Small molecular weight fluorescent dyes, methods of synthesis and use thereof

Abstract

This invention achieves a series of compact small-molecule organic dyes with emission wavelengths ranging from the visible light window to the second near-infrared window through a rational strategy of balancing the molecular weight and emission wavelength of the dyes. These dyes exhibit good versatility and excellent blood-brain barrier crossing ability. This invention creatively selects low-molecular-weight flexible conjugated polyene chains as π-electron bridges, which effectively expands the π-conjugated system while maintaining the small molecular weight of the dyes. Furthermore, by extending the π-conjugated structure through conjugated polyene chains to enhance the ICT effect, a redshift in emission wavelength can be achieved while maintaining a small molecular weight. Therefore, based on the p-hydroxybenzylidene-imidazolinone chromophore, this invention integrates molecular design strategies to synergistically enhance the ICT effect, constructing a series of small-molecular-weight fluorescent dyes with D-π-A1 or D-π-A1-π-A2 structures, with wavelengths reaching up to 1200 nm or higher. This ensures the blood-brain barrier permeability of the dyes as probes, enabling their application in in vivo in situ brain imaging.

Description

Technical Field

[0001] This invention relates to the field of fluorescent dyes and biomedical interdisciplinary technologies, specifically to a series of low molecular weight fluorescent dyes with emission wavelengths ranging from the visible light window to the second near-infrared window, their synthesis methods, and their applications. Background Technology

[0002] In recent years, the incidence of neurodegenerative diseases has been rising, accounting for a significant proportion of human diseases, but the specific pathogenic mechanisms of these diseases remain unclear. The blood-brain barrier (BBB) ​​prevents most exogenous substances, including drugs and dyes, from entering the brain, and is one of the major obstacles hindering the development of brain science research. Fluorescence imaging technology, especially near-infrared second window (NIR-II, 1000–1700 nm) fluorescence imaging technology, has received increasing attention in brain science research. This is mainly because the longer excitation and emission wavelengths of NIR-II fluorescence imaging technology can effectively suppress the inherent background fluorescence interference and light scattering of biological tissues. Therefore, the tissue penetration during imaging is deeper, and the spatial resolution and signal-to-noise ratio are higher, greatly improving the reliability of brain imaging results. However, against the backdrop of the new demand for precise imaging, the development of NIR-II fluorescent dyes suitable for brain science research remains slow. A significant bottleneck in brain optical imaging is the lack of NIR-II emitting dyes capable of penetrating the BBB. Therefore, there is an urgent need to develop a class of dyes / probes that possess both NIR-II emission and BBB penetration capabilities.

[0003] To date, several typical NIR-II molecular dyes have been reported, including polymethyl dyes, benzothiadiazole dyes, BODIPY dyes, and rhodamine-modified dyes. While these classic dyes have shown promising results in angiography, respiratory rate measurement, and fluorescence-guided tumor resection, their large molecular weight (MW > 600 Da), inherent macromolecular framework structure, and positive charge make them unsuitable for crossing the brain-body barrier (BBB) ​​and difficult to apply to in vivo in situ brain imaging. This is because these dyes are generally designed to achieve NIR-II emission by expanding the π-conjugated system of the dye to obtain a large rigid plane or conjugated framework, which inadvertently increases the molecular weight and lipophilicity of the dye. Therefore, there is an urgent need to develop a new class of compact NIR-II fluorescent dyes to resolve the long-standing contradiction between maintaining a small molecular weight and possessing a long NIR-II emission wavelength, thus better meeting the needs of brain imaging research. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a series of low molecular weight fluorescent dyes with emission wavelengths from the visible light window to the second near-infrared window and their synthesis methods, which are in response to the shortcomings of the above-mentioned prior art. They have good versatility, molecular weight <500Da, and emission wavelengths from 444nm to 1218nm. In particular, dyes with wavelengths in the NIR-II region can better meet the needs of brain imaging research.

[0005] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows:

[0006] A series of low molecular weight fluorescent dyes with emission wavelengths ranging from the visible light window to the second near-infrared window, satisfying the D-π1-A1 or D-π1-A1-π2-A2 structure, with the general structural formula shown in equation (I):

[0007]

[0008] In formula (I): n is 0 to 3;

[0009] R1- is selected from -H, -N(CH3)2, etc.;

[0010] R2- is -H, and the low molecular weight fluorescent dye satisfies the D-π1-A1 structure; or R2 is selected from... One or more of them, and connected to formula (1) by the leftmost carbon bond on the side chain, satisfying the D-π1-A1-π2-A2 structure.

[0011] According to the above scheme, the small-volume fluorescent dye includes one or more dyes with the structure shown in formula (I).

[0012] According to the above scheme, the synthesis method of the low molecular weight fluorescent dye varies depending on the groups selected for R1- and R2-, and mainly includes the following five synthetic routes:

[0013] Route 1: n is one of 0 to 3, R1 is selected from -H, -N(CH3)2, etc.; when R2 is -H, the synthesis method includes the following steps:

[0014] Trans-cinnamaldehyde and its derivatives were reacted with N-acetylglycine and anhydrous sodium acetate in acetic anhydride to prepare intermediate 1. Intermediate 1 was then reacted with anhydrous potassium carbonate and an aqueous solution of methylamine (30%-40% by mass of methylamine) in ethanol to prepare compounds of general formula (I) (specifically, compounds I-1 to I-5 as described in "Specific Embodiments"). The reaction route of this synthetic method is as follows:

[0015]

[0016] More specifically, the synthesis process of Route 1 includes the following steps:

[0017] 1) In a round-bottom flask, trans-cinnamaldehyde and its derivatives, N-acetylglycine, and anhydrous sodium acetate were dissolved in acetic anhydride to obtain a reaction solution. The reaction solution was then reacted at 90-110℃ for 4-6 hours. The mixture was then extracted by vacuum distillation, and intermediate 1 was directly used in the next step. The molar ratio of benzaldehyde and its derivatives, N-acetylglycine, and anhydrous sodium acetate was 1:(3-8):(3-8), and the concentration of benzaldehyde and its derivatives in acetic anhydride was in the range of 0.5-1 mol / L.

[0018] 2) Dissolve intermediate 1, anhydrous potassium carbonate, and an aqueous solution of methylamine in ethanol and react under reflux for 2-5 hours. After the reflux reaction is complete, cool to room temperature and adjust the pH of the system to neutral with an ethanol solution containing hydrochloric acid. Finally, cool the crystals and crystallize at 0°C for 4 hours to obtain compounds of general formula (I) (specifically, compounds I-1 to I-5 in the "Specific Embodiments"). The molar ratio of intermediate 1, anhydrous potassium carbonate, and methylamine is 1:(5-8):(7-10), and the concentration of intermediate 1 in ethanol is in the range of 0.2-0.3 mol / L.

[0019] Route 2: When R1 is -N(CH3)2 and R2 is When n is one of 0 to 3, the synthesis method includes the following steps:

[0020] p-Dimethylaminobenzyl-imidazolinone and its derivatives were dissolved in 1,4-dioxane with selenium dioxide (SeO2) to prepare intermediate 2; intermediate 2 was dissolved in anhydrous ethanol with benzothiazol-2-acetonitrile and tetrabutylammonium bromide to prepare compounds of general formula (I) (specifically, compounds I-6 to I-9 as described in "Specific Embodiments"). The reaction route of this synthetic method is as follows:

[0021]

[0022] More specifically, the synthesis process of Route 2 includes the following steps:

[0023] 1) Add p-dimethylaminobenzyl-imidazolinone and its derivatives, selenium dioxide (SeO2), and 1,4-dioxane solvent to a round-bottom flask. Stir and heat to 90-110℃ for 4-8 hours. Cool the reaction to room temperature, filter off the solid SeO2, combine the obtained intermediate 2, concentrate it, and use it for the next step without purification. The molar ratio of p-dimethylaminobenzyl-imidazolinone and its derivatives to selenium dioxide is 1:(1-1.3), and the concentration of p-dimethylaminobenzyl-imidazolinone and its derivatives in 1,4-dioxane is in the range of 0.1-0.3 mol / L.

[0024] 2) Benzothiazol-2-acetonitrile and tetrabutylammonium bromide were added to anhydrous ethanol, followed by intermediate 2. The reaction temperature was raised to 70-90℃ under stirring and the reaction was continued for 8-12 h. After the reaction was completed, the mixture was cooled to room temperature, extracted and concentrated with dichloromethane, and finally separated by column chromatography to obtain a dark green solid powder, yielding a compound of general formula (I). The molar ratio of intermediate 2, benzothiazol-2-acetonitrile, and tetrabutylammonium bromide was 1:(1-3):(0.01-0.02), and the concentration of intermediate 2 in anhydrous ethanol was in the range of 0.03-0.05 mol / L.

[0025] Route 3: When n is any one of 0 to 2, R1 is -N(CH3)2; R2 is... The synthesis method includes the following steps:

[0026] Dimethylaminobenzyl-imidazolinone and its derivatives are dissolved in tetrahydrofuran with 4-quinoline carboxaldehyde and zinc chloride to prepare intermediate 3 (compounds I-10 to I-12 in the "Specific Embodiments" are one of intermediate 3); intermediate 3 is reacted with iodomethane (CH3I) dissolved in toluene to prepare compounds of general formula (I) (specifically, compounds I-13 to I-15 in the "Specific Embodiments"). The reaction route of this synthetic method is as follows:

[0027]

[0028] More specifically, the synthesis process of Route 3 includes the following steps:

[0029] 1) One of dimethylaminobenzylidene-imidazolinone and its derivatives, 4-quinoline carboxaldehyde and zinc chloride are dissolved in tetrahydrofuran, heated to 70-90℃ with stirring, and reacted for 4-8 hours; after the reaction is completed, it is cooled to room temperature, extracted and concentrated with dichloromethane, and finally separated by column chromatography to obtain a reddish-brown solid powder, namely intermediate 3 (one of compounds I-10 to I-12 in "Specific Embodiments"); wherein, the molar ratio of dimethylaminobenzylidene-imidazolinone and its derivatives, 4-quinoline carboxaldehyde and zinc chloride is 1:(1-1.5):(1-1.1), and the concentration of dimethylaminobenzylidene-imidazolinone and its derivatives in tetrahydrofuran is in the range of 0.03-0.04 mol / L.

[0030] 2) Intermediate 3 and iodomethane (CH3I) are added to toluene solvent, heated to 100-120°C, and stirred for 8-12 hours. After the reaction is complete, the mixture is cooled to room temperature to precipitate a solid. Finally, the solid is obtained by column chromatography (specifically, compounds I-13 to I-15 in the "Specific Embodiments"). The molar ratio of intermediate 3 to iodomethane is 1:(5-15), and the concentration of intermediate 3 in toluene is 5-15 mmol / L.

[0031] Route 4: When n is 0, R1 is -N(CH3)2, R2 is... or The synthesis method includes the following steps:

[0032] (E)-3-(4-(dimethylamino)phenyl)propenal (i.e., compound 3 in the "Specific Embodiments") or 5-(4-(dimethylamino)phenyl)thiophene-2-carboxaldehyde was reacted with compound I-2 (named (Z)-5-(4-(dimethylamino)benzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one) in ethanol to prepare compounds of general formula (I) (specifically, such as compound I-16 or I-17 in the "Specific Embodiments").

[0033]

[0034] More specifically, the synthesis process of Route 4 includes the following steps:

[0035] (E)-3-(4-(dimethylamino)phenyl)propenal or 5-(4-(dimethylamino)phenyl)thiophen-2-carboxaldehyde were added to an ethanol solvent with compound I-2, and then piperidine was added under stirring. The mixture was heated to 60°C-80°C and refluxed for 3-5 hours to obtain compounds of general formula (I) (specifically, compounds I-16 or I-17 in the "Specific Embodiments"). The molar ratio of (E)-3-(4-(dimethylamino)phenyl)propenal to compound I-2 and pyridine was 1:(0.7-0.9):(0.1-0.3); the molar ratio of 5-(4-(dimethylamino)phenyl)thiophen-2-carboxaldehyde to compound I-2 and pyridine was 1:(0.7-0.9):(0.1-0.3); and the concentration of compound I-2 in the ethanol solvent was in the range of 0.01-0.02 mol / L.

[0036] Route 5: When n is 0, R1 is -N(CH3)2, R2 is... The synthesis method includes the following steps:

[0037] Compound I-3, ZnCl2 and (E)-3-(quinolin-4-yl)propenal were added to the solvent tetrahydrofuran to react and prepare compounds of general formula (I) (specifically, such as compound I-18 in "Specific Embodiments").

[0038]

[0039] More specifically, the synthesis process of Route 5 includes the following steps:

[0040] Compound I-3, (E)-3-(quinoline-4-yl)propenal, ZnCl2, and tetrahydrofuran solvent were added to a round-bottom flask and heated to 60-80°C with stirring for 6-8 hours. After the reaction was completed, the mixture was cooled to room temperature, extracted and concentrated, and separated by column chromatography to obtain the compound of general formula (I) (specifically, as compound I-18 in "Specific Embodiments"). The molar ratio of compound I-3, 3-(quinoline-4-yl)propenal to ZnCl2 was 1:(1-1.5):(0.9-1.1), and the concentration of (E)-3-(quinoline-4-yl)propenal in the tetrahydrofuran solvent was 0.02-0.04 mol / L.

[0041] The technical concept of this invention is as follows:

[0042] As is well known, push-pull (D-π-A) dyes possess unique intramolecular charge transfer (ICT) properties. Structurally, the absorption and emission wavelengths of the fluorophore can be tuned by altering its push-pull effect and / or effective π-conjugation. On one hand, the introduction of strong electron donor (EDG) and electron acceptor (EWG) groups can increase the energy level of the highest occupied molecular orbital (HOMO) and decrease the energy level of the lowest unoccupied molecular orbital (LUMO). The narrowing of the HOMO-LUMO band gap is proportional to a redshift in the dye's absorption and emission wavelengths. On the other hand, under strong intramolecular interactions, the molecular conformation may undergo intramolecular torsion accompanied by electron transfer, leading to enhanced polarization and complete charge separation between the HOMO and LUMO bands, resulting in a significant Stokes shift and redshifted fluorescence. Simultaneously, modulating the push-pull electron capabilities of the donor / acceptor units to enhance the ICT effect may achieve a redshift in the emission wavelength. Another feasible method to promote a redshift in the dye's absorption / emission wavelengths is to expand the conjugated structure of the molecular backbone. However, it should be noted that this method often involves complex synthetic processes to obtain dyes with large conjugated rigid planar structures and high molecular weights. This may not only lead to the enhancement of harmful π-π stacking interactions, but also to undesirable lipophilicity, reduced BBB permeability, and decreased brain delivery efficiency of the dye.

[0043] This invention creatively selects low-molecular-weight flexible conjugated polyolefin chains as π-electron bridges, rather than blindly extending the conjugated structure. This effectively expands the π-conjugated system while maintaining the small molecular weight of the dye. Furthermore, by simultaneously adjusting the electron-pulling ability of the donor / acceptor and extending the π-conjugated structure through conjugated polyolefin chains to enhance the ICT effect, redshift emission (e.g., ...) can be achieved while maintaining a small molecular weight. Figure 1 (As shown in Figure A). Therefore, based on the p-hydroxybenzylidene-imidazolinone (p-HBD) chromophore, this invention utilizes an integrated molecular design strategy to synergistically enhance the ICT effect, including improving the electron push-pull capability of the donor / acceptor and extending the π-conjugation length of the dye. This has led to the construction of a series of derivatives with D-π-A1 or D-π-A1-π-A2 structures (i.e., low molecular weight fluorescent dyes represented by the general structural formula (I)), with wavelengths reaching up to 1200 nm or higher (e.g., ...). Figure 1 (As shown in B). The D-π-A1-π-A2 framework structure synergistically enhances the ICT effect, thereby achieving maximum redshift emission of NIR-II while retaining the small molecular weight of the molecule, ensuring the probe's BBB permeability.

[0044] Compared with the prior art, the beneficial effects of the present invention are:

[0045] This invention achieves a series of compact, low-molecular-weight organic dyes with emission wavelengths ranging from the visible light window to the second near-infrared window by balancing the molecular weight and emission wavelength of the dyes using a rational strategy. In particular, the dyes exhibiting NIR-II fluorescence emission can effectively increase the penetration depth of light in the skull and reduce light scattering and absorption in brain tissue, thus enabling better application in imaging brain diseases. Attached Figure Description

[0046] Figure 1 This is a technical concept diagram of the dye described in this invention;

[0047] Figure 2 The UV-Vis absorption spectra of dyes I-1 to I-18 (i.e., compounds I-1 to I-18) described in this invention;

[0048] Figure 3 The fluorescence spectra of dyes I-1 to I-18 described in this invention;

[0049] Figure 4 The Stokes shift spectra of dyes I-1 to I-18 described in this invention;

[0050] Figure 5 The graphs show a comparison of the molecular weights of dyes I-1 to I-18 described in this invention with those of reported or commercial dyes.

[0051] Figure 6The above are comparative spectra of the Stokes shifts of dyes I-1 to I-18 described in this invention with those of reported or commercial dyes.

[0052] Figure 7 The photostability spectra of dyes I-6 to I-9 and I-13 to I-15 (>900 nm) with longer emission wavelengths as described in this invention are shown.

[0053] Figure 8 The NIRII fluorescence signal acquisition spectra of the dyes I-6 to I-9 and I-13 to I-15 (>900nm) with longer emission wavelengths as described in this invention;

[0054] Figure 9 The dye I-7 described in this invention is used to visualize the in vivo brain images of Alzheimer's disease mice.

[0055] Figures 10 to 27 The following are high-resolution mass spectra of dyes I-1 to I-18 described in this invention. Detailed Implementation

[0056] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the present invention is not limited to the following embodiments.

[0057] In this invention, a series of compact, low-molecular-weight fluorescent dyes with emission wavelengths ranging from the visible light window to the second near-infrared window are selected from one or more compounds I-1 to I-18 with the following structures:

[0058]

[0059] This invention provides a specific synthetic route for the above-mentioned low molecular weight fluorescent dyes, as shown below:

[0060]

[0061] Furthermore, the reaction conditions 1)-10) involved in the above synthetic route are as follows: 1) N-acetylglycine, sodium acetate, acetic anhydride, 100℃, 5 hours;

[0062] 2) Methylamine, K2CO3, EtOH, reflux at 80℃ for 3 hours;

[0063] 3) SeO2, 1,4-dioxane, 100℃, 6 hours;

[0064] 4) Benzothiazol-2-acetonitrile, TBAB, EtOH, 80℃, 10 hours;

[0065] 5) 4-Quinoline formaldehyde, ZnCl2, THF, 80℃, 6 hours;

[0066] 6) CH3I, toluene, 110℃, 10 hours;

[0067] 7) Piperidine, EtOH, 80℃, 3 hours;

[0068] 8) Piperidine, EtOH, 80℃, 3 hours;

[0069] 9) Compound 7, ZnCl2, THF, 80℃, 6 hours.

[0070] Example 1

[0071] Synthesis of compound I-1:

[0072] Trans-cinnamaldehyde (4 g 0.03 mol, i.e., compound 1 in the aforementioned specific synthetic route), N-acetylglycine (17.7 g 0.151 mol), and anhydrous sodium acetate (12.3 g 0.151 mol) were dissolved in acetic anhydride (50 mL) and placed in a round-bottom flask. The reaction solution was then reacted at 100 °C for 5 h. After cooling the product to room temperature, the acetic anhydride was removed under reduced pressure, the product was extracted with dichloromethane, and then concentrated under reduced pressure. The resulting intermediate 1 was used directly in the next step.

[0073] Intermediate 1 (1 g 0.0047 mol), anhydrous potassium carbonate (4.15 g 0.03 mol), and a 33% aqueous solution of methylamine (4 ml, containing 0.04 mol of methylamine) were dissolved in 20 mL of ethanol and reacted under reflux for 3 hours. After the reaction was complete, the mixture was cooled to room temperature, and the pH of the reaction solution was adjusted to neutral using an ethanol solution containing 10% hydrochloric acid. Finally, the crystals were cooled and crystallized at 0 °C for 4 hours to obtain compound I-1, i.e., dye I-1. The results of the 1H NMR and high-resolution mass spectrometry of compound I-1 are as follows, confirming the existence of compound I-1:

[0074] 1 H NMR (400MHz, CDCl3) δ7.57–7.48(m,3H),7.27(ddd,J=12.5,8.4,4.3Hz,4H),6.95(dd,J=13.7,9.1Hz,2H),3.10(s,3H),2.30(s,3H). 13 C NMR (101MHz, CDCl3) δ160.85 (s), 142.42 (s), 129.37 (s), 128.83 (d, J = 4.0Hz) ,127.71(s),123.15(s),77.36(s),77.04(s),76.72(s),26.61(s),15.49(s).

[0075] HRMS(ESI):calcd for C 14H 15 N2O + 227.1179, found 227.1178 [M+H] + .

[0076] Example 2

[0077] Synthesis of compound I-2:

[0078] The synthesis procedure for compound I-1 was followed, except that compound 1 was replaced with compound 2. The 1H NMR and high-resolution mass spectrometry results for compound I-2 are shown below, confirming the formation of compound I-2:

[0079] 1 H NMR (400MHz, DMSO) δ8.05(d,J=8.9Hz,2H),6.86(s,1H),6.74(d,J=9.0Hz,2H),3.08(s,3H),3.00(s,6H),2.32(s,3H).

[0080] HRMS(ESI):calcd for C 14 H 18 N3O + 244.1444, found 244.1445[M+H] + .

[0081] Example 3

[0082] Synthesis of compound I-3:

[0083] The synthesis procedure for compound I-1 was followed, except that compound 1 was replaced with compound 3. The 1H NMR and high-resolution mass spectrometry results for compound I-3 are shown below, confirming the formation of compound I-3:

[0084] 1 H NMR (400MHz, DMSO) δ7.44(d,J=8.8Hz,2H),7.30–7.19(m,1H),7.14–7.06(m,1H),6.84( d,J=11.4Hz,1H),6.74(dd,J=16.3,7.4Hz,2H),3.06(s,3H),2.98(s,7H),2.29(s,3H).

[0085] HRMS(ESI):calcd for C 16 H 20 N3O + 270.1601, found 270.1603[M+H] + .

[0086] Example 4

[0087] Synthesis of compound I-4:

[0088] The synthesis procedure for compound I-1 was followed, except that compound 1 was replaced with compound 4. The 1H NMR and high-resolution mass spectrometry results for compound I-4 are shown below, confirming the yield of compound I-4:

[0089] 1 H NMR (400MHz, CDCl3) δ7.43–7.25(m,2H),7.03–6.76(m,2H),6.76–6.55(m,4H),3.10(d,J=5.2Hz,3H),2.94(d,J=4.0Hz,6H),2.28(d,J=3.4Hz,3H).

[0090] HRMS(ESI):calcd for C 18 H 22 N3O + 296.1757, found 296.1755[M+H] + .

[0091] Example 5

[0092] Synthesis of compound I-5:

[0093] The synthesis procedure for compound I-1 was followed, except that compound 1 was replaced with compound 5. The 1H NMR and high-resolution mass spectrometry results for compound I-5 are shown below, confirming the formation of compound I-5:

[0094] 1 H NMR (400MHz, DMSO) δ7.65(s,1H),7.61(dd,J=7.6,3.8Hz,4H),7.55(dd,J=9.9,4.4Hz,4H),3.96(dd,J=16.8,10.2Hz,3H),1.34(s,3H),1.23(s,6H). 13 C NMR(101MHz,DMSO)δ132.51(d,J=2.6Hz),131.95(d,J=9.8Hz),129.23(d,J=11.8Hz),40.61(s),4 0.40(s),40.20(s),40.23–39.76(m),39.76–39.72(m),39.57(s),39.36(s),32.00(s),29.89(s).

[0095] HRMS(ESI):calcd for C18 H 22 N3O + 296.1757, found 296.1755[M+H] + .

[0096] Example 6

[0097] Synthesis of compound I-6:

[0098] Compound I-2 (500 mg 2.06 mmol), SeO2 (274 mg 2.46 mmol), and 10 mL of 1,4-dioxane solvent were added to a round-bottom flask. The mixture was heated to 100 °C with stirring for 6 hours. After the reaction was complete, the mixture was cooled to room temperature, the solid SeO2 was filtered off, and the resulting intermediate 2 was combined, concentrated, and used in the next step without further purification.

[0099] Intermediate 2 (200 mg 0.78 mmol) was added to 20 mL of anhydrous ethanol pre-contained with benzothiazol-2-acetonitrile (271 mg 1.56 mmol) and tetrabutylammonium bromide (38 mg 0.117 mmol). The mixture was then heated to 80 °C with stirring and reacted for 10 hours. After the reaction was complete, the mixture was cooled to room temperature, extracted with dichloromethane, and concentrated. Finally, a dark green solid powder, compound I-6, was obtained by column chromatography. The 1H NMR and high-resolution mass spectrometry results of compound I-6 are shown below, confirming the existence of compound I-6:

[0100] 1 H NMR(400MHz,TFA-D)δ8.75(d,J=8.7Hz,2H),8.42(d,J=8.4Hz,1H),8.36(d,J=8.2Hz,1H),8.08(s, 1H),8.01(s,1H),7.94(s,1H),7.89(d,J=8.8Hz,3H),3.66(s,3H),3.61(s,6H).HRMS(ESI):calcd for C 23 H 20 N5OS+414.1383,found 414.1380[M+H]+.

[0101] Example 7

[0102] Synthesis of compound I-7:

[0103] The synthesis of compound I-6 was carried out following the same steps, except that compound I-2 was replaced with compound I-3. The 1H NMR and high-resolution mass spectrometry results of compound I-7 are shown below, confirming the formation of compound I-7:

[0104] 1 H NMR(400MHz,TFA-D)δ8.40(d,J=8.3Hz,1H),8.27(d,J=8.0Hz,1H),8.11–8. 02(m,3H),7.91–7.79(m,5H),7.73–7.66(m,2H),3.77(s,3H),3.54(s,6H). 13 C NMR(101MHz,TFA-D)δ162.43(s),161.99(s),161.56(s),161.12(s),150.25(s),149 .77(s),131.82(s),128.95(s),118.43(s),115.61(s),115.08(s),114.48(s),112. 80(s),111.65(s),109.99(s),70.56(s),65.62(s),51.92(s),46.90(s),38.59(s), 29.80(s),29.08(s),28.40(s),23.05(s),22.19(s),12.12(s),11.69(s),9.12(s).

[0105] HRMS(ESI):calcd for C 25 H 22 N5OS + 440.1540, found 440.1540[M+H] + .

[0106] Example 8

[0107] Synthesis of compound I-8:

[0108] The synthesis of compound I-6 was carried out following the same steps, except that compound I-2 was replaced with compound I-4. The 1H NMR and high-resolution mass spectrometry results of compound I-8 are shown below, confirming the yield of compound I-8:

[0109] 1 H NMR(400MHz,TFA-D)δ8.46(d,J=8.3Hz,1H),8.32(d,J=8.2Hz,1H),8.06–7.92(m,6H),7.7 6(d,J=8.5Hz,3H),7.51(s,1H),5.29(s,2H),3.82(s,3H),3.56(s,6H).HRMS(ESI):calcd for C 27 H 24 N5OS +466.1696, found 466.1691[M+H] + .

[0110] Example 9

[0111] Synthesis of compound I-9:

[0112] The synthesis of compound I-6 was carried out following the same steps, except that compound I-2 was replaced with compound I-5. The 1H NMR and high-resolution mass spectrometry results of compound I-9 are shown below, confirming the formation of compound I-9:

[0113] 1 H NMR (400MHz, TFA-D) δ8.46(d,J=8.3Hz,1H),8.32(d,J=8.2Hz,2H),8.16–7.86(m,8H),7.76(d,J=8.5Hz,4H),7.51(s,1H),3.82(s,3H),3.56(s,6H).

[0114] HRMS(ESI):calcd for C 29 H 26 N5OS + 492.1853, found 492.1859 [M+H] + .

[0115] Example 10

[0116] Synthesis of compound I-10:

[0117] Compound I-2 (83 mg 0.34 mmol), 4-quinolinecarbaldehyde (64 mg 0.41 mmol), zinc chloride (46 mg 0.34 mmol), and 10 mL of tetrahydrofuran solvent were added to a round-bottom flask. The mixture was heated to 80 °C with stirring and reacted for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, extracted with dichloromethane, and concentrated. Finally, a reddish-brown solid powder, the corresponding compound I-10, was obtained by column chromatography. The 1H NMR and high-resolution mass spectrometry results of compound I-10 are shown below, confirming the existence of compound I-10:

[0118] 1 H NMR(400MHz, CDCl3)δ8.90(d,J=4.5Hz,1H),8.63(d,J=15.6Hz,1H),8.23(d,J=8.4Hz,1H),8.20–8.0 9(m,3H),7.80–7.54(m,3H),7.01(d,J=15.6Hz,1H),6.69(d,J=8.8Hz,2H),3.31(s,3H),3.03(s,6H).13 C NMR(101MHz, CDCl3)δ170.53(s),155.33(s),151.96(s),135.35(s),135.05(s),132.88(s),130.97(s),130.02(s),127.35(s) ),123.59(s),122.66(s),120.08(s),117.69(s),111.91(s),77.35(s),77.03(s),76.71(s),40.10(s),29.71(s),26.74(s).

[0119] HRMS(ESI):calcd for C 24 H 23 N4O + 483.1866, found 483.1865[M+H] + .

[0120] Example 11

[0121] Synthesis of compound I-11:

[0122] The synthesis of compound I-10 was carried out following the same steps, except that compound I-2 was replaced with compound I-3. The 1H NMR and high-resolution mass spectrometry results of compound I-11 are shown below, confirming the formation of compound I-11:

[0123] 1 H NMR (400MHz, CDCl3) δ8.98(t,J=5.3Hz,1H),8.77(d,J=15.7Hz,1H),8.37(d,J=8.2Hz,1H),8.21(d,J=7.6Hz,1H),7.82(ddd,J=8.4,6.9,1.3Hz,1H),7 .73–7.62(m,3H),7.56(t,J=8.3Hz,2H),7.23(d,J=12.0Hz,1H),7.08(dt, J=15.7,9.8Hz,2H),6.71(dd,J=8.8,6.9Hz,2H),3.39(s,3H),3.07(s,6H).

[0124] HRMS(ESI):calcd for C 26 H 25 N4O + 409.2023, found 409.2025 [M+H] + .

[0125] Example 12

[0126] Synthesis of compound I-12:

[0127] The synthesis of compound I-10 was carried out following the same steps, except that compound I-2 was replaced with compound I-4. The 1H NMR and high-resolution mass spectrometry results of compound I-12 are shown below, confirming the yield of compound I-12:

[0128] 1 H NMR (400MHz, CDCl3) δ8.88(t,J=5.0Hz,1H),8.60(dd,J=36.6,15.7Hz,1H),8.23(t,J=8.9Hz,1H),8.12(dd,J=8.1,4.6Hz,1H),7.79–7.68(m,1H ),7.59(dt,J=9.3,2.8Hz,2H),7.31(d,J=8.8Hz,2H),7.20(d,J=4.7Hz, 1H),7.10–6.67(m,5H),6.60(d,J=8.8Hz,2H),3.28(s,3H),2.94(s,6H). 13 C NMR(101MHz, CDCl3)δ169.24(s),155.60(s),150.90(s),149.87(s),148.58(s),145.63(s),141.0 4(s),139.73(s),133.71(s),131.43(s),130.00(d,J=10.3Hz),128.82(d,J=7.5Hz),127.37(s),12 6.90(s),126.15(s),125.74(s),124.73(d,J=12.5Hz),123.54(s),119.34(s),117.50(d,J=12.4Hz ),112.16(s),77.30(d,J=11.5Hz),77.04(s),76.72(s),40.24(s),29.71(s),26.72(s),26.48(s).

[0129] HRMS(ESI):calcd for C 28 H 27 N4O + 435.2179, found 435.2169 [M+H] + .

[0130] Example 13

[0131] Synthesis of compound I-13:

[0132] Compound I-10 (99.5 mg 0.26 mmol) was added to a round-bottom flask, followed by CH3I (371 mg 2.6 mmol) and 30 mL of toluene solvent. The mixture was heated to 110 °C and stirred for 10 hours. After the reaction was complete, the mixture was cooled to room temperature to precipitate a solid. Finally, a green solid powder, compound I-13, was obtained by column chromatography. The 1H NMR and high-resolution mass spectrometry results of compound I-13 are shown below, confirming the existence of compound I-13:

[0133] 1 H NMR (400MHz, DMSO) δ9.51(d,J=6.4Hz,1H),8.87(d,J=8.5Hz,1H),8.76–8.66(m,2H),8.53(d,J=9.0Hz,1H),8.35–8.25(m,3H ),8.15(d,J=7.8Hz,1H),7.92(d,J=15.5Hz,1H),7.16(s,1H),6.84(d,J=9.1Hz,2H),4.62(s,3H),3.38(s,3H),3.09(s,6H). 13 CNMR (101MHz, DMSO) δ 135.76 (d, J = 15.1Hz), 131.16 (s), 112.50 (s), 40.62 (s), 40.41 (s), 40.10 (d, J = 21.0Hz), 39.79 (s), 39.58 (s), 39.37 (s).

[0134] HRMS(ESI):calcd for C 25 H 25 N4O + 397.2023, found 397.2021 [M].

[0135] Example 14

[0136] Synthesis of compound I-14:

[0137] Following the synthetic steps of compound I-13, simply replace compound I-10 with compound I-11. The 1H NMR and high-resolution mass spectrometry results of compound I-14 are shown below, confirming the acquisition of compound I-14:

[0138] 1H NMR (400MHz, DMSO) δ9.51(d,J=6.4Hz,1H),8.90(d,J=8.6Hz,1H),8.73(dd,J=10 .9,4.3Hz,2H),8.53(d,J=8.7Hz,1H),8.39–8.29(m,1H),8.13(t,J=7.6Hz,2H),7 .93(d,J=15.7Hz,1H),7.55(dd,J=9.8,6.2Hz,2H),7.32(d,J=15.0Hz,2H),7.17 (d,J=11.8Hz,1H),6.77(d,J=8.4Hz,2H),4.62(s,3H),3.38(s,3H),3.03(s,6H). 13 C NMR (126MHz, DMSO) δ168.71 (s), 155.40 (s), 152.11 (s), 150.34 (s), 139.28 (s), 130.49 (d, J = 7.6Hz), 127. 08(s),124.28(s),112.55(s),45.66(s),31.69(d,J=17.2Hz),29.48(s),29.17(s),27.11(s),14.42(s).

[0139] HRMS(ESI):calcd for C 27 H 27 N4O + 423.2179, found 423.2188 [M] + .

[0140] Example 15

[0141] Synthesis of compound I-15:

[0142] Following the synthetic steps of compound I-13, simply replace compound I-10 with compound I-12. The 1H NMR and high-resolution mass spectrometry results of compound I-15 are shown below, confirming the acquisition of compound I-15:

[0143] 1H NMR (400MHz, DMSO) δ9.50(dd,J=10.7,6.5Hz,1H),8.87–8.49(m,4H),8.38–8.28(m,1H),8.12(dd,J=14.2,6.9Hz,1H),7.99–7.86(m,1H),7. 46(t,J=8.0Hz,2H),7.33–6.99(m,4H),6.91(d,J=15.1Hz,1H),6.72(dd,J=8.8,4.3Hz,2H),4.61(d,J=7.1Hz,3H),3.36(s,3H),2.99(s,6H). 13 C NMR(101MHz,DMSO)δ130.57(s),112.56(s),56.49(s),40.61(s),40.42(s) ,40.30(d,J=21.0Hz),39.99(s),39.78(s),39.57(s),39.36(s),19.03(s).

[0144] HRMS(ESI):calcd for C 29 H 29 N4O + 449.2336, found 449.2326 [M] + .

[0145] Example 16

[0146] Synthesis of compound I-16:

[0147] (E)-3-(4-(dimethylamino)phenyl)propenal (86 mg 0.49 mmol) and compound I-2 (100 mg 0.41 mmol) were added to 30 mL of ethanol and transferred to a round-bottom flask. Piperidine (8.3 mg 0.098 mol) was then added with stirring. The mixture was heated to 80 °C and refluxed for 3 hours. After cooling, the mixture was concentrated by rotary evaporation and separated by column chromatography to obtain compound I-16. The results of the 1H NMR and high-resolution mass spectrometry of compound I-16 are shown below, confirming the existence of compound I-16:

[0148] 1H NMR (400MHz, CDCl3) δ8.08(d,J=8.6Hz,2H),7.79(dd,J=14.8,10.8Hz,1H),7.32(d,J=8.5Hz,2H),7.01(s,1H), 6.88–6.76(m,2H),6.64(dd,J=13.1,6.8Hz,4H),6.20(d,J=14.9Hz,1H),3.19(s,3H),3.00(s,6H),2.94(s,6H). 13 C NMR(101MHz, CDCl3)δ157.07(s),151.39(s),150.90(s),134.38(s),128.67(s),127.55(s),123.35(s),11 2.17(s),111.88(s),77.35(s),77.03(s),76.72(s),58.48(s),40.19(d,J=15.8Hz),26.50(s),18.45(s).

[0149] HRMS(ESI):calcd for C 25 H 29 N4O + 401.2336, found 401.2323[M+H] + .

[0150] Example 17

[0151] Synthesis of compound I-17:

[0152] 5-(4-(dimethylamino)phenyl)thiophene-2-carboxaldehyde (113 mg 0.49 mmol) and compound I-2 (100 mg 0.41 mmol) were added to 30 mL of ethanol and transferred to a round-bottom flask. Piperidine (8.3 mg 0.098 mol) was then added with stirring. The mixture was heated to 80 °C and refluxed for 3 hours. After cooling, the mixture was concentrated by rotary evaporation and separated by column chromatography to obtain compound I-17. The 1H NMR and high-resolution mass spectrometry results of compound I-17 are shown below, confirming the existence of compound I-17:

[0153] 1 H NMR(400MHz, DMSO)δ8.16(d,J=8.5Hz,2H),8.04(d,J=15.4Hz,1H),7.64–7.51(m,3H),7.3 8(d,J=3.7Hz,1H),6.90(s,1H),6.84–6.65(m,6H),3.24(s,3H),3.04(s,6H),2.97(s,6H). 13C NMR(126MHz,DMSO)δ170.12(s),157.29(s),151.72(s),150.86(s),148.09(s),137.88(s),134.57(s) ,127.05(s),122.73(d,J=9.4Hz),121.31(s),112.83(s),112.28(s),56.50(s),55.37(s),26.69(s).

[0154] HRMS(ESI):calcd for C 27 H 29 N4OS + 457.2057, found 457.2043 [M+H] + .

[0155] Example 18

[0156] Synthesis of compound I-18:

[0157] Compound I-3 (67 mg, 0.25 mmol), 3-(quinolin-4-yl)propenal (55 mg, 0.30 mmol, i.e., compound 7), ZnCl2 (33 mg, 0.25 mmol), and 10 mL of THF solvent were added to a round-bottom flask and heated to 80 °C for 6 hours with stirring. After the reaction was completed, the mixture was cooled to room temperature, extracted with dichloromethane, and concentrated. Finally, 87 mg of a reddish-brown solid powder was obtained by column chromatography, which yielded compound I-18. The results of the 1H NMR and high-resolution mass spectrometry of compound I-18 are as follows, confirming the existence of compound I-18:

[0158] 1 H NMR (400MHz, CDCl3) δ8.84(d,J=4.3Hz,1H),8.08(t,J=7.9Hz,2H),7.97–7.86(m,1H),7.77–7.37(m,8H),7.07(d ,J=11.8Hz,1H),6.98(d,J=15.5Hz,1H),6.60(t,J=8.1Hz,2H),6.47(d,J=14.9Hz,1H),3.23(s,3H),2.97(s,6H). 13C NMR(101MHz, CDCl3)δ155.58(s),151.41(s),149.84(s),144.58(s),133.46(s),132.73(s),131.75(s),130.08(s),129.78(d,J=17.2Hz),127 .06(s),124.68(s),123.12(s),119.47(s),119.08(s),116.97(s),111 .98(s),77.35(s),77.03(s),76.72(s),40.18(s),29.71(s),26.59(s).

[0159] HRMS(ESI):calcd for C 28 H 27 N4O + 435.2179, found 435.2180 [M+H] + .

[0160] In summary, based on the above NMR and mass spectrometry test results, it can be determined that the target compounds I-1 to I-18 (i.e. dyes I-1 to I-18) synthesized in all embodiments are consistent with the structures shown in the synthetic routes.

[0161] Performance characterization and applications:

[0162] 1. Normalized UV absorption and fluorescence spectra of dyes I-1 to I-18

[0163] To better understand the photophysical properties of the dyes provided in this invention, the ultraviolet absorption and fluorescence spectra of these dyes were tested. Figure 2 and Figure 3 The normalized spectral results of all dyes in the organic solvent dimethyl sulfoxide are presented separately. For example... Figure 2 As shown, all dyes exhibit broad UV absorption spectra in DMSO, ranging from 300-950 nm; similarly, as Figure 3 The fluorescence emission of the dye shown extends from the visible region to the NIR-II range (414-1218 nm).

[0164] Therefore, the present invention provides a series of novel dyes with emission spectra extending from the visible light region to NIR-II, with a maximum emission wavelength exceeding 1200 nm.

[0165] 2. Stokes shift determination of dyes I-1 to I-18 and comparison with molecular weights of commercial or reported dyes.

[0166] As is well known, complete spectral separation of dyes helps reduce spectral self-absorption interference and crosstalk, thereby obtaining imaging results with higher contrast. From Figure 4 It is known that the dyes provided by this invention have large Stokes shifts, with the largest Stokes shift reaching 566 nm, which is greater than the Stokes shifts of existing known and commercial dyes. Figure 5 The large Stokes shift of the dye is attributed to strong intramolecular charge transfer and significant geometric relaxation during photoexcitation.

[0167] 3. Comparison of molecular weights of dyes I-1 to I-18 with commercial or reported dyes

[0168] The most valuable characteristic of dyes I-1 to I-18 is that, despite their long emission wavelengths, their molecular weights are all less than 500 g / mol, far smaller than those of reported commercial dyes and typical mainstream dyes. Figure 6 The above results demonstrate that the series of dyes provided by this invention effectively balances molecular weight and fluorescence emission wavelength, possesses potential BBB penetration capability, and meets the two key conditions for brain imaging (long wavelength and low molecular weight), thus demonstrating potential application in brain imaging.

[0169] 4. Evaluation of the photostability of dyes I-1 to I-18

[0170] To investigate the photostability of dyes I-1 to I-18, dyes I-6 to I-9 and I-13 to I-15 were used as examples. These dyes have longer fluorescence emission wavelengths and exhibit less tissue interference when applied to in vivo imaging. Compared to the commercial dye ICG, this invention monitors the photostability of the dyes in DMSO (808 nm, 1W cm⁻¹). -2 The fluorescence intensity in the sample was used to evaluate its photostability. For example... Figure 7 As shown, these dyes exhibit good photostability compared to ICG, indicating that this series of dyes can meet the requirements for long-term fluorescence imaging in in vivo imaging.

[0171] 5. NIR-II imaging capability test of dyes I-1 to I-18

[0172] To evaluate the NIR-II imaging capability of the dyes, 50 μM dye solutions were imaged at an excitation wavelength of 808. A series of long-pass filters covering the NIR-II window were used to capture the NIR-II fluorescence properties of the dyes, taking dyes I-6 to I-9 and I-13 to I-15 as examples. Figure 8 As shown, these dyes all exhibit significant NIR-II fluorescence signals in the wavelength range above 1000 nm, and can be used for NIR-II imaging.

[0173] 6. Applications of dye-based NIR-II in vivo imaging

[0174] The feasibility of in vivo imaging of Aβ plaques using dye I-7 was investigated as an example. The specific procedure was as follows: In vivo imaging studies were performed using 6-month-old and 12-month-old APP / PS1 transgenic AD model mice and 12-month-old wild-type mice, respectively. 10-300 minutes after intravenous injection of dye I-7 (0.5 mM, 200 μL), NIR-II fluorescence signals in the brains of AD model mice and wild-type mice were intermittently collected, obtaining the NIR-II fluorescence signals in the brains of AD model mice and wild-type mice, respectively. Figure 9 As shown in Figure A, compared to the AD model mouse group, the NIR-II fluorescence signals collected in the brains of wild-type mice were very small at all time points after injection of dye I-7. In contrast, clear fluorescence signals were observed in 6-month-old and 12-month-old AD mice as early as 10 minutes after injection. Furthermore, the results showed that the dye slowly entered the brain parenchyma from the initial cerebral blood vessels and clearly delineated the shape of the mouse brain over time. The experimental results indicate that dye I-7 can effectively cross the blood-brain barrier and specifically bind to Aβ plaques, prolonging the retention time of the probe in the brains of AD mice and improving the real-time imaging of Aβ plaques in vivo.

[0175] In addition, wild-type mice of the same age and AD mice injected with ICG via the tail vein were selected for NIR-II fluorescence imaging as controls. Figure 9 As shown in Figure B, two minutes after ICG injection, the ICG clearly delineated the blood vessels in the mouse head. However, over time, the fluorescence signal in the brain gradually weakened and eventually disappeared, likely because the ICG was slowly metabolized in the cerebral blood vessels. Therefore, even in AD mice, the dye ICG could not cross the blood-brain barrier to reach the brain parenchyma and bind to Aβ plaques. In situ fluorescence imaging results indicate that the compact fluorescent dyes prepared in this invention, represented by dye I-7, can successfully cross the blood-brain barrier, enabling non-invasive real-time imaging of Aβ plaques.

[0176] 7. Evaluation of the physicochemical parameters of dyes I-1 to I-18.

[0177] This embodiment evaluated the physicochemical parameters of dyes to better assess their blood-brain barrier permeability. Generally, molecules with a molecular weight less than 500 g / mol, log P < 5, H bond acceptors less than 10, and H bond donors less than 5 have potential blood-brain barrier permeability. Therefore, these key parameters of the dye fluorophores were studied using a combination of theoretical calculations and experiments. Taking dyes I-6 to I-12 as examples, their physicochemical properties are shown in Table 1. Moreover, it was found that dyes I-6 to I-9 exhibited better blood-brain barrier permeability compared to dyes I-10 to I-12.

[0178] Table 1

[0179]

[0180]

[0181] This invention is not limited to the above embodiments. For those skilled in the art, several improvements and modifications can be made without departing from the principle of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention.

Claims

1. A fluorescent dye, characterized in that, Molecular weight less than 500 Da, fluorescence emission wavelength in the range of 900-1250 nm, and general structural formula as shown in formula (I): In formula (I): n is 0~3; R1- is -N(CH3)2; R2- is or .

2. The fluorescent dye according to claim 1, characterized in that, The fluorescent dye includes one or more dyes with the structure shown in formula (I).

3. The method for synthesizing the fluorescent dye according to claim 1, characterized in that, When R1 is -N(CH3)2, R2 is When n is any one of 0 to 3, the synthesis method is as follows: The compound shown in formula S was dissolved in 1,4-dioxane with selenium dioxide to prepare intermediate 2; intermediate 2 was dissolved in anhydrous ethanol with benzothiazol-2-acetonitrile and tetrabutylammonium bromide to prepare the compound of general formula (I); the reaction route of this synthetic method is as follows: ； When n is any value between 0 and 2, R1 is -N(CH3)2, and R2 is... The synthesis method is as follows: The compound shown in formula S was dissolved in tetrahydrofuran with 4-quinoline carboxaldehyde and zinc chloride to prepare intermediate 3; intermediate 3 was reacted with iodomethane dissolved in toluene to prepare the compound of general formula (I); the reaction route of this synthetic method is as follows: 。 4. The method for synthesizing fluorescent dyes according to claim 3, characterized in that, When R1 is -N(CH3)2, R2 is When n is one of 0 to 3, the synthesis method includes the following steps: 1) Mix the compound shown in formula S, selenium dioxide (SeO2) and 1,4-dioxane solvent, stir and heat to 90-110℃ for 4-8 h, cool the reaction to room temperature, filter off the solid SeO2, and combine to obtain intermediate 2; 2) Add benzothiazole-2-acetonitrile and tetrabutylammonium bromide to anhydrous ethanol, then add intermediate 2, and raise the reaction temperature to 70-90 °C under stirring, and continue the reaction for 8-12 h; after the reaction is completed, cool to room temperature, extract and concentrate with dichloromethane, and finally separate by column chromatography to obtain a dark green solid powder, which is the compound of general formula (I).

5. The method for synthesizing fluorescent dyes according to claim 4, characterized in that, The molar ratio of the compound shown in Formula S to selenium dioxide is 1:(1-1.3), and the concentration of the compound shown in Formula S in 1,4-dioxane is in the range of 0.1-0.3 mol / L.

6. The method for synthesizing fluorescent dyes according to claim 4, characterized in that, The molar ratio of intermediate 2, benzothiazole-2-acetonitrile and tetrabutylammonium bromide is 1:(1-3):(0.01-0.02), and the concentration of intermediate 2 in anhydrous ethanol is in the range of 0.03-0.05 mol / L.

7. The method for synthesizing fluorescent dyes according to claim 3, characterized in that, When n is any value between 0 and 2, R1 is -N(CH3)2, and R2 is... The synthesis method includes the following steps: 1) The compound shown in formula S was dissolved in tetrahydrofuran with 4-quinoline carboxaldehyde and zinc chloride, and heated to 70-90 °C with stirring for 4-8 h. After the reaction was completed, it was cooled to room temperature, extracted and concentrated with dichloromethane, and finally separated by column chromatography to obtain a reddish-brown solid powder, namely intermediate 3. 2) Intermediate 3 and iodomethane were added to toluene solvent, heated to 100-120℃, and stirred for 8-12 h. After the reaction was completed, the mixture was cooled to room temperature to precipitate a solid. Finally, the compound of general formula (I) was obtained by column chromatography.

8. The method for synthesizing fluorescent dyes according to claim 7, characterized in that, The molar ratio of the compound shown in Formula S, 4-quinoline carboxaldehyde, and zinc chloride is 1:(1-1.5):(1-1.1), and the concentration of the compound shown in Formula S in tetrahydrofuran is in the range of 0.03-0.04 mol / L.

9. The method for synthesizing fluorescent dyes according to claim 7, characterized in that, The molar ratio of intermediate 3 to iodomethane is 1:(5-15), and the concentration of intermediate 3 in toluene is 5-15 mmol / L.

10. The use of the fluorescent dye according to claim 1 or 2 in the preparation of fluorescent imaging agents in vivo.

Images