Small-molecular-weight fluorescent dye as well as synthesis method and application thereof

Abstract

The molecular weight and the emission wavelength of the dye are balanced through a reasonable strategy, a series of compact organic small molecule dyes with the emission wavelength from a visible light window to a second near-infrared window are obtained, the universality is good, and the good blood brain barrier crossing capacity is achieved. According to the invention, a low-molecular-weight flexible conjugated polyene chain is creatively selected as a pi electron bridge, so that the extension of a pi conjugated system can be effectively realized, the small molecular weight of the dye can be maintained, and moreover, the conjugated polyene chain is used for prolonging a pi-conjugated structure to enhance an ICT effect, so that the dye has a good application prospect. The red shift of the emission wavelength can be realized while the molecular weight is kept small. Therefore, on the basis of the p-hydroxybenzylidene-imidazolinone chromophore, an ICT effect is synergistically enhanced through an integrated molecular design strategy, a series of small-molecular-weight fluorescent dyes with a D-pi-A1 or D-pi-A1-pi-A2 structure are constructed, the maximum wavelength can reach 1200 nm or above, the blood-brain barrier permeability of the dye as a probe is ensured, and the fluorescent dye can be used for detecting the blood-brain barrier permeability of the probe. The method can be applied to in-vivo in-situ brain imaging.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent dyes and biomedical cross-applications, and in particular to a series of small molecular weight fluorescent dyes with emission wavelengths ranging from the visible light window to the second near-infrared window, a synthesis method and applications thereof. Background Art

[0002] In recent years, the incidence of neurodegenerative diseases has continued to rise, accounting for a considerable 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 main obstacles to the slow development of brain science research. Fluorescence imaging technology, especially near-infrared second window (NIR-II, 1000-1700nm) fluorescence imaging technology, has received increasing attention in brain science research. The main reason is that the excitation and emission wavelengths of NIR-II fluorescence imaging technology are longer, which can effectively suppress the inherent background fluorescence interference and light scattering of biological tissues. Therefore, the tissue penetration of light during imaging is deeper, and the spatial resolution and signal-to-noise ratio are also higher, which greatly improves the reliability of brain imaging results. However, in the context of the new demand for precise imaging, the development of NIR-II fluorescent dyes that can be used in brain science research is still slow. An important bottleneck in brain optical imaging is the lack of NIR-II emitting dyes that can cross the BBB. Therefore, it is urgent to develop a class of dyes / probes that have both NIR-II emission and BBB penetration capabilities.

[0003] So far, several typical NIR-II molecular dyes have been reported, including polymethyl dyes, benzothiadiazole dyes, BODIPY dyes, and rhodamine-modified dyes. Although these classic dyes have shown very attractive effects in angiography, respiratory rate determination, and fluorescence-guided tumor resection. However, due to the large molecular weight (MW>600Da) of these dyes and their inherent macromolecular framework structure with positive charge, they are not suitable for crossing the BBB and are difficult to be applied to in situ brain imaging in vivo. The reason is that these dyes are generally designed to obtain a large rigid plane or conjugated skeleton by extending the π-conjugated system of the dye to achieve NIR-II emission, but this also increases the molecular weight and lipophilicity of the dye invisibly. Therefore, it is urgent to develop a new class of compact NIR-Ⅱ fluorescent dyes to solve the long-standing contradiction of NIR-Ⅱ dyes in maintaining a small molecular weight and having a long NIR-Ⅱ emission wavelength, so as to better meet 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 in view of the deficiencies in the above-mentioned prior art. The dyes have good universality, 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:

[0006] A series of low molecular weight fluorescent dyes with emission wavelengths from the visible light window to the second near infrared window, which satisfy the D-π1-A1 or D-π1-A1-π2-A2 structure, and the general structural formula is shown in formula (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 small molecular weight fluorescent dye satisfies the D-π1-A1 structure; or R2 is selected from etc., and connected to formula (1) via the leftmost carbon bond on the side chain, satisfying the D-π1-A1-π2-A2 structure.

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

[0012] According to the above scheme, the synthesis method of the small molecular weight fluorescent dye varies according to the different groups selected by R1- and R2-, and mainly includes the following five synthesis routes:

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

[0014] Trans-cinnamaldehyde and its derivatives are dissolved in acetic anhydride with N-acetylglycine and anhydrous sodium acetate to obtain intermediate 1; intermediate 1 is dissolved in ethanol with anhydrous potassium carbonate and methylamine aqueous solution (the mass fraction of methylamine in the methylamine aqueous solution is 30%-40%) to react to prepare compounds of general formula (I) (specifically, compounds I-1 to I-5 in the "Specific Implementation Method"). The reaction route of the synthesis 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 are dissolved in acetic anhydride to obtain a reaction solution, and then the reaction solution is reacted at 90-110°C for 4-6 hours; vacuum distillation and extraction are performed, and the obtained intermediate 1 is directly used in the next step. Among them, the molar ratio of benzaldehyde and its derivatives, N-acetylglycine, and anhydrous sodium acetate is 1:(3-8):(3-8), and the concentration of benzaldehyde and its derivatives in acetic anhydride is in the range of 0.5-1 mol / L;

[0018] 2) Dissolve the intermediate 1, anhydrous potassium carbonate and methylamine aqueous solution in ethanol and react under reflux for 2-5 hours; after the reflux reaction is completed, 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 the compound of general formula (I) (specifically, compounds I-1 to I-5 in the "Specific Implementation Method"). 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 comprises the following steps:

[0020] p-Dimethylaminobenzylidene-imidazolidinone and its derivatives are dissolved in 1,4-dioxane with selenium dioxide (SeO2) to obtain intermediate 2; intermediate 2 is dissolved in anhydrous ethanol with benzothiazole-2-acetonitrile and tetrabutylammonium bromide to prepare compounds of general formula (I) (specifically, compounds I-6 to I-9 in the "Specific Implementation Method"). The reaction route of the synthesis method is as follows:

[0021]

[0022] More specifically, the synthesis process of route 2 comprises the following steps:

[0023] 1) Add p-dimethylaminobenzylidene-imidazolidinone and its derivatives, selenium dioxide (SeO2) and 1,4-dioxane solvent to a round-bottom flask, stir and heat to 90-110°C for 4-8h, cool the reaction to room temperature, filter out the SeO2 solid, combine the obtained intermediate 2, concentrate it and use it in the next step without purification. The molar ratio of p-dimethylaminobenzylidene-imidazolidinone and its derivatives to selenium dioxide is 1:(1-1.3), and the concentration of p-dimethylaminobenzylidene-imidazolidinone and its derivatives in 1,4-dioxane is in the range of 0.1-0.3 mol / L;

[0024] 2) Add benzothiazole-2-acetonitrile and tetrabutylammonium bromide to anhydrous ethanol, then add intermediate 2, raise the reaction temperature to 70-90°C under stirring conditions, and continue the reaction for 8-12 hours; after the reaction is completed, cool to room temperature, extract with dichloromethane and concentrate, and finally separate by column chromatography to obtain a dark green solid powder to obtain a compound of general formula (I). 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.

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

[0026] Dimethylaminobenzylidene-imidazolidinone and its derivatives are dissolved in tetrahydrofuran with 4-quinolinecarboxaldehyde and zinc chloride to obtain intermediate 3 (compounds I-10 to I-12 in the "Specific Implementation Method" are one of intermediate 3); intermediate 3 is dissolved in toluene with iodomethane (CH3I) to react to obtain a compound of formula (I) (specifically, compounds I-13 to I-15 in the "Specific Implementation Method"). The reaction route of the synthesis method is as follows:

[0027]

[0028] More specifically, the synthesis process of route three comprises the following steps:

[0029] 1) dissolving dimethylaminobenzal-imidazolidinone and one of its derivatives, 4-quinolinecarboxaldehyde and zinc chloride in tetrahydrofuran, heating to 70-90°C with stirring, and reacting for 4-8h; after the reaction is completed, cooling to room temperature, extracting with dichloromethane and concentrating, and finally, separating by column chromatography to obtain a reddish-brown solid powder, namely intermediate 3 (one of compounds I-10 to I-12 in the "Specific Implementation Method"); wherein the molar ratio of dimethylaminobenzal-imidazolidinone and its derivatives, 4-quinolinecarboxaldehyde and zinc chloride is 1:(1-1.5):(1-1.1), and the addition concentration of dimethylaminobenzal-imidazolidinone and its derivatives in tetrahydrofuran is in the range of 0.03-0.04mol / L.

[0030] 2) Intermediate 3 and iodomethane (CH3I) are added to a toluene solvent, heated to 100-120°C, and stirred for reaction for 8-12 hours; after the reaction is completed, the solid is precipitated by cooling to room temperature, and finally, the compound of general formula (I) (specifically, compounds I-13 to I-15 in the "Specific Implementation Method") is obtained by column chromatography. 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 When, the synthesis method comprises the following steps:

[0032] (E)-3-(4-(dimethylamino)phenyl)acrolein (i.e., compound 3 in the "specific embodiment") or 5-(4-(dimethylamino)phenyl)thiophene-2-carboxaldehyde is reacted with compound I-2 (compound I-2 is named (Z)-5-(4-(dimethylamino)benzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazole-4-one) in solvent ethanol to obtain a compound of general formula (I) (specifically, compound I-16 or I-17 in the "specific embodiment").

[0033]

[0034] More specifically, the synthesis process of route 4 comprises the following steps:

[0035] (E)-3-(4-(dimethylamino)phenyl)propenal or 5-(4-(dimethylamino)phenyl)thiophene-2-carboxaldehyde and compound I-2 are added to an ethanol solvent, and then piperidine is added under stirring, and the temperature is raised to 60°C-80°C for reflux reaction for 3-5 hours to obtain a compound of general formula (I) (specifically, compound I-16 or I-17 in the "Specific Implementation Method"). Wherein, the molar ratio of (E)-3-(4-(dimethylamino)phenyl)propenal to compound I-2 and pyridine is 1:(0.7-0.9):(0.1-0.3); the molar ratio of 5-(4-(dimethylamino)phenyl)thiophene-2-carboxaldehyde to compound I-2 and pyridine is 1:(0.7-0.9):(0.1-0.3), and the concentration of compound I-2 in the ethanol solvent is in the range of 0.01-0.02 mol / L.

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

[0037] Compound I-3, ZnCl2 and (E)-3-(quinoline-4-yl)acrolein are added to tetrahydrofuran solvent to react to obtain a compound of general formula (I) (specifically, compound I-18 in the "Specific Implementation Method").

[0038]

[0039] More specifically, the synthesis process of route 5 comprises the following steps:

[0040] Add compound I-3, (E)-3-(quinoline-4-yl)propenal, ZnCl2 and tetrahydrofuran solvent into a round-bottom flask, heat to 60-80°C and react for 6-8 hours under stirring; after the reaction is completed, cool to room temperature, extract and concentrate, and separate by column chromatography to obtain a compound of general formula (I) (specifically, compound I-18 in the "specific implementation method"); wherein the molar ratio of compound I-3, 3-(quinoline-4-yl)propenal and ZnCl2 is 1:(1-1.5):(0.9-1.1), and the concentration of (E)-3-(quinoline-4-yl)propenal in the solvent tetrahydrofuran is 0.02-0.04 mol / L.

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

[0042] It is well known that push-pull (D-π-A) dyes have unique intramolecular charge transfer (ICT) properties. Structurally, the absorption and emission wavelengths of fluorophores can be tuned by changing their push-pull effect and / or effective π conjugation effect. On the one hand, the introduction of strong electron donor groups (EDG) and electron acceptor groups (EWG) can increase the energy level of the highest occupied molecular orbital (HOMO) and reduce the energy level of the lowest unoccupied molecular orbital (LUMO). The reduction of the HOMO-LUMO energy gap is proportional to the red shift of the dye absorption and emission wavelengths. On the other hand, under strong intramolecular interactions, the molecular conformation may undergo intramolecular twisting accompanied by electron transfer, resulting in enhanced polarization and complete charge separation between HOMO-LUMO, resulting in a large Stokes shift and red-shifted fluorescence. At the same time, regulating the push-pull electron ability of the donor / acceptor units to enhance the ICT effect may achieve a red shift in the emission wavelength. Another feasible method to promote the red shift of the dye absorption / emission wavelength is to expand the conjugated structure of the molecular skeleton. However, it should be noted that this approach often involves complex synthetic processes to obtain dyes with large conjugated rigid planar structures and high molecular weights, which may not only lead to enhanced detrimental π-π stacking interactions but also result in undesirable lipophilicity, reduced BBB permeability, and decreased brain delivery efficiency of the dyes.

[0043] The present invention creatively selects to use low molecular weight flexible conjugated polyene chain as π electron bridge, instead of blindly extending the conjugated structure, so that the π conjugated system can be effectively extended while maintaining the small molecular weight of the dye. In addition, by simultaneously adjusting the push-pull electron ability of the donor / acceptor and extending the π-conjugated structure through the conjugated polyene chain to enhance the ICT effect, it is possible to achieve red-shifted emission (such as Figure 1 Therefore, based on the p-hydroxybenzylidene-imidazolidinone (p-HBD) chromophore, the present invention integrates molecular design strategies to synergistically enhance the ICT effect, including improving the electron push-pull ability of the donor / acceptor and extending the π-conjugation length of the dye, and constructs a series of derivatives with D-π-A1 or D-π-A1-π-A2 structures (i.e., low molecular weight fluorescent dyes shown in the general structural formula (I)), the maximum wavelength of which can reach more than 1200nm (such as Figure 1 B). Among them, the framework structure of D-π-A1-π-A2 synergistically enhances the ICT effect, thereby obtaining the maximum red-shifted emission of NIR-II while retaining the small molecular weight of the molecule, ensuring the BBB permeability of the probe.

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

[0045] The present invention balances the molecular weight and emission wavelength of the dye through a reasonable strategy, and obtains a series of compact small molecular weight organic small molecule dyes with emission wavelengths from the visible light window to the second near infrared window. In particular, the dyes with NIR-II fluorescence emission can effectively increase the penetration depth of light in the brain skull and reduce light scattering and absorption of brain tissue, and can be better applied to the imaging of brain diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is a technical concept diagram of the dye of the present invention;

[0047] Figure 2 is the UV-visible absorption spectrum of the dyes I-1 to I-18 (i.e. compounds I-1 to I-18) of the present invention;

[0048] Figure 3 is the fluorescence spectra of the dyes I-1 to I-18 described in the present invention;

[0049] Figure 4 Stokes shift spectra of dyes I-1 to I-18 of the present invention;

[0050] Figure 5 A comparison spectrum of the molecular weights of the dyes I-1 to I-18 of the present invention and those of reported or commercial dyes;

[0051] Figure 6The comparison spectra of the Stokes shifts of the dyes I-1 to I-18 of the present invention and those of reported or commercial dyes;

[0052] Figure 7 The photostability spectra of the dyes I-6 to I-9 and I-13 to I-15 (>900nm) with longer emission wavelengths described in the present invention;

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

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

[0055] Figures 10 to 27 The high-resolution mass spectra of dyes I-1 to I-18 described in the present invention are shown in sequence. DETAILED DESCRIPTION

[0056] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the present invention is not limited to the following embodiments.

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

[0058]

[0059] The present invention provides a specific synthesis route of the above-mentioned small molecular weight fluorescent dye, as shown below:

[0060]

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

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

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

[0064] 4) Benzothiazole-2-acetonitrile, TBAB, EtOH, 80°C, 10 hours;

[0065] 5) 4-quinolinecarboxaldehyde, ZnCl2, THF, 80°C, 6 hours;

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

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

[0068] 8) piperidine, EtOH, 80°C, 3 hours;

[0069] 9) Compound 7, ZnCl2, THF, 80°C, 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 above-mentioned 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 the product was cooled to room temperature, acetic anhydride was removed under reduced pressure, extracted with dichloromethane, and then concentrated under reduced pressure. The intermediate 1 obtained was directly used in the next step.

[0073] The above intermediate 1 (1g 0.0047mol), anhydrous potassium carbonate (4.15g 0.03mol) and 33% methylamine aqueous solution (4ml, containing 0.04mol of methylamine) were dissolved in 20mL of ethanol and reacted under reflux for 3 hours. After the reaction was completed, it was cooled to room temperature and the pH value of the reaction solution was adjusted to neutral with an ethanol solution containing 10% hydrochloric acid. Finally, the crystals were cooled and crystallized at 0°C for 4 hours, i.e., compound I-1, i.e., dye I-1. The results of the nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrometry of compound I-1 are as follows, confirming that compound I-1 is obtained:

[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] According to the synthesis steps of compound I-1, compound 1 is replaced by compound 2. The results of H NMR and high-resolution mass spectrometry of compound I-2 are as follows, confirming that compound I-2 is obtained:

[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] According to the synthesis steps of compound I-1, compound 1 is replaced by compound 3. The results of H NMR and high-resolution mass spectrometry of compound I-3 are as follows, confirming that compound I-3 is obtained:

[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] According to the synthesis steps of compound I-1, compound 1 is replaced by compound 4. The results of H NMR and high-resolution mass spectrometry of compound I-4 are as follows, confirming that compound I-4 is obtained:

[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] According to the synthesis steps of compound I-1, compound 1 is replaced by compound 5. The results of H NMR and high-resolution mass spectrometry of compound I-5 are as follows, confirming that compound I-5 is obtained:

[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 and heated to 100 ° C with stirring for 6 hours. After the reaction was completed, the reaction was cooled to room temperature, the SeO2 solid was filtered off, and the intermediate 2 was combined and concentrated for the next step without purification.

[0099] The above intermediate 2 (200mg 0.78mmol) was added to anhydrous ethanol (20ml) containing benzothiazole-2-acetonitrile (271mg1.56mmol) and tetrabutylammonium bromide (38mg 0.117mmol), and then heated to 80°C under stirring and continued to react for 10 hours. After the reaction was completed, it was cooled to room temperature, extracted with dichloromethane and concentrated. Finally, a dark green solid powder was obtained by column chromatography, namely compound I-6. The results of the nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrometry of compound I-6 are as follows, confirming that compound I-6 was obtained:

[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] According to the synthesis steps of compound I-6, compound I-2 is replaced by compound I-3. The results of H NMR and high-resolution mass spectrometry of compound I-7 are as follows, confirming that compound I-7 is obtained:

[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] According to the synthesis steps of compound I-6, compound I-2 is replaced by compound I-4. The results of H NMR and high-resolution mass spectrometry of compound I-8 are as follows, confirming that compound I-8 is obtained:

[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] According to the synthesis steps of compound I-6, compound I-2 is replaced by compound I-5. The results of H NMR and high-resolution mass spectrometry of compound I-9 are as follows, confirming that compound I-9 is obtained:

[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-quinolinecarboxaldehyde (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, heated to 80 ° C with stirring, and reacted for 6 hours. After the reaction was completed, it was cooled to room temperature, extracted with dichloromethane and concentrated. Finally, a reddish-brown solid powder was obtained by column chromatography, namely the corresponding compound I-10. The results of the nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrometry of compound I-10 are as follows, confirming that compound I-10 was obtained:

[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] Embodiment 11

[0121] Synthesis of compound I-11:

[0122] According to the synthesis steps of compound I-10, compound I-2 is replaced by compound I-3. The results of H NMR and high-resolution mass spectrometry of compound I-11 are as follows, confirming that compound I-11 is obtained:

[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] According to the synthesis steps of compound I-10, compound I-2 is replaced by compound I-4. The results of H NMR and high-resolution mass spectrometry of compound I-12 are as follows, confirming that compound I-12 is obtained:

[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] Embodiment 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, heated to 110°C, and stirred for 10 hours. After the reaction was completed, the mixture was cooled to room temperature to precipitate a solid. Finally, a green solid powder, i.e., compound I-13, was obtained by column chromatography. The results of the H NMR spectrum and high-resolution mass spectrometry of compound I-13 are as follows, confirming that compound I-13 was obtained:

[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) δ135.76 (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] Embodiment 14

[0136] Synthesis of compound I-14:

[0137] According to the synthesis steps of compound I-13, compound I-10 is replaced by compound I-11. The results of H NMR and high-resolution mass spectrometry of compound I-14 are as follows, confirming that compound I-14 is obtained:

[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] Embodiment 15

[0141] Synthesis of compound I-15:

[0142] According to the synthesis steps of compound I-13, compound I-10 is replaced by compound I-12. The results of H NMR and high-resolution mass spectrometry of compound I-15 are as follows, confirming that compound I-15 is obtained:

[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 (86mg 0.49mmol) and compound I-2 (100mg 0.41mmol) were added to 30mL of ethanol and transferred to a round-bottom flask, and then piperidine (8.3mg 0.098mol) was added under stirring, and finally the temperature was raised to 80°C, refluxed for 3 hours, and concentrated by rotary evaporation after cooling and separated by column chromatography to obtain compound I-16. The results of the nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrometry of compound I-16 are as follows, confirming that compound I-16 is obtained:

[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] Embodiment 17

[0151] Synthesis of compound I-17:

[0152] 5-(4-(dimethylamino)phenyl)thiophene-2-carboxaldehyde (113mg 0.49mmol) and compound I-2 (100mg 0.41mmol) were added to 30mL of ethanol and transferred to a round-bottom flask, and then piperidine (8.3mg 0.098mol) was added under stirring, and finally the temperature was raised to 80°C, refluxed for 3 hours, and concentrated by rotary evaporation after cooling and separated by column chromatography to obtain compound I-17. The results of the nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrometry of compound I-17 are as follows, confirming that compound I-17 is obtained:

[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] Embodiment 18

[0156] Synthesis of compound I-18:

[0157] Compound I-3 (67 mg, 0.25 mmol), 3-(quinolin-4-yl) acrolein (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 under stirring. After the reaction was completed, it was cooled to room temperature, extracted with dichloromethane and concentrated. Finally, 87 mg of reddish brown solid powder was obtained by column chromatography, i.e., compound I-18 was obtained. The results of the nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrometry of compound I-18 are as follows, confirming that compound I-18 was obtained:

[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, it can be determined from the above NMR and mass spectrometry test results 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 synthesis route.

[0161] Performance characterization and application:

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

[0163] In order to better understand the photophysical properties of the dyes provided by the present invention, the ultraviolet absorption and fluorescence spectra of these dyes were tested. Figure 2 and Figure 3 The normalized results of the spectra of all dyes in the organic solvent dimethyl sulfoxide are shown. Figure 2 As shown, all dyes exhibited a broad UV absorption spectrum in DMSO, ranging from 300-950 nm; similarly, Figure 3 The fluorescence emission of the dyes 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 region to NIR-II, with a maximum emission wavelength exceeding 1200 nm.

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

[0166] It is well known that complete spectral separation of dyes is beneficial to reduce spectral self-absorption interference and crosstalk, thereby obtaining imaging results with higher contrast. Figure 4 It can be seen that the dyes provided by the present invention have a large Stokes shift, with the largest Stokes shift reaching 566nm, which is larger than the Stokes shift of existing known and commercial dyes ( Figure 5 The large Stokes shift of the dye is attributed to the strong intramolecular charge transfer and the large amount of geometric relaxation during the photoexcitation process.

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

[0168] The most valuable property of dyes I-1 to I-18 is that despite their long emission wavelengths, their molecular weights are all less than 500 g / mol, which is much smaller than that of reported commercial dyes and typical mainstream dyes ( Figure 6 ). The above results show that the series of dyes provided by the present invention have a good balance between molecular weight and fluorescence emission wavelength, have potential BBB penetration ability, and meet the two key conditions of brain imaging (long wavelength and small molecular weight), and have the potential to be used in brain imaging.

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

[0170] In order 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 have less tissue interference when used for in vivo imaging. The commercial dye ICG was used as a comparison. The present invention monitored the photostability of the dyes in DMSO (808 nm, 1 W cm -2 ) to evaluate its photostability. Figure 7 As shown, compared with ICG, these dyes have good photostability, indicating that this series of dyes of the present invention can meet the needs of long-term fluorescence imaging in living body 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, such as dyes I-6 to I-9 and I-13 to I-15. Figure 8 As shown, these dyes all exhibit obvious NIR-II fluorescence signals in the band above 1000nm and can be used for NIR-II imaging.

[0173] 6. NIR-II in vivo imaging applications of dyes

[0174] The feasibility of in vivo imaging of Aβ plaques was investigated using dye I-7 as an example. The specific process was as follows: in vivo imaging studies were conducted using 6-month-old and 12-month-old APP / PS1 transgenic AD model mice and 12-month-old wild-type mice. 10-300 minutes after intravenous injection of dye I-7 (0.5mM, 200μL), the NIR-II fluorescence signals in the brains of AD model mice and wild-type mice were obtained by intermittently collecting the NIR-II fluorescence signals in the brains of AD model mice and wild-type mice. Fig. 9 As shown in A, compared with 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 the injection of dye I-7. In contrast, clear fluorescence signals could be observed in 6-month-old and 12-month-old AD mice at the 10th minute after injection. And the results showed that the dye slowly entered the brain parenchyma from the initial cerebral blood vessels and clearly outlined the shape of the mouse brain over time. The experimental results show 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 brain 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 the dye ICG via the tail vein were selected for NIR-II fluorescence imaging as controls. Fig. 9 As shown in B, in the second minute after ICG injection, ICG can clearly depict the blood vessels in the mouse head. However, as time goes by, the fluorescent signal in the brain gradually weakens until it disappears, which may be because ICG is slowly metabolized in the cerebral blood vessels. Therefore, even in AD mice, the dye ICG cannot cross the blood-brain barrier to reach the brain parenchyma and bind to Aβ plaques. The results of in situ fluorescence imaging show that the compact fluorescent dye represented by dye I-7 prepared by the present invention can smoothly cross the blood-brain barrier and achieve 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 evaluates the physicochemical parameters of dyes to better evaluate whether they have BBB permeability. In general, molecules with molecular weight less than 500g / mol, log P<5, H-bond acceptors less than 10 and H-bond donors less than 5 have potential BBB permeability. Therefore, these key parameters of dye fluorescence fluorophores were studied by a method combining theoretical calculation and experiment, and dyes I-6 to I-12 were used as examples, and their physicochemical parameter properties are shown in Table 1. Moreover, it can be found that compared with dyes I-10 to I-12, dyes I-6 to I-9 have better blood-brain barrier permeability.

[0178] Table 1

[0179]

[0180]

[0181] The present invention is not limited to the above-mentioned embodiments. For those skilled in the art, several improvements and modifications may be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as within the protection scope of the present invention.

Claims

1. A series of small molecular weight fluorescent dyes, characterized in that: The molecular weight is less than 500Da, the fluorescence emission wavelength is in the range of 440-1250nm, and the general structural formula is shown in formula (I): In formula (I): n is 0 to 3; R1- is selected from one or two of -H, -N(CH3)2; R2- is -H, or R2 One or more of the .

2. The low molecular weight fluorescent dye according to claim 1, characterized in that: The compact fluorescent dye comprises one or more dyes having the structure shown in formula (I).

3. The method for synthesizing a low molecular weight fluorescent dye according to claim 1, characterized in that: Depending on the groups selected for R1- and R2-, there are mainly five synthetic routes: Route 1: When n is one of 0 to 3, R1- is selected from one of -H and -N(CH3)2, and R2- is -H, the synthesis method is as follows: Trans-cinnamaldehyde and its derivatives are dissolved in acetic anhydride with N-acetylglycine and anhydrous sodium acetate to obtain intermediate 1; intermediate 1 is reacted with anhydrous potassium carbonate and methylamine aqueous solution in ethanol to prepare a compound of general formula (I); the reaction route of the synthesis method is as follows: Route 2: When R1 is -N(CH3)2 and R2 is When n is one of 0 to 3, the synthesis method is as follows: p-Dimethylaminobenzylidene-imidazolidinone and its derivatives and selenium dioxide are dissolved in 1,4-dioxane to prepare intermediate 2; intermediate 2 is dissolved in anhydrous ethanol with benzothiazole-2-acetonitrile and tetrabutylammonium bromide to prepare a compound of general formula (I); the reaction route of the synthesis method is as follows: Route 3: When n is one of 0 to 2, R1 is -N(CH3)2; R2- is The synthesis method is as follows: Dimethylaminobenzylidene-imidazolidinone and its derivatives are dissolved in tetrahydrofuran with 4-quinolinecarboxaldehyde and zinc chloride to obtain intermediate 3; intermediate 3 is reacted with iodomethane dissolved in toluene to obtain a compound of general formula (I); the reaction route of the synthesis method is as follows: Route 4: When n is 0, R1 is -N(CH3)2, R2- is or When, the synthesis method comprises the following steps: Relative reaction of (E)-3-(4-(dimethylamino)phenyl)acrolein or 5-(4-(dimethylamino)phenyl)thiophene-2-carboxaldehyde with (Z)-5-(4-(dimethylamino)benzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one in ethanol as a solvent to obtain a compound of general formula (I); Route 5: When n is 0, R1 is -N(CH3)2, R2- is The synthesis method is as follows: Compound I-3, ZnCl2 and (E)-3-(quinolin-4-yl)acrolein are added to tetrahydrofuran solvent for reaction to obtain a compound of general formula (I); compound I-3 is (Z)-5-((E)-3-(4-(dimethylamino)phenyl)allylmethylene)-2,3-dimethyl-3,5-dihydro-4H-imidazole-4-one.

4. The method for synthesizing a low molecular weight fluorescent dye according to claim 3, characterized in that: The synthesis process of route 1 includes the following steps: 1) dissolving trans-cinnamaldehyde and its derivatives, N-acetylglycine and anhydrous sodium acetate in acetic anhydride to obtain a reaction solution, and then reacting the reaction solution at 90-110° C. for 4-6 hours; performing reduced pressure distillation and extraction to obtain an intermediate 1; wherein the molar ratio of benzaldehyde and its derivatives, N-acetylglycine and anhydrous sodium acetate is 1:(3-8):(3-8), and the concentration of benzaldehyde and its derivatives in acetic anhydride is in the range of 0.5-1 mol / L; 2) dissolving the intermediate 1, anhydrous potassium carbonate and methylamine aqueous solution in ethanol and reacting them under reflux for 2-5 hours; after the reflux reaction is completed, cooling to room temperature, and adjusting the pH of the system to neutral with an ethanol solution containing hydrochloric acid; finally, cooling and crystallizing the crystals to obtain a compound of general formula (I); wherein the molar ratio of the intermediate 1, anhydrous potassium carbonate and methylamine is 1:(5-8):(7-10), and the concentration of the intermediate 1 in ethanol is in the range of 0.2-0.3 mol / L.

5. The method for synthesizing a low molecular weight fluorescent dye according to claim 3, characterized in that: The synthesis process of route 2 includes the following steps: 1) mixing p-dimethylaminobenzylidene-imidazolidinone and its derivatives, selenium dioxide (SeO2) and 1,4-dioxane solvent, stirring and heating to 90-110°C for 4-8h, cooling the reaction to room temperature, filtering out SeO2 solid, and combining the obtained intermediate 2; wherein the molar ratio of dimethylaminobenzylidene-imidazolidinone and its derivatives to selenium dioxide is 1:(1-1.3), and the concentration of dimethylaminobenzylidene-imidazolidinone and its derivatives in 1,4-dioxane is in the range of 0.1-0.3 mol / L; 2) adding benzothiazole-2-acetonitrile and tetrabutylammonium bromide to anhydrous ethanol, and then adding intermediate 2, raising the reaction temperature to 70-90°C under stirring conditions, and continuing the reaction for 8-12 hours; after the reaction is completed, cooling to room temperature, extracting with dichloromethane and concentrating, and finally, separating by column chromatography to obtain a dark green solid powder, i.e., obtaining a compound of general formula (I); wherein 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.

6. The method for synthesizing a low molecular weight fluorescent dye according to claim 3, characterized in that: The synthesis process of route three includes the following steps: 1) dissolving one of dimethylaminobenzylidene-imidazolidinone and its derivatives, 4-quinolinecarboxaldehyde and zinc chloride in tetrahydrofuran, heating to 70-90° C. with stirring, and reacting for 4-8 hours; after the reaction is completed, cooling to room temperature, extracting with dichloromethane and concentrating, and finally separating by column chromatography to obtain a reddish brown solid powder, i.e., intermediate 3; wherein the molar ratio of dimethylaminobenzylidene-imidazolidinone and its derivatives, 4-quinolinecarboxaldehyde and zinc chloride is 1:(1-1.5):(1-1.1), and the addition concentration of dimethylaminobenzylidene-imidazolidinone and its derivatives in tetrahydrofuran is in the range of 0.03-0.04 mol / L; 2) intermediate 3 and iodomethane are added to a toluene solvent, heated to 100-120° C., and stirred for reaction for 8-12 hours; after the reaction is completed, the mixture is cooled to room temperature to precipitate a solid, and finally, a compound of formula (I) is obtained by column chromatography; wherein 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.

7. The method for synthesizing a low molecular weight fluorescent dye according to claim 3, characterized in that: The synthesis process of route 4 comprises the following steps: (E)-3-(4-(dimethylamino)phenyl)propenal or 5-(4-(dimethylamino)phenyl)thiophene-2-carboxaldehyde and compound I-2 are added to an ethanol solvent respectively, and then piperidine is added under stirring, and the temperature is raised to 60°C-80°C and refluxed for reaction for 3-5 hours to obtain a compound of general formula (I); wherein the molar ratio of (E)-3-(4-(dimethylamino)phenyl)propenal to compound I-2 and pyridine is 1:(0.7-0.9):(0.1-0.3); the molar ratio of 5-(4-(dimethylamino)phenyl)thiophene-2-carboxaldehyde to compound I-2 and pyridine is 1:(0.7-0.9):(0.1-0.3), and the concentration of compound I-2 in the ethanol solvent is in the range of 0.01-0.02 mol / L.

8. The method for synthesizing a low molecular weight fluorescent dye according to claim 3, characterized in that: The synthesis process of route 5 includes the following steps: Compound I-3, (E)-3-(quinoline-4-yl)acrolein and ZnCl2 are dissolved in tetrahydrofuran, heated to 60-80°C with stirring and reacted for 6-8 hours; after the reaction is completed, it is cooled to room temperature, extracted and concentrated, and separated by column chromatography to obtain a compound of general formula (I); wherein the molar ratio of compound I-3, 3-(quinoline-4-yl)acrolein and ZnCl2 is 1:(1-1.5):(0.9-1.1), and the concentration of (E)-3-(quinoline-4-yl)acrolein in the solvent tetrahydrofuran is 0.02-0.04 mol / L.

9. Use of the low molecular weight fluorescent dye according to claim 1 or 2 in in vivo fluorescence imaging.

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