Boron coordination salicylhydrazone double-state emission fluorophore as well as preparation method and application thereof

Abstract

The invention relates to the technical field of organic synthesis and the technical field of fluorescent materials, in particular to a boron coordination salicylhydrazone double-state emission fluorophore as well as a preparation method and application of the boron coordination salicylhydrazone double-state emission fluorophore. The structure of the boron coordination salicylazone double-state emission fluorophore is shown as I, II or III; wherein R1 is selected from one of H, halogen atoms, diethylamino and thienyl, R2 is selected from one of H, halogen atoms, diethylamino and thienyl, and R3 is selected from one of aryl, 9, 9 '-spirobifluorenyl, alkoxy and halogen atoms. The boron coordination salicylazone double-state emission fluorophore is diversified in structure and good in solubility, has excellent photophysical properties and extremely high quantum yield, and also shows excellent biocompatibility and remarkable liposome targeting property at the same time. Moreover, the preparation method of the boron-coordinated salicylazone double-state emission fluorophore adopts a one-pot two-step method, is simple and convenient, is high in yield, is green and environment-friendly, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic synthesis and the technical field of fluorescent materials, in particular, relates to a boron-coordinated salicylhydrazone-based dual-state emission fluorophore and a preparation method and application thereof. BACKGROUND

[0002] As a key functional material, fluorescent small molecules have irreplaceable application value in the fields of material science and biomedical science. Among them, boron-containing fluorophores have attracted much attention due to their unique structural characteristics. Typical representatives include boron-dipyrromethene (BODIPY) and its derivatives. The four-coordinated structure of BODIPY can effectively enhance the rigidity of the pi-conjugated system and suppress non-radiative transitions through steric hindrance, thereby significantly improving the luminescent performance.

[0003] However, the traditional BODIPY fluorophore has a significant solid-state pi-pi stacking problem, which leads to a serious aggregation-caused quenching (ACQ) effect. Although materials with aggregation-induced emission characteristics (AIE) can overcome the ACQ effect, their flexible molecular structure often leads to insufficient fluorescence intensity in solution state, and there are few reports of materials that can achieve nearly 100% solid-state quantum yield. This technical contradiction makes it an important challenge in the field of material chemistry to develop dual-state emission (DSE) fluorescent molecules with high efficient solid-state and solution-state luminescent performance. SUMMARY

[0004] To solve the above technical problems, the present application provides a boron-coordinated salicylhydrazone-based dual-state emission fluorophore (BOSHY) and a preparation method and application thereof. The boron-coordinated salicylhydrazone-based dual-state emission fluorophore has a diverse structure, good solubility, excellent photophysical properties, and extremely high quantum yield. In addition, it also exhibits excellent biocompatibility and significant liposome targeting. Furthermore, the preparation method of the boron-coordinated salicylhydrazone-based dual-state emission fluorophore adopts a one-pot two-step method, which is simple, convenient, high-yield, green, and environmentally friendly, and has a broad application prospect.

[0005] To achieve the above purpose, in a first aspect, the present application provides a boron-coordinated salicylhydrazone-based dual-state emission fluorophore, the structure of which 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.

[0006] ​Further, 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'-spirobifluorene, methoxy, F, Cl, Br and I.

[0007] Further, 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'-spirobifluorene, methoxy, F, Cl, Br and I.

[0008] Further, the structure of the boron-coordinated salicydhydrazone-based dual-state emitting fluorophore is shown in 1a-j, 2a-b and 3a-b,

[0009]

[0010] .

[0011] In a second aspect, the present application provides a preparation method of the boron-coordinated salicydhydrazone-based dual-state emitting fluorophore, which comprises: 1) condensing a precursor with hydrazine hydrate in a solvent under an acidic environment to generate a hydrazone ligand; 2) coordinating the hydrazone ligand with an organic boron compound in a solvent to generate the boron-coordinated salicydhydrazone-based dual-state emitting fluorophore; In step 1), the precursor is a salicylaldehyde derivative and / or a gulonin derivative; In step 2), the organic boron compound is a boronic acid derivative and / or a boron trifluoride diethyl ether complex.

[0012] Further, the preparation method further comprises: continuing the Stille coupling reaction of the boron-coordinated salicydhydrazone-based dual-state emitting fluorophore.

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

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

[0015] Further, 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] Further, the julolidine derivative is 8-hydroxyjulolidine-9-carboxaldehyde.

[0017] Further, 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] Further, in step 1), the conditions of the condensation reaction include: being carried out in a metal bath, the temperature being 80-140℃, the time being 10-15h, and the stirring rate being 200-2000 rpm.

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

[0020] Further, the boronic acid derivative is selected from one or more of phenylboronic acid, 4-trifluoromethylphenylboronic acid, 4-(bromomethyl)phenylboronic acid, 4-methoxyphenylboronic acid, 4-(diphenylamino)phenylboronic acid, 9,9'-spirobifluorene-2-boronic acid, 3-thiopheneboronic acid, 1,4-benzenediboronic acid, 2-biphenylboronic acid and trimethylborate.

[0021] Further, in step 2), the conditions of the coordination reaction include: the temperature being 105-145℃, and the refluxing time being 10-15h.

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

[0023] Further, the conditions of the Stille coupling reaction include: under an argon atmosphere, coupling the palladium catalyst, the organotin reagent and the boron-coordinated salicylhydrazone bistate emitting fluorophore in toluene, the temperature being 80-140℃, and the oil bath time being 20-30h.

[0024] In a third aspect, the present application provides a boron-coordinated salicylhydrazone bistate emitting fluorophore prepared by the preparation method of the second aspect.

[0025] In a fourth aspect, the present application provides a fluorescent probe prepared from the boron-coordinated salicylhydrazone bistate emitting fluorophore of the first or third aspect. The fluorescent probe has targeting property to liposomes.

[0026] In the above technical solution, the application innovatively designs a kind of boronic acid salicyl hydrazone fluorescent molecules (BOSHY).The molecule system is constructed by one-pot condensation of commercially available salicylaldehyde, salicylaldehyde derivative or gurjon derivative with hydrazine and aryl boronic acid. By introducing a stereogenic boron center in the 6,7-fused ring skeleton, the four-coordinated boron substitution simultaneously achieves the following technical effects: (1) enhances the molecular rigidity; (2) provides the necessary steric hindrance to inhibit the solid-state aggregation quenching; (3) maintains the intramolecular charge transfer (ICT) effect between the amino donor and the boron-coordinated salicyl hydrazone nucleus, ensuring the solution-state fluorescence intensity. This strategy successfully breaks through the performance limitations of traditional boron dyes, providing a new idea for the development of dual-state emission fluorescent materials.

[0027] The boron-coordinated salicyl hydrazone dual-state emission fluorophores of the application exhibit significant DSE characteristics, with high quantum yield (up to 95% in solution and up to 74% in solid state), and a large Stokes shift of up to 7200 cm -1 In addition, the introduction of axial aryl not only improves the solubility, but also effectively prevents aggregation, while the twisted molecular geometry significantly reduces the π-π stacking interaction. These BOSHYs exhibit excellent lipid droplet specificity, with extremely high targeting accuracy and excellent biocompatibility. These outstanding characteristics 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 salicyl hydrazone dual-state emission fluorophore of the application is simple in operation, few in steps, and easy in raw material, and has a broad application prospect.

[0029] Other features and advantages of the application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0030] The accompanying drawings are included to provide a further understanding of the application, and constitute a part of the specification, and together with the following specific embodiments, serve to explain the application, but do not constitute a limitation on the application. In the drawings: Figure 1 The X-ray crystal structure of the boron-coordinated salicyl hydrazone dual-state emission fluorophore of the application, (a) is the X-ray crystal structure of 1c, (b) is the X-ray crystal structure of 1f, (c) is the X-ray crystal structure of 1i and (d) is 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; F atoms are green; Figure 2Normalized absorption (a) and emission (b, c) spectra of the boron-coordinated salicyhydrazone-based dual-emissive fluorophore of the present application in dichloromethane and solid state; (d) is the emission intensity of 1a (excitation wavelength is 440 nm, respectively) in buffer system with pH from 1 to 8; Figure 3 Transient absorption (a) and decay kinetics (b) curves of the boron-coordinated salicyhydrazone-based dual-emissive fluorophore of the present application 1b and transient absorption (c) and decay kinetics (d) curves of 1i; Figure 4 Cell viability of HeLa cells treated with different concentrations of 1i (a) and 1j (b) for 24 hours; Figure 5 Confocal fluorescence images of HeLa cells stained by the boron-coordinated salicyhydrazone-based dual-emissive fluorophore of the present application 1i (1.0 μΜ) and DAPI, (a) bright field image; (b) fluorescence image after DAPI staining; (c) fluorescence image of 1i after incubation for 2.5 hours; (d) merged image of figure b and figure c, scale bar: 50 μm; Lipid droplet co-localization study of 1i (1.0 μΜ) in HeLa cells, (e) bright field image; (f) fluorescence image of Lipi-blue staining; (g) fluorescence image of 1i; (h) merged image of figure f and figure g; (i) intensity profile of 1i and Lipi-Blue in the line region of HeLa cells, Pearson correlation coefficient Rr = 0.96 ± 0.02; (j) correlation scatter plot of Lipi-Blue and 1i intensity, Pearson correlation coefficient Rr = 0.98 ± 0.01, overlap coefficient R = 0.99 ± 0.01; scale bar: 50 μm; (k) cell viability of HeLa cells incubated with 1i at different concentrations for 24 hours; Figure 6 Confocal fluorescence images of HeLa cells stained by the boron-coordinated salicyhydrazone-based dual-emissive fluorophore of the present application 1j (1.0 μΜ) and DAPI, (a) bright field image; (b) fluorescence image after DAPI staining; (c) fluorescence image of 1j after incubation for 2.5 hours; (d) merged image of figure b and figure c, scale bar: 50 μm; 1j (1.0 µM) lipid droplet co-localization study in HeLa cells, (e) bright field image; (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 boxed region of HeLa cells, Pearson correlation coefficient Rr = 0.88 ± 0.05; (j) Correlation scatter plot of 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 DESCRIPTION

[0031] The specific embodiments of the present application will be described in detail below. It should be understood that the detailed description and specific examples, while indicating preferred embodiments of the application, are given by way of illustration only, and are not by way of limitation.

[0032] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and any values are provided as approximate descriptions of the ranges and are understood to be open-ended. Each range disclosed herein is to be understood to include individual values, and sub-ranges, falling within the stated range. In other words, any numerical range disclosed herein is intended to include all derived numerical ranges falling within the stated range; for example, to include any value falling within the stated range, as well as to include any value that can be obtained by applying a modifier such as "about," "approximately," "substantially," or equivalents thereof, to any of the values in the stated range.

[0033] In a first aspect, the present application provides a boron-coordinated salicylaldimine-based dual-state emissive fluorophore, the structure of the boron-coordinated salicylaldimine-based dual-state emissive fluorophore is shown as I, II or III, ; wherein R1 is selected from one of H, a halogen atom, diethylamino and a thienyl group, R2 is selected from one of H, a halogen atom, diethylamino and a thienyl group, and R3 is selected from one of an aryl group, a 9,9'-spirobifluorene group, an alkoxy group and a halogen atom.

[0034] The boron-coordinated salicylhydrazone bistate emitting fluorophore of the present application selects the NO2 tridentate coordination mode of the salicylaldehyde hydrazone ligand, and through the asymmetric coordination engineering between the ligand molecule and the boric acid, an asymmetric topological dye containing a six-membered ring or a seven-membered ring at the boron center is constructed, and the excited state structure relaxation energy is regulated by means of ring strain. The substituents of the boron site protrude vertically outward from the π surface, and the ligand presents a slightly curved state due to the structural strain generated by the ring-fused tetrahedral geometry. This structure can effectively solve the solubility problem of the large conjugate plane structure. In addition, through the synergistic effect of the electronic push-pull effect and the steric hindrance of the ligand, the limitation of mutual exclusion between solubility and solid-state luminescent efficiency is broken through. Compared with the traditional bidentate system, the strong electron-donating group such as diethylamino-substituted NO2 type tridentate ligand can produce significant ring strain effect by forming a six-membered or seven-membered chelate ring, which not only can enhance the radiation transition, but also can inhibit the non-radiative decay. The structure of the complex has diversity, which can be achieved by improving the substituents of salicylaldehyde or boric acid derivatives, or carrying out post-modification derivatization reaction.

[0035] In a preferred embodiment of the present application, 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'-spirobifluorene, methoxy, F, Cl, Br and I.

[0036] In a preferred embodiment of the present application, 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'-spirobifluorene, F and methoxy.

[0037] In a preferred embodiment of the present application, the structure of the boron-coordinated salicylhydrazone bistate emitting fluorophore is shown in 1a-j, 2a-b and 3a-b,

[0038]

[0039] .

[0040] In a second aspect, the present application provides a preparation method of a boron-coordinated salicylhydrazone bistate emitting fluorophore, which comprises: 1) condensation reaction of precursor substances and hydrazine hydrate in a solvent under acidic environment to generate a hydrazone ligand; 2) subjecting the hydrazone ligand to a coordination reaction with an organic boron compound in a solvent to generate the boron-coordinated salicyhydrazone bistate emissive fluorophore; In step 1), the precursor is a salicylaldehyde derivative and / or a gularonidine derivative; In step 2), the organic boron compound is a boric acid derivative and / or a boron trifluoride diethyl ether complex.

[0041] The boron-coordinated salicyhydrazone bistate emissive fluorophore of the present application is synthesized by condensation of salicylaldehyde, a salicylaldehyde derivative or a gularonidine derivative with hydrazine hydrate under acidic conditions to obtain a ligand, and then coordination with boron trifluoride, various boric acids or boric acid derivatives. A series of BOSHYs fluorophores with good solubility are successfully synthesized by one-pot two-step method. The fluorophore exhibits excellent photophysical properties. For example, introduction of a strong electron-donating group or increase of the conjugated system on the basis of the skeleton causes a significant red shift of the absorption spectrum in solution, and in particular, the liquid fluorescence quantum yield of the compound is as high as 95%. The solid-state quantum yield can also be as high as 74%. Cell experiments show that the series of dyes exhibit excellent biocompatibility (cell survival rate > 90%) and significant liposome targeting (co-localization coefficient with commercial dye up to 0.96). The molecular design paradigm established by the present application and the structure-activity relationship exhibited thereby provide a theoretical basis and practical reference for the development of new BOPPY biological probes, and have a broad application prospect.

[0042] In a preferred embodiment of the present application, the preparation method further comprises: subjecting the boron-coordinated salicyhydrazone bistate emissive fluorophore to a Stille coupling reaction.

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

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

[0045] In a preferred embodiment of the present application, the salicylaldehyde derivative is selected from one or two 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 application, the gularonidine derivative is 8-hydroxygularonidine-9-carboxaldehyde.

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

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

[0049] In a preferred embodiment of the present application, the boronic acid derivative is selected from the group consisting of phenylboronic acid, 4-trifluoromethylphenylboronic acid, 4-(bromomethyl)phenylboronic acid, 4-methoxyphenylboronic acid, 4-(diphenylamino)phenylboronic acid, 9,9'-spirobifluorene-2-boronic acid, 3-thiopheneboronic acid, 1,4-benzenediboronic acid, 2-biphenylboronic acid, trimethyl borate.

[0050] In a preferred embodiment of the present application, in step 2), the conditions of the coordination reaction include: temperature being 105-145℃, and refluxing time being 10-15h.

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

[0052] In a preferred embodiment of the present application, the conditions of the Stille coupling reaction include: under argon atmosphere, coupling the palladium catalyst, the organotin reagent, and the boron-coordinated salicydrazine bistate emitting fluorophore in toluene, temperature being 80-140℃, and oil bath time being 20-30h.

[0053] In a third aspect, the present application provides a boron-coordinated salicydrazine bistate emitting fluorophore prepared by the preparation method of the second aspect.

[0054] In a fourth aspect, the present application provides a fluorescent probe prepared from the boron-coordinated salicydrazine bistate emitting fluorophore of the first or third aspect. The fluorescent probe has targeting property to liposome.

[0055] In a preferred embodiment of the present application, the room temperature is 15-30℃.

[0056] The present application will be described in detail below by way of examples. In the following examples, the drugs and reagents are all conventional commercially available products.

[0057] Example 1 The synthesis route of 1a is specifically as follows:

[0058] In a 100 mL pressure 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 successively, and the system was heated and stirred in a 105 °C metal bath for 12 hours. After confirming complete conversion of the starting material by TLC monitoring, benzeneboronic acid (1.1 g, 9 mmol, 9 equiv.) was added, and the reaction system was warmed to 130 °C and stirred for 12 hours. After the reaction was completed, it was cooled to room temperature, extracted with dichloromethane (3 x 20 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product 1a (239 mg, 51%) was obtained as an orange-yellow solid by column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v).

[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 105 °C metal bath for 12 h. TLC monitoring showed complete conversion of starting material after which 4-trifluoromethylbenzeneboronic acid (760 mg, 4 mmol, 4 equiv.) was added and the reaction was warmed to 130 °C and stirred at reflux for 12 h. After completion of the reaction, it was cooled to room temperature and extracted with dichloromethane (3 x 20 mL) and the organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product 1b was purified by column chromatography (eluent: dichloromethane / petroleum ether = 1:2, v / v) to give an orange yellow solid (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,F-C), 132.1, 125.2 (q, J = 30.4 Hz, F-C), 124.0 (q, J = 3.8 Hz, F-C), 123.5,105.9, 105.7, 104.7, 104.5, 102.3, 102.2, 100.9, 45.1, 44.8, 12.9, 12.8. 11B NMR (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 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 successively and the system was heated in a 105 °C metal bath with stirring for 12 hours. After confirming complete conversion of the starting material by TLC monitoring, 4-(bromomethyl)benzeneboronic acid (430 mg, 2 mmol, 1 equiv.) was added and the reaction system was warmed to 130 °C and stirred for 12 hours. After the reaction was completed, it was cooled to room temperature, extracted with dichloromethane (3 x 20 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, column chromatography was used for purification (eluent: dichloromethane / petroleum ether = 1:1, v / v) to obtain the 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 1d: In a 100 mL pressure reaction bottle, 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 successively, and the system was heated and stirred in a 105 °C metal bath for 12 hours. After confirming complete conversion of the starting material by TLC monitoring, 4-methoxyphenylboronic acid (608 mg, 4 mmol, 2 equiv.) was added, and the reaction system was warmed to 130 °C and stirred for 12 hours. After the reaction was completed, it was cooled to room temperature, extracted with dichloromethane (3 x 20 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product 1d was obtained as an orange-yellow solid (194 mg, 39%) after purification by column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v).

[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 reaction bottle, 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 successively and the system was heated in a 105 °C metal bath with stirring for 12 hours. After confirming complete conversion of the starting material by TLC monitoring, 4-(diphenylamino)benzeneboronic acid (578 mg, 2 mmol, 1 equiv.) was added and the reaction system was warmed to 130 °C and stirred for 12 hours. After the reaction was completed, it was cooled to room temperature, extracted with dichloromethane (3 x 20 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product 1e (460 mg, 72%) was obtained as an orange-yellow solid by column chromatography (eluent: ethyl acetate / petroleum ether = 1:3, v / v).

[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, CDC13) 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, CDC13) 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 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 successively, and the system was heated and stirred in a 105 °C metal bath for 12 hours. After confirming complete conversion of the starting material by TLC monitoring, 9,9-spirobiluorene-2-boronic acid (720 mg, 2 mmol, 1 equiv.) was added, and the reaction system was warmed to 130 °C and stirred for 12 hours. After the reaction was completed, it was cooled to room temperature, extracted with dichloromethane (3 x 20 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v) was used to purify the product, which was obtained as an orange solid (450 mg, 64%).

[0064] 1 H NMR (400 MHz, CDC13) 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, 1.1 Hz, 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, 126.8,124.4, 123.9, 123.7, 119.6, 119.4, 119.3, 118.9, 105.6, 104.4, 98.8, 53.4,44.9, 29.7, 12.7. HRMS (ESI) calcd for C 47 H 44 BN4O2[M + H] + : 707.3557, found707.3586. Example 7 1g of synthesis: In a 100 mL pressure reaction bottle, 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 successively, and the system was heated and stirred in a 105 °C metal bath for 12 hours. After confirming complete conversion of the starting material by TLC monitoring, 1,4-benzenediboronic acid (166 mg, 1 mmol, 0.5 equiv.) was added, and the reaction system was warmed to 130 °C and stirred for 12 hours. After the reaction was completed, it was cooled to room temperature, extracted with dichloromethane (3 x 20 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, column chromatography was performed (eluent: dichloromethane / petroleum ether = 1:1, v / v) to give the orange solid product 1g (171 mg, 20%).

[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 1h: In a 100 mL pressure reaction bottle, 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 successively, and the system was heated and stirred in a 105 °C metal bath for 12 hours. After confirming complete conversion of the raw material by TLC monitoring, N,N-diisopropylethylamine (DIPEA, 3 mL) and boron trifluoride etherate (BF3·OEt2, 3 mL) were added successively under argon protection, and the reaction system was warmed to 130 °C and refluxed and stirred for 12 hours. After cooling to room temperature, the reaction was extracted with dichloromethane (3 × 20 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, column chromatography purification (eluent: dichloromethane / petroleum ether = 1:1, v / v) gave the 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 reaction flask, 8-hydroxyjulolidine-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 successively, and the system was heated and stirred in a 105 °C metal bath for 12 hours. After confirming complete conversion of the starting material by TLC monitoring, 4-trifluoromethylphenylboronic acid (760 mg, 4 mmol, 4 equiv.) was added, and the reaction system was warmed to 130 °C and stirred for 12 hours. After the reaction was completed, it was cooled to room temperature, extracted with dichloromethane (3 x 20 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product 1i (300 mg, 52%) was obtained as an orange-yellow solid by column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v).

[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, F-C),125.0 (q, J = 273.4 Hz, F-C), 123.8 (q, J = 2.5 Hz, F-C), 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 reaction bottle, 8-hydroxyjulolidine-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 successively. The reaction system was heated and stirred in a 105 °C metal bath for 12 hours, and TLC monitoring confirmed that the starting material was completely converted. Then trimethyl borate (1.1 mL, 4 mmol, 2 equiv.) was added, and the reaction system was heated to 130 °C and stirred for 12 hours. After the reaction was completed, it was cooled to room temperature, extracted with dichloromethane (3 x 20 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product 1j (235 mg, 49%) was obtained as an orange solid by column chromatography (eluent: ethyl acetate / petroleum ether = 1:1, v / v).

[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 synthetic route of 2a and 2b is as follows:

[0069] In a 250 mL round-bottom flask, 4-(diethylamino)-2-hydroxybenzaldehyde (386 mg, 2 mmol) and salicylidene hydrazine (272 mg, 2 mmol) were dissolved in 20 mL of anhydrous 1,2-dichloroethane, stirred at 50 °C oil bath for 1 hour, and after TLC monitoring confirmed complete conversion of the raw material, 4-trifluoromethylphenylboronic acid (950 mg, 5 mmol) was added at 120 °C, and the reaction was continued until TLC showed that the reaction was complete. After cooling to room temperature, the crude product was separated by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 1:2, v / v) to obtain orange-yellow solid 2a (214 mg, 23%) and 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 synthetic route of 3a is as follows: In a 100 mL pressure reaction bottle, 4-bromo-2-hydroxybenzaldehyde (402 mg, 2 mmol) was dissolved in 20 mL of anhydrous chlorobenzene, and hydrazine hydrate (60 μL, 1 mmol) and glacial acetic acid (5 drops) were added in turn, and stirred at 105 °C in a metal bath for 12 hours. After TLC monitoring confirmed that the starting material was completely converted, 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 system was heated to 130 °C and stirred for 12 hours. After the reaction was completed, it 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, column chromatography was used for purification (eluent: dichloromethane / petroleum ether = 2:3, v / v), and a yellow solid product 3a (207 mg, 43%) was obtained.

[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 synthetic route of compound 3b is as follows:

[0071] In a 50 mL Schlenk 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 the system was recycled for three times of vacuum-purging with argon, the reaction was continuously stirred at 130 °C oil bath for 24 hours, and the reaction progress was monitored in real time by thin layer chromatography (TLC). After the starting material was completely converted, the reaction system was cooled to room temperature, extracted with dichloromethane (3 × 20 mL). After the organic phases were combined, they were dried over anhydrous sodium sulfate, and the solvent was removed by reduced pressure concentration. The obtained crude product was purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v) to obtain 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 (101 MHz, 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 The single crystals of 1c, 1f, 1i and 2b were successfully cultivated in dichloromethane / n-hexane mixed solvent system by solvent slow evaporation method. The molecular structure was accurately analyzed by X-ray single crystal diffraction analysis (Bruker D8 Venture diffractometer), and the crystal structure diagram of 1c, 1f, 1i and 2b is shown in Figure 1 Table 1.

[0073] Table 1

[0074] From the data in Table 1 and the crystal structure diagram of Figure 1 , it can be seen that the boron-coordinated salicylidene class of dual-state fluorophores of the present application 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) slender B-N bonds demonstrate flexible bond connectivity compared to rigid BODIPY analogs (about 1.50 Å); (3) tetrahedral coordination environment at the boron center varies significantly. The molecular structure includes four rigid components: a central seven-membered chelate ring, an adjacent six-membered ring (showing 0.02-0.03 Å longer B-O bonds than the seven-membered para portion), and two peripheral benzene rings. The dihedral angles between the benzene rings on the salicylidene segment exhibit significant variation (134.9°~140.2°) indicating substitution group-dependent π-conjugation. All particles crystallize in the form of racemic mixtures, with sp 3The hybrid boron centers adopt a distorted tetrahedral geometry (THCDA = 0.85-0.92) with the O2-B1-N1 pair showing particular sensitivity to substitution reactions (109.2° in 1c vs. 118.8° in 2b). Notably, the near orthogonal arrangement between the axial and equatorial phenyl rings (80.94°-85.96°) minimizes gross π-stacking. For example, the dihedral angle between the phenyl ring of the axial boron atom and the phenylacetylene ring adjacent to the newly formed six-membered ring is 81.45° in 1c, 85.96° in 1f, 80.94° in 1i, and 83.90° in 2b.

[0075] Test Example 2 We systematically investigated the photophysical properties of 1a-j, 2a-b and 3a-b in toluene, dichloromethane and acetonitrile, and their photophysical properties in solid state, and the results are shown in Tables 2-3 and 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 °C)

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

[0078] From the data in Tables 2-3 and Figure 2 (a)-(c), these BOSHYs exhibit broad absorption ranges and super-bright fluorescence properties. These BOSHYs display broad absorption bands (A abs = 396-500 nm) with high molar extinction coefficients (ε max ≈ 3.7-4.9 x 10 -4 M -1 cm -1 ). For example, 1a shows significant absorption properties in toluene with a peak at 444 nm and a high molar extinction coefficient of 3.81 x 10 -4 M -1 cm -1 . In addition, 1a emits at 538 nm in toluene, exhibiting a relatively large Stokes shift of about 4900 cm -1 , which is larger than that of typical BODIPY dyes. Similar spectral shapes are also observed for other BOSHYs. The emission maxima (λ em ) are significantly red-shifted (Δ λ = 65-160 nm) compared to the absorption, resulting in large Stokes shifts (3500-7200 cm-1 ). This suggests the presence of significant structural relaxation in the excited state, most likely due to the ICT effect facilitated by the donor-acceptor structure of the BOSHY core.

[0079] In solution, the electron-donating substituents on boron have a smaller effect on the bathochromic emission spectrum than the electron-withdrawing substituents. For example, the electron-donor group (such as 1b) shifts the emission further to 570 nm, while the electron-donor group (such as NMe2 in 1e) results in a bathochromic emission shift (Δλ em = +12 nm υs In contrast, the electron-donor groups attached at the corresponding positions show a more significant red-shift effect than the substituents attached to the aromatic ring of the salicylhydrazone moiety. After forming an additional ethyl anchor group on the salicyloyl moiety, a 22 nm absorption and 48 nm emission bathochromic shift of 1j was observed in dichloromethane compared to 1a. The absorption and deep red emission of 1i with F groups in acetonitrile are centered at 463 / 593 nm, and most of the BOSHYs in this invention exhibit excellent fluorescence performance (Ф F = 63-95%) with 1b (95%) and 1i (89%) standing out. This performance exceeds that of classic BODIPYs (typically Ф F = 60-80%) and approaches the BOPHY benchmark (Ф F ≈ 90-100%), such a high fluorescence quantum yield is attributed to: (1) rigidity by tetrahedral boron coordination, which suppresses non-radiative decay; (2) balanced ICT from the diethylamino donor to the boron acceptor core, which minimizes energy loss.

[0080] At the same time, we observed that these BOSHYs exhibit strong solid-state emission with fluorescence maxima at 515-607 nm, significant solid-state Ф F up to 74%. Their solid-state emission bands can be tuned by changing the boron-coordinating substituents and the groups of the salicyloyl hydrazone. For example, 1a and 1e exhibit strong solid-state emission at 565 nm (Ф F = 603%) and 577 nm (Ф F = 74%), respectively.

[0081] Notably, the main solid-state emission bands of most BOSHYs have a bathochromic shift relative to their solution counterparts. For example, 1j shows a maximum emission at 590 nm (Ф F = 52%) in the powder and 572 nm (Ф F = 74%) in dichloromethane). The strong solid-state fluorescence of these BOSHYs is consistent with their crystal packing structures, which are characterized by the formation of sliding stack arrays JAggregates (intermolecular distance of 4.31-6.33 A, sliding angle of 19.1°-33.8°). This misaligned molecular structure follows Kasha's exciton model, which is evidenced by the characteristic hypsochromic shift in the solid-state emission spectrum. This arrangement helps to delocalize the exciton, thereby suppressing the quenching effect, and thus achieving a high solid-state fluorescence efficiency.

[0082] Test Example 3 To further investigate the pH-responsive properties of 1a, we performed a systematic quantitative analysis by preparing a series of PBS buffer solutions with different pH values. The results are shown in Figure 2 (d).

[0083] Figure 2 The results of (d) show that 1a exhibits a significant pH-dependent fluorescence behavior: under acidic conditions (pH = 1-4), the system shows a significant fluorescence quenching effect as the pH value decreases; while when (pH = 4-8), a gradual increase in fluorescence intensity is observed. This reversible pH-responsive property indicates 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 track the excited-state dynamics of 1b and 1i, and the results are shown in Figure 3

[0085] Figure 3 shows the spectral evolution of 1b in toluene within 7 ns after excitation at 400 nm. After photoexcitation, both molecules show significant redshift in stimulated emission, indicating that the structure of the excited state relaxes to the excited-state potential minimum within the first 5 ps. After this structural relaxation, the TA spectrum decays back to the baseline, and no further spectral evolution occurs, indicating that radiative transition is the main deactivation pathway. This observation is consistent with theoretical predictions.

[0086] In addition, the thienyl-substituted BOSHY 3b derived from 3a has a solution emission maximum at 517 nm with a quantum yield of 52%. In contrast, 3b shows a moderate blue shift relative to the analogous compound containing an amino group (such as 1b, with an emission wavelength of 570 nm), but its quantum yield exceeds that of the derivative without an amino group (such as 3a, with a fluorescence quantum yield of 4%), highlighting the synergistic effect of the thienyl conjugate providing an electron donor and the inherent fluorescence enhancement effect of the BOSHY core. The results show that the introduction of a strong electron donor group at the 4-position of the BOSHY skeleton is a crucial structural determinant for simultaneously achieving increased excitation spectral redshift and enhanced fluorescence quantum yield.

[0087] Test Example 5 ​The cytotoxicity of 1i and 1j was systematically investigated by CCK-8 method, and the results are shown in Figure 4 .

[0088] The experimental results show that the cell survival rate after treatment of 1i and 1j remains above 90% even at a high concentration of 50 μM, which fully proves the excellent biocompatibility and low cytotoxicity of the two compounds, and lays an important foundation for subsequent liposome positioning research.

[0089] Application Example 1 Liposome polarity is closely related to the occurrence and development of metabolic diseases such as fatty liver, which makes the development of liposome polarity sensitive probes important in the field of disease diagnosis. In order to systematically evaluate the lipid droplet (LD) targeting specificity of low polarity 1i, we used probe 1i and nuclear counterstaining agent DAPI (4', 6-diamidino-2-phenylindole) for co-localization study in HeLa cells, and the results are shown in Figure 5 (a)-(d).

[0090] The confocal microscopy images of Figure 5 (a)-(d) show that the nuclear region presents the characteristic blue fluorescence of DAPI (λ ex =405 nm, λ em =425-475 nm), while the liposomes in the cytoplasm are specifically labeled by 1i, emitting 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 positioning specificity of 1i, we used commercial liposome dye Lipi-Blue (1 μM) as a reference for double-channel co-localization experiment, and the experimental settings are as follows: 1) Blue channel (Blue Channel) (λ ex =405 nm, λ em =425-475 nm) to collect Lipi-Blue signal; 2) Green channel (Green Channel) (λ ex =488 nm, λ em =500-550 nm) to record 1i fluorescence.

[0092] Figure 5 The results of (f)-(h) show that the fluorescence distribution of the two probes is highly overlapped, and the co-localization analysis shows that the Pearson correlation coefficient is 0.96 ( Figure 5 (h)), and the overlap coefficient is 0.99 ( Figure 5(j)). By selecting typical regions for linear scanning analysis of fluorescence intensity (Fig. 1i, white line) Figure 5 (f)-(h) white line region), it was found that the fluorescence intensity distribution curve of 1i was highly consistent with that of Lipi-Blue (Fig. 1i, white line) Figure 5 (i).

[0093] 1j, which is similar in structure to 1i, also showed the same LD labeling performance (Fig. 1j, white line) Figure 6 ), and the fluorescence signals of the two were highly consistent, with Pearson correlation coefficients and overlap coefficients reaching 0.93 and 0.95, respectively. These data fully demonstrate that 1j has excellent liposome targeting specificity, and its positioning effect is comparable to that of commercial probes. This further confirms the universality and reliability of BOSHY series compounds in subcellular organelle imaging, and provides important experimental basis for the development of new liposome fluorescent probes.

[0094] In summary, the boron-coordinated salicylhydrazone bistate emitting fluorophore of the present application selects the NO2 tridentate coordination mode of the salicylaldehyde hydrazone ligand, and through the asymmetric coordination engineering between the ligand molecule and the boronic acid, a non-symmetric topological dye containing a six-membered ring or a seven-membered ring at the boron center is constructed, and the excited state structure relaxation energy is regulated by means of ring strain. The substituents at the boron site protrude vertically outward from the π surface, and due to the structural strain generated by the ring-fused tetrahedral geometry, the ligand exhibits a slightly curved state. This structure can effectively solve the solubility problem of large conjugated planar structures. In addition, through the synergistic effect of electronic push-pull effect and steric hindrance of the ligand, the mutual exclusion between solubility and solid-state luminescent efficiency is broken through. Compared with traditional bidentate systems, the NO2 type tridentate ligand with strong electron-donating groups such as diethylamino substituents can produce significant ring strain effect by forming six-membered or seven-membered chelate rings, which not only can enhance the radiation transition, but also can inhibit non-radiative decay. The structure of such complexes has diversity, which can be achieved by improving the substituents of salicylaldehyde or boronic acid derivatives, or carrying out post-modification derivatization reactions.

[0095] Meanwhile, the boron-coordinated salicyhydrazide bistate emitting fluorophore of the application is synthesized by condensation of salicylaldehyde, salicylaldehyde derivative or gurjon derivative with hydrazine hydrate under acidic condition to obtain a ligand, and then coordination with boron trifluoride, various boric acid or boric acid derivatives, and a series of BOSHYs fluorophore with good solubility is successfully synthesized by one-pot two-step method. The fluorophore shows excellent photophysical properties. For example, introduction of a strong electron-donating group or increase of the conjugated system on the basis of the skeleton causes obvious red shift of the absorption spectrum of the solution, and in particular, the liquid fluorescence quantum yield of the compound is as high as 95%. And the solid quantum yield can also be as high as 74%. The enantiomers after chiral resolution show clear mirror image relationship. Cell experiments show that the series of dyes exhibit excellent biocompatibility (cell survival rate > 90%) and significant liposome targeting (co-localization coefficient with commercial dye up to 0.96). The molecular design paradigm established by the application and the structure-activity relationship exhibited thereby provide a theoretical basis and practical reference for developing new BOPPY biological probes, and have broad application prospects.

[0096] The above describes the preferred embodiments of the application in detail, but the application is not limited to the specific details in the above-described embodiments, and various simple modifications can be made to the technical solutions of the application within the technical concept of the application, and the simple modifications all belong to the protection scope of the application.

[0097] In addition, it should be noted that various specific technical features described in the above-described specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the application will not further describe various possible combination manners.

[0098] In addition, various different embodiments of the application can also be combined in any appropriate manner, as long as it does not deviate from the idea of the application, and it should also be considered as disclosed by the application.

Claims

1. A boron-coordinated salicylhydrazone-like dual-state fluorophore, characterized in that, The structure of the boron-coordinated salicylhydrazone-like dual-state fluorophore 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.

2. The boron-coordinated salicylhydrazone-like dual-state fluorophore according to claim 1, characterized in that, 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. Preferably, 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.

3. The boron-coordinated salicylhydrazone-like dual-state fluorophore according to claim 1 or 2, characterized in that, The structural formulas of the boron-coordinated salicylhydrazone-like dual-state fluorophores are shown in 1a-j, 2a-b, and 3a-b. 。 4. A method for preparing a boron-coordinated salicylhydrazone-like dual-state fluorophore, 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 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.

5. The preparation method according to claim 4, characterized in that, The preparation method further includes: subjecting the boron-coordinated salicylhydrazone-like dual-state fluorophore to Stille coupling reaction.

6. The preparation method according to claim 4, characterized in that, In step 1), the acidic environment is provided by a Lewis acid; Preferably, the Lewis acid is selected from one or more of acetic acid, p-toluenesulfonic acid, hydrochloric acid, nitric acid, and sulfuric acid; More preferably, the salicylaldehyde derivative is selected from one or more of 4-bromo-2-hydroxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, 4-(diethylamino)-2-hydroxybenzaldehyde, 2,4-dihydroxybenzaldehyde and 5-bromo-2-hydroxybenzaldehyde; More preferably, the julonidine derivative is 8-hydroxyjulonidine-9-carboxaldehyde; More preferably, 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; More preferably, 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. More preferably, in step 1), the molar ratio of the precursor substance to hydrazine hydrate is 2 to 4:

1.

7. The preparation method according to claim 4, characterized in that, 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. Preferably, in step 2), the conditions for the coordination reaction include: a temperature of 105~145℃ and a reflux time of 10~15h; More preferably, in step 2), the molar ratio of the organoboron compound to the hydrazone ligand is 2 to 10:

1.

8. The preparation method according to claim 5, characterized in that, 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℃ and an oil bath time of 20~30h.

9. A boron-coordinated salicylhydrazone-like dual-state fluorophore prepared by the preparation method according to any one of claims 4-8.

10. A fluorescent probe, characterized in that, The fluorescent probe is prepared from the boron-coordinated salicylhydrazone dual-state fluorophore as described in any one of claims 1-3 or the boron-coordinated salicylhydrazone dual-state fluorophore as described in claim 9; The fluorescent probe is specifically designed to target liposomes.

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