3H), 4.49 (s, 2H), 3.44 – 3.36 (m, 8H), 1.20 (td, J = 7.4, 2.3 Hz, 12H). 13 C NMR (126 MHz, CDCl3) δ 161.3, 160.5, 156.0, 155.3, 153.4, 137.3, 135.3, 132.8, 132.1,127.5, 105.6, 104.4, 98.7, 46.9, 44.9, 12.7. 11 B NMR (128 MHz, CDCl3) δ 4.11.HRMS (ESI) calcd for C 29H 35 BBrN4O2[M + H] + : 561.2036, found 561.2052. Example 4 Synthesis of product 1d: In a 100 mL pressure-resistant reaction flask, 4-(diethylamino)-2-hydroxybenzaldehyde (435 mg, 2 mmol) was dissolved in 20 mL of anhydrous chlorobenzene. Hydrazine hydrate (60 μL, 1 mmol) and glacial acetic acid (5 drops) were added sequentially, and the system was heated and stirred in a metal bath at 105 °C for 12 h. After confirming complete conversion of the starting material by TLC, 4-methoxyphenylboronic acid (608 mg, 4 mmol, 2 equiv.) was added, and the reaction system was heated to 130 °C and refluxed for 12 h. After the reaction was complete, the mixture was cooled to room temperature and extracted with dichloromethane (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product was purified by column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v) to give an orange-yellow solid product 1d (194 mg, 39%).

[0062] 1 H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H), 7.22 (d, J = 8.5 Hz, 2H), 7.14(d, J = 8.8 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 6.69 (d, J = 8.6 Hz, 2H), 6.55 (s,1H), 6.28 – 6.23 (m, 2H), 6.20 (s, 1H), 3.70 (s, 3H), 3.43 – 3.34 (m, 8H), 1.19 (q, J = 7.1 Hz, 12H). 13 C NMR (126 MHz, Chloroform- d ) δ 161.6, 160.7, 158.5,155.2, 153.3, 137.3, 133.0, 132.8, 112.8, 105.4, 104.5, 98.8, 54.8, 44.9,12.8. 11 B NMR (128 MHz, CDCl3) δ 4.20. HRMS (ESI) calcd for C 29 H 36 BN4O3[M + H]+ :499.2880, found 499.2899. Example 5 Synthesis of 1e: In a 100 mL pressure-resistant reaction flask, 4-(diethylamino)-2-hydroxybenzaldehyde (435 mg, 2 mmol) was dissolved in 20 mL of anhydrous chlorobenzene. Hydrazine hydrate (60 μL, 1 mmol) and glacial acetic acid (5 drops) were added sequentially, and the system was heated and stirred in a metal bath at 105 °C for 12 h. After confirming complete conversion of the starting material by TLC, 4-(diphenylamino)phenylboronic acid (578 mg, 2 mmol, 1 equiv.) was added, and the reaction system was heated to 130 °C and refluxed for 12 h. After the reaction was complete, the mixture was cooled to room temperature and extracted with dichloromethane (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was purified by column chromatography (eluent: ethyl acetate / petroleum ether = 1:3, v / v) to give an orange-yellow solid product 1e (460 mg, 72%).

[0063] 1 H NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 7.35 (s, 1H), 7.17 (d, J = 2.2Hz, 2H), 7.16 – 7.14 (m, 3H), 7.14 (d, J = 1.7 Hz, 1H), 7.11 (d, J = 12.9 Hz, 1H), 7.03 (d, J = 1.3 Hz, 2H), 7.01 (d, J = 1.2 Hz, 2H), 6.98 (d, J = 8.9 Hz, 1H), 6.94 – 6.88 (m, 2H), 6.85 – 6.80 (m, 2H), 6.51 (d, J = 2.4 Hz, 1H), 6.30 – 6.24(m, 2H), 6.20 (d, J = 9.1 Hz, 1H), 3.43 – 3.34 (m, 8H), 1.19 (td, J = 7.1, 5.5Hz, 12H). 13C NMR (126 MHz, CDCl3) δ 161.4, 160.6, 156.6, 155.1, 153.2, 152.3,148.1, 145.9, 137.2, 132.7, 129.2, 128.9, 124.0, 123.0, 122.0, 110.8, 105.4,104.5, 104.1, 102.0, 98.8, 44.9, 44.5, 12.8. 11 B NMR (128 MHz, CDCl3) δ 4.27(brs). HRMS (ESI) calcd for C 40 H 43 BN5O2[M + H] + : 636.3510, found 636.3520. Example 6 Synthesis of 1f: In a 100 mL pressure-resistant reaction flask, 4-(diethylamino)-2-hydroxybenzaldehyde (435 mg, 2 mmol) was dissolved in 20 mL of anhydrous chlorobenzene. Hydrazine hydrate (60 μL, 1 mmol) and glacial acetic acid (5 drops) were added sequentially, and the system was heated and stirred in a metal bath at 105 °C for 12 h. After confirming complete conversion of the starting material by TLC, 9,9-spirodifluorene-2-boric acid (720 mg, 2 mmol, 1 equiv.) was added, and the reaction system was heated to 130 °C and refluxed for 12 h. After the reaction was complete, the mixture was cooled to room temperature and extracted with dichloromethane (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product was purified by column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v) to give an orange solid product 1f (450 mg, 64%).

[0064] 1 H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.73 – 7.60 (m, 5H), 7.32 –7.27 (m, 2H), 7.25 – 7.17 (m, 2H), 7.13 – 7.09 (m, 2H), 7.04 (td, J = 7.5, 1.1Hz, 1H), 6.98 (td, J = 7.5, 1.1 Hz, 1H), 6.88 (dd, J = 7.5, 1.1 Hz, 1H), 6.72 –6.65 (m, 3H), 6.60 (d, J= 7.6 Hz, 1H), 6.41 (d, J = 7.6 Hz, 1H), 6.24 (dd, J =9.0, 2.5 Hz, 1H), 6.19 – 6.12 (m, 2H), 3.35 (q, J = 7.3 Hz, 8H), 1.12 (dd, J =15.3, 7.1 Hz, 12H). 13 C NMR (126 MHz, CDCl3) δ 160.4, 153.0, 149.3, 142.6,141.5, 140.2, 136.9, 132.8, 131.5, 128.0, 127.5, 127.3, 127.2, 126.9, HRMS (ESI) calcd for C 47 H 44 BN4O2[M + H] + : 707.3557, found707.3586. Example 7 Synthesis of 1 g: In a 100 mL pressure-resistant reaction flask, 4-(diethylamino)-2-hydroxybenzaldehyde (435 mg, 2 mmol) was dissolved in 20 mL of anhydrous chlorobenzene. Hydrazine hydrate (60 μL, 1 mmol) and glacial acetic acid (5 drops) were added sequentially, and the system was heated and stirred in a metal bath at 105 °C for 12 h. After confirming complete conversion of the starting material by TLC, 1,4-phenylenediboric acid (166 mg, 1 mmol, 0.5 equiv.) was added, and the reaction system was heated to 130 °C and refluxed for 12 h. After the reaction was complete, the mixture was cooled to room temperature and extracted with dichloromethane (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was purified by column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v) to give 1 g (171 mg, 20%) of orange solid product.

[0065] 1 H NMR (400 MHz, CDCl3) δ 8.22 (s, 1H), 7.22 (s, 1H), 7.07 (d, J = 8.8Hz, 1H), 7.01 (s, 2H), 6.87 (d,J = 8.9 Hz, 1H), 6.47 (d, J = 2.4 Hz, 1H), 6.25 –6.12 (m, 3H), 3.36 (tt, J = 7.2, 3.4 Hz, 8H), 1.17 (d, J = 7.3 Hz, 12H). 13 C NMR (126 MHz, CDCl3) δ 161.6, 160.7, 155.0, 153.1, 137.2, 132.6, 130.6, 105.2,104.6, 98.8, 44.8, 12.8. 11 B NMR (128 MHz, CDCl3) δ 4.68 (brs). HRMS (ESI)calcd for C 50 H 61 B2N8O4[M + H] + : 859.5027, found 859.5041. Example 8 Synthesis in 1 h: In a 100 mL pressure-resistant reaction flask, 4-(diethylamino)-2-hydroxybenzaldehyde (435 mg, 2 mmol) was dissolved in 20 mL of anhydrous chlorobenzene. Hydrazine hydrate (60 μL, 1 mmol) and glacial acetic acid (5 drops) were added sequentially, and the system was heated and stirred in a metal bath at 105 °C for 12 h. After confirming complete conversion of the starting material by TLC, N,N-diisopropylethylamine (DIPEA, 3 mL) and boron trifluoride diethyl ether complex (BF3·OEt2, 3 mL) were added sequentially under argon protection. The reaction system was heated to 130 °C and refluxed for 12 h. After the reaction was complete, the mixture was cooled to room temperature, extracted with dichloromethane (3 × 20 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product was purified by column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v) to give an orange solid product 1h (220 mg, 40%).

[0066] 1 H NMR (400 MHz, CDCl3) δ 11.09 (s, 1H), 8.85 (s, 1H), 8.16 (d, J = 4.0Hz, 1H), 7.16 (dd, J = 11.9, 8.9 Hz, 2H), 6.35 (dd, J= 9.0, 2.4 Hz, 1H), 6.27 –6.20 (m, 2H), 3.45 – 3.37 (m, 8H), 1.24 – 1.17 (m, 12H). 13 C NMR (126 MHz, CDCl3) δ 161.4, 160.2, 155.7, 155.3, 134.8, 133.1, 106.6, 104.7, 98.1, 45.1,12.7. 11 B NMR (128 MHz, CDCl3) δ 0.72. 19 F NMR (376 MHz, CDCl3) δ -134.62 (dd, J =36.8, 15.8 Hz, 1F). HRMS (ESI) calcd for C 22 H 28 BFN4O2[M + H] + : 411.2368, found411.2375. Example 9 Synthesis of 1i: In a 100 mL pressure-resistant reaction flask, 8-hydroxyjulonidine-9-carboxaldehyde (435 mg, 2 mmol) was dissolved in 20 mL of anhydrous chlorobenzene. Hydrazine hydrate (60 μL, 1 mmol) and glacial acetic acid (5 drops) were added sequentially, and the system was heated and stirred in a metal bath at 105 °C for 12 h. After confirming complete conversion of the starting material by TLC, 4-trifluoromethylphenylboronic acid (760 mg, 4 mmol, 4 equiv.) was added, and the reaction system was heated to 130 °C and refluxed for 12 h. After the reaction was complete, the mixture was cooled to room temperature and extracted with dichloromethane (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was purified by column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v) to give an orange-yellow solid product 1i (300 mg, 52%).

[0067] 1 H NMR (500 MHz, CDCl3) δ 8.21 (s, 1H), 7.34 – 7.30 (m, 4H), 7.20 (s,1H), 6.70 (s, 1H), 6.51 (s, 1H), 3.28 – 3.20 (m, 8H), 3.00 – 2.90 (m, 2H),2.81 – 2.75 (m, 1H), 2.64 (t, J = 6.5 Hz, 3H), 2.59 (t, J= 6.3 Hz, 2H), 1.97 (t, J = 6.1 Hz, 2H), 1.93 – 1.85 (m, 6H). 13 C NMR (126 MHz, CDCl3) δ 156.1, 156.0,153.3, 153.1, 150.7, 147.6, 133.4, 131.6, 128.6, 128.3 (q, J = 31.5 Hz, FC), 125.0 (q, J = 273.4 Hz, FC), 123.8 (q, J = 2.5 Hz, FC), 115.0, 113.6, 110.6,110.1, 106.9, 104.2, 50.4, 50.1, 50.0, 49.8, 27.2, 27.0, 21.8, 21.7, 21.6,21.3, 20.6, 20.2. 11 B NMR (128 MHz, CDCl3) δ 4.42 (brs). 19 F NMR (376 MHz, CDCl3)δ -62.16 (s). HRMS (ESI) calcd for C 33 H 33 BF3N4O2[M + H] + : 585.2643, found585.2651. Example 10 Synthesis of 1j: In a 100 mL pressure-resistant reaction flask, 8-hydroxyjulonidine-9-carboxaldehyde (435 mg, 2 mmol) was dissolved in 20 mL of anhydrous chlorobenzene, followed by the addition of hydrazine hydrate (60 μL, 1 mmol) and glacial acetic acid (5 drops). The reaction system was heated and stirred in a metal bath at 105 °C for 12 hours. After complete conversion of the starting material was confirmed by TLC, trimethyl borate (1.1 mL, 4 mmol, 2 equiv.) was added, and the reaction system was heated to 130 °C and refluxed for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and extracted with dichloromethane (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product was purified by column chromatography (eluent: ethyl acetate / petroleum ether = 1:1, v / v) to give an orange-yellow solid product 1j (235 mg, 49%).

[0068] 1H NMR (500 MHz, CDCl3) δ 8.09 (s, 1H), 7.40 (s, 1H), 6.71 (s, 2H), 3.30 – 3.27 (m, 4H), 3.21 – 3.17 (m, 4H), 3.09 (s, 3H), 2.95 – 2.89 (m, 2H),2.83 – 2.69 (m, 2H), 2.66 (t, J = 6.4 Hz, 3H), 1.97 – 1.89 (m, 9H). 13 C NMR (126MHz, CDCl3) δ 157.0, 155.3, 155.2, 153.8, 150.0, 133.6, 128.3, 114.8, 113.4,110.7, 110.6, 106.8, 104.4, 50.4, 50.2, 50.1, 49.8, 49.1, 27.4, 27.3, 22.0,21.9, 21.6, 21.4, 20.9, 20.5. HRMS (ESI) calcd for C 27 H 32 BN4O3[M + H] + :471.2567, found 471.2586. Example 11 The specific synthesis routes for 2a and 2b are as follows:

[0069] In a 250 mL round-bottom flask, 4-(diethylamino)-2-hydroxybenzaldehyde (386 mg, 2 mmol) and salicylaldehyde hydrazone (272 mg, 2 mmol) were dissolved in 20 mL of anhydrous 1,2-dichloroethane. The mixture was stirred in an oil bath at 50 °C for 1 hour. After the complete conversion of the starting material was confirmed by TLC, the temperature was raised to 120 °C and 4-trifluoromethylphenylboronic acid (950 mg, 5 mmol) was added. The reaction was continued until the TLC showed complete reaction. After cooling to room temperature, the mixture was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 1:2, v / v) to obtain an orange-yellow solid 2a (214 mg, 23%) and an orange-red solid 2b (485 mg, 52%). 2a: 1 H NMR (400 MHz, CDCl3) δ 8.58 (s, 1H), 7.52 (t, J= 7.4 Hz, 1H),7.42 – 7.37 (m, 6H), 7.11 (d, J = 8.4 Hz, 1H), 7.00 – 6.94 (m, 2H), 6.55 (t, J =2.2 Hz, 1H), 6.28 – 6.25 (m, 1H), 3.41 (q, J = 7.1 Hz, 4H), 1.21 (t, J = 7.1 Hz,6H). 13 C NMR (126 MHz, CDCl3) δ 162.0, 159.5, 158.8, 155.4, 153.4, 138.3,137.7, 132.4, 131.5, 129.3 (q, J = 31.5 Hz, F-C), 129.0, 125.0 (q, J = 273.4 Hz,F-C), 124.3 (q, J = 2.5 Hz, F-C), 120.3, 119.8, 114.7, 110.7, 105.5, 102.1,45.2, 13.1. 11 B NMR (128 MHz, CDCl3) δ -3.78 (brs). 19 F NMR (376 MHz, CDCl3) δ -62.42 – -62.44 (m, 3F). HRMS (ESI) calcd for C 25 H 24 BF3N3O2[M + H] + : 466.1914,found 466.1921. 2b: 1 H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.56 (s, 1H), 7.42 (t, J =7.7 Hz, 1H), 7.38 – 7.29 (m, 5H), 7.24 (d, J = 8.7 Hz, 1H), 7.16 (d, J = 7.8 Hz,1H), 6.89 (t, J = 7.4 Hz, 1H), 6.47 (d, J= 8.9 Hz, 1H), 6.31 (s, 1H), 3.42 (d, J =7.1 Hz, 4H), 1.26 (s, 3H), 1.22 – 1.20 (m, 3H). 13 C NMR (126 MHz, CDCl3) δ160.8, 159.5, 156.9, 155.1, 135.5, 134.3, 133.5, 132.0, 124.0, 122.0, 121.0,119.8, 45.9, 29.7, 12.5. 11 B NMR (128 MHz, CDCl3) δ 4.02. HRMS (ESI) calcd forC 25 H 24 BF3N3O2[M + H] + : 466.1914, found 466.1920. Example 12 The specific synthetic route for 3a is as follows: In a 100 mL pressure-resistant reaction flask, 4-bromo-2-hydroxybenzaldehyde (402 mg, 2 mmol) was dissolved in 20 mL of anhydrous chlorobenzene. Hydrazine hydrate (60 μL, 1 mmol) and glacial acetic acid (5 drops) were added sequentially, and the mixture was heated and stirred in a metal bath at 105 °C for 12 hours. After complete conversion of the starting material was confirmed by TLC, potassium phosphate (K3PO4, 690 mg, 3.0 mmol, 1.5 equiv.) and phenylboronic acid (1.1 g, 9.0 mmol, 4.5 equiv.) were added, and the reaction mixture was heated to 130 °C and refluxed for 12 hours. After the reaction was complete, the mixture was cooled to room temperature and extracted with dichloromethane (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was purified by column chromatography (eluent: dichloromethane / petroleum ether = 2:3, v / v) to give a yellow solid product 3a (207 mg, 43%).

[0070] 1 H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 7.67 (s, 1H), 7.50 (s, 1H), 7.33 (d, J = 1.8 Hz, 1H), 7.26 (d, J = 2.0 Hz, 1H), 7.23 – 7.10 (m, 6H), 7.08 –7.00 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 160.3, 159.8, 159.2, 156.2, 136.5,133.4, 132.0, 131.7, 129.6, 127.6, 127.5, 125.1, 123.8, 123.4, 123.1, 119.4,112.8. 11 B NMR (128 MHz, CDCl3) δ 4.20. HRMS (ESI) calcd for C 20 H 14 BBr2N2O2[M +H] + : 482.9515, found 482.9526. Example 13 The specific synthetic route for 3b is as follows:

[0071] In a 50 mL Shrek tube, compound 3a (100 mg, 0.21 mmol), tetrakis(triphenylphosphine)palladium (10 mg, 0.0086 mmol, 4 mol%), and tributyl(2-thienyl)tin reagent (157 mg, 0.42 mmol, 2 equiv.) were dissolved in toluene (20 mL). After three cycles of vacuum purging and argon purging, the reaction was continuously stirred in an oil bath at 130 °C for 24 hours, and the reaction progress was monitored in real time by thin-layer chromatography (TLC). After the starting materials were completely converted, the reaction system was cooled to room temperature and extracted with dichloromethane (3 × 20 mL). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v) to give an orange-yellow solid product 3b (88 mg, 85%).

[0072] 1 H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 7.69 (s, 1H), 7.66 (s, 1H), 7.48 – 7.37 (m, 6H), 7.28 – 7.25 (m, 4H), 7.20 – 7.11 (m, 6H). 13C NMR (101MHz, CDCl3) δ 160.3, 159.4, 159.3, 155.8, 143.7, 142.7, 140.4, 139.4, 136.4,131.9, 131.8, 128.5, 128.4, 127.6, 127.4, 127.4, 126.8, 125.6, 125.0, 119.5,118.3, 117.7, 117.2, 115.7, 113.0. 11 B NMR (128 MHz, CDCl3) δ 4.94. HRMS (ESI)calcd for C 28 H 20 BN2O2S2[M + H] + : 491.1059, found 491.1070. Test Example 1 Single crystals of 1c, 1f, 1i, and 2b were successfully cultured in a dichloromethane / n-hexane mixed solvent system using a slow solvent evaporation method. Their molecular structures were precisely resolved by X-ray single-crystal diffraction analysis (Bruker D8 Venture diffractometer). The crystal structure diagrams of 1c, 1f, 1i, and 2b are shown below. Figure 1 As shown, the analytical parameters of the single-crystal X-ray diffraction structures of 1c, 1f, 1i and 2b are shown in Table 1.

[0073] Table 1

[0074] From the data in Table 1 and Figure 1 As can be seen from the crystal structure diagram, the boron-coordinated salicylhydrazone-like dual-state fluorophore of the present invention has three key features: (1) significantly consistent N-N (1.40-1.41 Å) and B-N (1.58-1.59 Å) bond lengths, confirming robust coordination geometry; (2) the slender B-N bonds, compared to rigid BODIPY analogs (approximately 1.50 Å), demonstrate flexible bond connections; and (3) a significantly different tetrahedral coordination environment at the boron center. The molecular structure comprises four rigid components: a central seven-membered chelate ring, an adjacent six-membered ring (showing a 0.02-0.03 Å longer BO bond than the seven-membered para-part), and two peripheral benzene rings. The dihedral angles between the benzene rings on the salicylhydrazone segments exhibit significant variations (134.9°~140.2°), indicating substituent-dependent π-conjugation. All particles crystallize in the form of a racemic mixture, with sp... 3The hybrid boron centers employ a distorted tetrahedral geometry (THCDA = 0.85–0.92), with the O₂-B₁-N₁ structure exhibiting particular sensitivity to substitution reactions (109.2° in 1c and 118.8° in 2b). Notably, the near-orthogonal arrangement (80.94°–85.96°) between the axial and equatorial benzene rings minimizes coarse π-packing. For example, the dihedral angles between the phenyl ring of the axial boron atom and the adjacent benzoyne ring of the newly formed six-membered ring are 81.45° in 1c, 85.96° in 1f, 80.94° in 1i, and 83.90° in 2b.

[0075] Test Example 2 We systematically studied the photophysical properties of 1a-j, 2a-b, and 3a-b in three solvents of different polarities: toluene, dichloromethane, and acetonitrile, as well as their photophysical properties in the solid state. The test results are shown in Tables 2-3 and 3a-j. Figure 2 (a)-(c).

[0076] Table 2. Photophysical data of 1a-j, 2a-b and 3a-b in different solvents at room temperature (15-30℃).

[0077] Table 3. Photophysical data of 1a-j, 2a-b and 3a-b at room temperature (15-30℃)

[0078] From Tables 2-3 and Figure 2 Data from (a)-(c) show that these BOSHYs exhibit a broad absorption range and ultra-bright fluorescence. These BOSHYs display a broad absorption band (A... abs =396-500 nm), and also has a very high molar extinction coefficient (ε). max ≈3.7-4.9×10 -4 M -1 cm -1 For example, 1a exhibits significant absorption characteristics in toluene. It reaches a peak at 444 nm with a wavelength of 3.81 × 10⁻⁶. -4 M -1 cm -1 It exhibits a high molar extinction coefficient. Furthermore, 1a emits at 538 nm in toluene, displaying a molar extinction coefficient of approximately 4900 cm⁻¹. -1 The relatively large Stokes shift is greater than that of typical BODIPY dyes. Similar spectral shapes are also observed in other BOSHYS dyes. The emission maximum (λ) em A significant redshift (Δ) compared to absorption. λ =65-160nm), thus producing a large Stokes shift (3500-7200cm).-1 This suggests the presence of significant structural relaxation in the excited state, likely due to the ICT effect facilitated by the donor-acceptor structure of the Boshy core.

[0079] In solution, electron-donating substituents on boron have a smaller effect on hyperchromic emission spectra than electron-depleting substituents. For example, electron-donating groups (such as 1b) shift emission further towards 570 nm, while electron-donating groups (such as NMe2 in 1e) result in a hyperchromic emission shift (Δλ). em =+12 nm υs (1a). In contrast, the electron-donating groups attached to the corresponding positions exhibit a more significant redshift effect than the substituents attached to the aromatic ring of the salicylaldehyde hydrazone moiety. Following the formation of an additional ethyl stationary group on the salicylyl moiety, a large bath color shift of 22 nm absorption and 48 nm emission of 1j was observed in dichloromethane compared to 1a. The absorption and deep red emission of 1i with the F group in acetonitrile are concentrated at 463 / 593 nm, and most of the BOSHYs in this invention exhibit excellent fluorescence properties in toluene (Ф). F =63-95%), among which 1b (95%) and 1i (89%) stand out. This performance surpasses that of classic BODIPYs (typically Ф). F =60-80%, close to the BOPHY benchmark (Ф) F ≈90-100%), such a high fluorescence quantum yield is due to: (1) rigidification by tetrahedral boron coordination to suppress nonradiative decay; (2) balanced ICT from diethylamino donor to boron acceptor core to minimize energy loss.

[0080] Meanwhile, we observed that these boshys exhibit strong solid-state emission, with fluorescence maxima in the 515-607 nm range, and significant solid-state Φ. F Up to 74%. Their solid-state emission bands can be tuned by changing the boron coordinating substituents and the salicylhydrazone groups. For example, 1a and 1e at 565 nm (Ф F =603%) and 577nm(Ф) F Strong solid-state emission was observed at 74%).

[0081] It is noteworthy that most BOSHY main solid-state emission bands exhibit a dark shift relative to their solution counterparts. For example, 1j at 590 nm (mid-Ф in powder) F =52%) and 572nm (Ф in dichloromethane) F The maximum emission is observed at 74%). The intense solid-state fluorescence of these BOSHYs is highly consistent with their crystal-filled structures, characterized by formation via sliding stacked arrays. J- Aggregates (intermolecular distances of 4.31–6.33 Å, sliding angles of 19.1°–33.8°). This staggered molecular structure follows Kasha's exciton model, as evidenced by the characteristic hyperchromic shifts in solid-state emission spectra. This arrangement facilitates exciton delocalization, thereby suppressing quenching effects and achieving high solid-state fluorescence efficiency.

[0082] Test Example 3 To further investigate the pH response characteristics of 1a, we conducted a systematic quantitative analysis by preparing PBS buffer solutions with a series of pH values. The results are shown below. Figure 2 (d)

[0083] Figure 2 The results in (d) show that 1a exhibits significant pH-dependent fluorescence behavior: under acidic conditions (pH=1-4), the system exhibits a significant fluorescence quenching effect as the pH decreases; while at pH=4-8, a gradual increase in fluorescence intensity is observed. This reversible pH-responsive characteristic suggests that 1a has the potential to be used as a pH-sensitive fluorescent probe.

[0084] Test Example 4 We used femtosecond time-resolved transient absorption (TA) spectroscopy to trace the excited-state dynamics of 1b and 1i, and the results are as follows: Figure 3 As shown.

[0085] Figure 3 The spectral evolution of 1b in toluene after excitation at 400 nm over 7 ns is shown. Following photoexcitation, both molecules exhibit a significant redshift in stimulated emission, indicating that the excited state structure undergoes relaxation towards the minimum excited state energy within the initial 5 ps. After this structural relaxation, the TA spectrum decays back to baseline and ceases further spectral evolution, suggesting that radiative transition is the primary deactivation pathway. This observation is consistent with theoretical predictions.

[0086] Furthermore, the thiophene-substituted BOSHY3b derived from 3a exhibits a solution emission maximum at 517 nm and a quantum yield of 52%. In contrast, 3b shows a modest blue shift relative to amino-containing analogs (such as 1b, with an emission wavelength of 570 nm), but its quantum yield exceeds that of non-amino-containing derivatives (such as 3a, with a fluorescence quantum yield of 4%). This highlights the synergistic effect of the thiophene conjugation providing the electron donor, as well as the inherent fluorescence enhancement effect of the BOSHY core. The results indicate that the introduction of a strong electron donor group at the 4-position of the BOSHY backbone is a crucial structural determinant for simultaneously achieving an increased red shift in the excitation spectrum and enhanced fluorescence quantum yield.

[0087] Test Example 5 The cytotoxicity of 1i and 1j was systematically examined using the CCK-8 assay, and the results are as follows: Figure 4 As shown.

[0088] Experimental results showed that even at a high concentration of 50 μM, the cell viability after treatment with 1i and 1j remained above 90%, which fully demonstrated the excellent biocompatibility and low cytotoxicity of these two compounds, laying an important foundation for their subsequent liposome localization research.

[0089] Application Example 1 Liposome polarity abnormalities are closely related to the development and progression of metabolic diseases such as fatty liver, making the development of liposome polarity-sensitive probes of great significance in the field of disease diagnosis. To systematically evaluate the lipid droplet (LD) targeting specificity of low-polarity probe 1i, we conducted a co-localization study in HeLa cells using probe 1i and the nucleoside pair DAPI (4',6-diamidindo-2-phenylindole). The results are shown in [Figure number missing]. Figure 5 (a)-(d).

[0090] Depend on Figure 5 The confocal microscopy images (a)-(d) show that the nuclear region exhibits the characteristic blue fluorescence (λ) of DAPI. ex =405 nm, λ em =425-475 nm), while cytoplasmic lipid bodies are specifically labeled with 1i and emit bright yellow-green fluorescence (λ). ex =488 nm, λ em =500-550 nm), showing obvious lipid droplet morphology.

[0091] Application Example 2 To further verify the localization specificity of 1i, we used the commercially available liposome dye Lipi-Blue (1 μM) as a reference to perform a dual-channel co-localization experiment. The experimental setup is as follows: 1) Blue Channel (λ) ex =405 nm, λ em Lipi-Blue signals were acquired at 425-475 nm. 2) Green Channel (λ) ex =488 nm, λ em =500-550 nm) to record 1i fluorescence.

[0092] Figure 5 Results (f)-(h) showed that the fluorescence distributions of the two probes highly overlapped, and colocalization analysis showed a Pearson correlation coefficient of 0.96. Figure 5 (h)), with an overlap coefficient of 0.99 ( Figure 5(j)). Fluorescence intensity linear scanning analysis was performed on selected typical regions ( Figure 5 (f)-(h) regions shown by white lines), it was found that the fluorescence intensity distribution curves of 1i and Lipi-Blue are highly consistent ( Figure 5 (i)).

[0093] Similar to the 1i structure, 1j exhibits the same LD marking performance ( Figure 6 The fluorescence signals of the two molecules showed a high degree of consistency, with Pearson correlation coefficients and overlap coefficients reaching 0.93 and 0.95, respectively. These data fully demonstrate that 1j possesses excellent liposome targeting specificity, and its localization effect is comparable to that of commercially available probes. This further confirms the versatility and reliability of the BOSHY series compounds in subcellular organelle imaging, providing important experimental evidence for the development of novel liposome fluorescent probes.

[0094] In summary, the boron-coordinated salicylhydrazone-type dual-state fluorophores of this invention utilize the NO2 tridentate coordination mode of the salicylaldehyde hydrazone ligand. Through asymmetric coordination engineering between the ligand molecule and boric acid, an asymmetric topological dye containing a six- or seven-membered ring at the boron center is constructed, and the relaxation energy of the excited state structure is modulated by ring strain. The substituents at the boron sites protrude vertically outward from the π surface, and the ligand exhibits a slight bending state due to the structural strain caused by the tetrahedral geometry of the ring fusion. This structure can effectively solve the solubility problem of large conjugated planar structures. Furthermore, through the synergistic effect of the electronic push-pull effect of the ligand and steric hindrance, the mutual repulsion between solubility and solid-state luminescence efficiency is overcome. Compared with traditional bidentate systems, NO2-type tridentate ligands with strong electron-donating groups such as diethylamino substituted ligands can generate a significant ring strain effect by forming a six- or seven-membered chelate ring, which can not only enhance radiative transitions but also suppress non-radiative decay. The structures of these complexes are diverse and can be achieved by modifying the substituents of salicylaldehyde or boric acid derivatives, or by carrying out post-modification derivatization reactions.

[0095] Meanwhile, the boron-coordinated salicylic acid hydrazone-like dual-state fluorophores of this invention utilize the condensation of salicylaldehyde, salicylaldehyde derivatives, or julonidine derivatives with hydrazine hydrate under acidic conditions to obtain ligands, which are then coordinated with boron trifluoride, various boric acids, or boric acid derivatives. A series of BOSHY fluorophores with good solubility were successfully synthesized using a one-pot, two-step method. These fluorophores exhibit excellent photophysical properties. For example, introducing strong electron-donating groups or increasing the conjugation system on the framework results in a significant red shift in the absorption spectrum of the solution, particularly with a liquid-state fluorescence quantum yield as high as 95%. The solid-state quantum yield can also reach 74%. The enantiomers after chiral resolution exhibit a clear mirror-image relationship. Cell experiments show that this series of dyes exhibits excellent biocompatibility (cell viability >90%) and significant liposome targeting (co-localization coefficient with commercial dyes reaches 0.96). The molecular design paradigm established by this invention and the structure-activity relationship it demonstrates provide a theoretical basis and practical reference for the development of novel BOPPY-like biological probes, and have broad application prospects.

[0096] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0097] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0098] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A boron-coordinated salicylhydrazone-like dual-state fluorophore, characterized in that, The structures of the boron-coordinated salicylhydrazone-like dual-state fluorophores are shown in 1i and 1j: 。 2. A method for preparing a boron-coordinated salicylhydrazone-like dual-state fluorophore as described in claim 1, characterized in that, The preparation method includes: 1) Under acidic conditions, the precursor is condensed with hydrazine hydrate in a solvent to generate hydrazone ligands; 2) The hydrazone ligands are coordinated with organoboron compounds in a solvent to generate boron-coordinated salicylhydrazone dual-state fluorophores; In step 1), the precursor substance is a julonidine derivative; the julonidine derivative is 8-hydroxyjulonidine-9-carboxaldehyde; In step 2), the organoboron compound is a boric acid derivative; the boric acid derivative is 4-trifluoromethylphenylboronic acid or trimethyl borate.

3. The preparation method according to claim 2, characterized in that, In step 1), the acidic environment is provided by a Lewis acid; the Lewis acid is selected from one or more of acetic acid, p-toluenesulfonic acid, hydrochloric acid, nitric acid, and sulfuric acid.

4. The preparation method according to claim 2, characterized in that, In step 1), the solvent is selected from one or more of acetonitrile, chloroform, 1,2-dichloromethane, toluene, chlorobenzene, o-dichlorobenzene, p-dichlorobenzene and m-dichlorobenzene; The conditions for the condensation reaction include: being carried out in a metal bath at a temperature of 80-140°C for 10-15 hours and a stirring rate of 200-2000 rpm. The molar ratio of the precursor to hydrazine hydrate is 2-4:

1.

5. The preparation method according to claim 2, characterized in that, In step 2), the conditions for the coordination reaction include: a temperature of 105~145℃ and a reflux time of 10~15h; The molar ratio of organoboron compounds to hydrazone ligands is 2 to 10:

1.

6. A fluorescent probe, characterized in that, The fluorescent probe is prepared from the boron-coordinated salicylhydrazone-type dual-state fluorophore as described in claim 1; The fluorescent probe is specifically designed to target liposomes.

Images