Coumarin hemicyanine dye with rigid structure and preparation method and application thereof

Abstract

The present invention belongs to the field of fluorescent probes, and in particular to coumarin hemicyanine dyes and preparation methods and applications of structural rigidity. To solve the problem of low fluorescence quantum yield and small Stokes shift in existing fluorescent dyes, the present invention, by introducing a dicyclic system in classical coumarin hemicyanines (CHCs), constructs a series of structural rigid coumarin hemicyanine dyes (RCHC1‑3), and the series of dyes not only retains the large Stokes shift of classical CHCs dyes, but also importantly, the rigid structure effectively suppresses the non-radiative transition process induced by double bond isomerization, therefore with improved fluorescence quantum yield. In view of the above advantages, the series of dyes not only show improved fluorescence brightness during cell imaging, but also can avoid the interference of biological autofluorescence and excitation light scattering, therefore with potential application value.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent probes, and in particular relates to a structurally rigid coumarin hemicyanine dye and a preparation method and application thereof. Background Art

[0002] Due to its high sensitivity, visualization, biocompatibility, no radiation, and real-time detection, fluorescence technology has become an indispensable research tool in the fields of biology and medicine. In fact, the core and material basis of fluorescence technology is fluorescent dyes, and their photophysical properties, such as molar absorption coefficient, fluorescence quantum yield, excitation and emission wavelength, largely determine the application effect of this technology.

[0003] At present, the fluorescent dyes widely used in the field of fluorescence technology mainly include rhodamines, boron-dipyrromethene (Bodipys), cyanines (Cyanine) and their derivatives. However, these dyes still have some problems in practical use. For example, rhodamine dyes have non-radiative transitions induced by twisted charge transfer (TICT) of the excited state, resulting in a low fluorescence quantum yield in aqueous solution; boron-dipyrromethene is inherently highly lipid-soluble and difficult to be compatible with aqueous biological systems, and is prone to non-specific staining and increases background signals, thereby reducing the sensitivity of the detection of research targets; cyanine dyes are prone to non-radiative transitions induced by double bond isomerization, so they usually have low fluorescence quantum yields. In addition, these dyes usually have a small Stokes shift (<20nm), which will inevitably be interfered by self-quenching, backscattering of excitation light and autofluorescence of biological molecules.

[0004] As a member of cyanine dyes, coumarin hemicyanine dyes have a relatively large Stokes shift (~80nm), but they are also prone to non-radiative transitions induced by double bond isomerization, so they usually have low fluorescence quantum yields. The current increasing image quality requirements and the advanced imaging technology developed in recent years have posed new challenges to various dyes. Therefore, the development of biocompatible, bright fluorescent dyes or probes has always been a research topic in the field of fluorescence technology. Summary of the invention

[0005] In view of the above problems, the present invention introduces a bicyclic system into the classic coumarin hemicyanines (CHCs) to construct a series of structurally rigid coumarin hemicyanine dyes (RCHC1-3). The rigid structure of this series of dyes effectively inhibits the non-radiative transition process induced by double bond isomerization, and thus has an improved fluorescence quantum yield compared with CHCs.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The structurally rigid coumarin hemicyanine dye has the following structural formula:

[0008]

[0009] The comparative compound of the rigid coumarin hemicyanine dye has the structural formula:

[0010]

[0011] The method for preparing a structurally rigid coumarin hemicyanine dye comprises the following steps:

[0012]

[0013] (1) At 0°C, 4-bromo-2-hydroxyacetophenone (Compound 1a) is dissolved in anhydrous toluene, and then a toluene solution of NaH is added dropwise. When no gas is generated, diethyl carbonate dissolved in toluene is added dropwise, and reflux reaction is performed. After the reaction is completed, the reaction solution is cooled and dried to obtain a crude product. The crude product is dissolved in anhydrous toluene and reflux reaction is continued. After the reaction solution is cooled, a large amount of solid is obtained. The solid is filtered, washed and dried to obtain 4-hydroxy-7-bromocoumarin (Compound 1b);

[0014] (2) 4-Hydroxy-7-bromocoumarin (Compound 1b) and K2CO3 were dissolved in anhydrous acetonitrile respectively, reacted under reflux conditions for 1 hour, then benzyl bromide dissolved in acetonitrile was added dropwise, and the reaction was continued under reflux conditions for 6 hours. After the reaction was completed, K2CO3 was filtered out to terminate the reaction, and the reaction solution was cooled to -20°C to generate a large amount of solid. The obtained solid was filtered, washed and dried to obtain 4-benzyloxy-7-bromocoumarin (Compound 1c);

[0015] (3) Under N2 protection, PdCl2, Cs2CO3, 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP), thiomorpholine-1,1-dioxide and 4-benzyloxy-7-bromocoumarin (Compound 1c) were dissolved in anhydrous 1,4-dioxane, respectively, and stirred for reaction. After the reaction was completed, the reaction solution was cooled to room temperature and filtered. The filter cake was recrystallized from ethyl acetate to obtain Compound 1d;

[0016] (4) Compound 1d was dissolved in anhydrous dichloromethane at 0°C, and then a solution of BBr3 in anhydrous dichloromethane was added dropwise. The reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction solution was poured into ice water to generate a large amount of solid. The large amount of solid was filtered, washed with water, and dried to obtain compound A3;

[0017] (5) Under the protection of N2, redistilled DMF was added dropwise to POCl3 to react to obtain a red solution, and then a DMF solution of a 7-hydroxycoumarin derivative (the 7-hydroxycoumarin derivative is defined as A1-A3 according to the different substituents on the N of compound A3) was added dropwise to continue the reaction. After the reaction was completed, the reaction solution was poured into ice water, and the pH was adjusted with NaOH solution until a large amount of solid precipitated. The solid was filtered, washed with water, dried, and recrystallized from anhydrous ethanol to obtain 3-aldehyde-4-chlorocoumarin derivatives B1-B3;

[0018] (6) Under nitrogen protection, 2,3,3-trimethyl-3H-indole (Compound 1), 2-(2-bromoethyl)-1,3-dioxane (Compound 2), NaHCO3 and KI were dissolved in anhydrous acetonitrile, stirred for reaction, and after the reaction, the reaction solution was cooled to room temperature, the solvent was dried, and the crude product was separated and purified by column chromatography to obtain Compound 3;

[0019] (7) 3-formyl-4-chlorocoumarin derivative B1-B3, compound 3, triethylamine hydrochloride and piperidine were dissolved in acetonitrile in sequence, and refluxed under vigorous stirring. After the reaction, the reaction solution was cooled to room temperature, and the solvent was dried to obtain an intermediate; the intermediate was redissolved in a mixed solvent of dichloromethane and concentrated hydrochloric acid and continued to react until the reaction of the raw materials was complete as monitored by TLC. After the reaction, the dichloromethane was dried, and the remaining acid solution was adjusted to pH 6-7 with a Na2CO3 aqueous solution, extracted, and dried to obtain a crude product. The crude product was separated and purified by column chromatography to obtain the target compound RCHC1-3.

[0020] Furthermore, in the step (1), the molar ratio of 4-bromo-2-hydroxyacetophenone to NaH is 1:3; the temperature of the reflux reaction is 110° C., and the time is 24 h; the temperature of the continued reflux reaction is 110° C., and the time is 36 h.

[0021] Furthermore, in the step (2), the molar ratio of 4-hydroxy-7-bromocoumarin, K2CO3 and benzyl bromide is 1:2:2; the temperature of the reaction under reflux conditions is 82°C and the time is 1 hour; the temperature of the reaction solution continuing to react under reflux conditions is 82°C and the time is 6 hours.

[0022] Furthermore, in the step (3), the molar ratio of PdCl2, Cs2CO3, 1,1'-binaphthyl-2,2'-bis(diphenylphosphine), thiomorpholine-1,1-dioxide and 4-benzyloxy-7-bromocoumarin is 0.05:1.2:0.1:1.1:1; the temperature of the stirring reaction is 90°C and the time is overnight.

[0023] Furthermore, in the step (4), the molar ratio of compound 1d to BBr3 is 1:2.

[0024] Furthermore, in the step (5), the volume molar ratio of DMF, POCl3 and 7-hydroxycoumarin derivative A1-A3 is 1 mL: 1 mL: 5 mmol; the reaction temperature is 50°C and the reaction time is 30 minutes; the reaction temperature is 70°C and the reaction time is overnight.

[0025] Furthermore, in the step (6), the molar ratio of 2,3,3-trimethyl-3H-indole, 2-(2-bromoethyl)-1,3-dioxane, NaHCO3 and KI is 2:3:4:3; the stirring reaction temperature is 95°C and the time is 18 hours; and the developing solvent for the column chromatography separation and purification is CH2Cl2 / MeOH=9:1 (v / v).

[0026] Furthermore, in the step (7), the molar ratio of 3-formyl-4-chlorocoumarin derivatives B1-B3, compound 3, and triethylamine hydrochloride is 1:1:1; the volume ratio of dichloromethane and concentrated hydrochloric acid is 50 mL:15 mL; the temperature of the reflux reaction is 82°C and the time is 12 h; the temperature of the continued reaction is 25°C; and the developing solvent for the column chromatography separation and purification is CH2Cl2:MeOH:TFA=400:10:1 (v / v / v).

[0027] The application of structurally rigid coumarin hemicyanine dyes in verifying starvation-induced cell autophagy and in cell imaging.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] Traditional coumarin hemicyanine dyes (CHCs) usually have low fluorescence quantum yield in solvents due to the prevalence of non-radiative transition processes induced by double bond cis-trans isomerization in the molecules. The structurally rigid coumarin hemicyanine dyes (RCHCs) developed by the present invention not only retain the excellent photophysical properties of CHCs, such as absorption and emission wavelengths in the red to near-infrared region, large Stokes shift, good biocompatibility, etc., but more importantly, the fluorescence quantum yield of these dyes has been greatly improved, which is mainly attributed to the fact that their rigid molecular structure effectively inhibits the non-radiative transition process caused by double bond isomerization. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 NMR and HRMS diagrams of compound 1b;

[0031] Figure 2 is the NMR spectrum of compound 1c;

[0032] Figure 3 is the NMR spectrum of compound 1d;

[0033] Figure 4NMR and HRMS diagrams of compound A3;

[0034] Figure 5 is the NMR diagram of compound B1;

[0035] Figure 6 is the NMR diagram of compound B2;

[0036] Figure 7 NMR and HRMS diagrams of compound B3;

[0037] Figure 8 NMR and HRMS diagrams of compound 3;

[0038] Fig. 9 NMR and HRMS diagrams of compound RCHC1;

[0039] Fig.10 NMR and HRMS diagrams of compound RCHC2;

[0040] Fig.11 NMR and HRMS diagrams of compound RCHC3;

[0041] Fig.12 Normalized absorption and emission spectra of CHC1 and RCHC1-3 in (A) CH3CN and (B) PBS;

[0042] Fig.13 The frontier molecular orbitals of the ground state (S0, left) and excited state (S1, right) of (A) RCHC1, (B) RCHC2, and (C) RCHC3 calculated by density functional theory;

[0043] Fig.14 Cytotoxicity experiments after A549 cells were treated with different concentrations (0 μM, 1.0 μM, 2.0 μM, 3.0 μM, 4.0 μM and 5.0 μM) of (A) RCHC1, (B) RCHC2 and (C) RCHC3 for 24 hours;

[0044] Fig.15 Cell images of A549 cells treated with CHC1 (0.2 μM), RCHC1 (0.2 μM), RCHC2 (0.2 μM) and RCHC3 (0.2 μM), respectively, with an excitation wavelength of 561 nm, a collection wavelength of 570-740 nm, and a scale bar of 20 μm;

[0045] Fig.16 Cell images of A549 cells loaded with Rh B, RCHC1 and RCHC2 respectively; the excitation wavelength is 561 nm, the collection wavelength is shown on the right side of the figure, scale: 20 μm;

[0046] Fig.17 Cell images of A549 cells after incubation with RCHC1 (0.2 μM) and Lyso-Tracker Green (0.2 μM); or RCHC1 (0.2 μM) and Mito-Tracker Deep Red (0.2 μM); or RCHC2 (0.2 μM) and Lyso-Tracker Green (0.2 μM); or RCHC2 (0.2 μM) and Mito-Tracker Deep Red (0.2 μM). For RCHC1 and RCHC2, the wavelength was 570-630 nm (λ ex =561nm), for Lyso-Tracker Green and Mito-Tracker Deep Red, the collection wavelengths were 490-560nm (λ ex =488nm) and 640-750nm (λ ex =633nm), scale bar: 10μm.

[0047] Fig.18 Cell imaging of A549 cells preloaded with RCHC2 (0.2 μM) and LTG (0.2 μM) in a D-Hanks buffer system during autophagy induced by starvation. For RCHC2, the wavelength of 630-740 nm (λ ex =561nm), for Lyso-Tracker Green, the collection wavelength is 490-560nm (λ ex =488nm), scale bar: 20μm. DETAILED DESCRIPTION

[0048] Example 1

[0049] The structurally rigid coumarin hemicyanine dye has the following structural formula:

[0050]

[0051] The method for preparing a structurally rigid coumarin hemicyanine dye comprises the following steps:

[0052] (1) At 0°C, 4-bromo-2-hydroxyacetophenone (Compound 1a, 20 g, 93 mmol) was dissolved in anhydrous toluene (200 mL), and then a toluene solution (70 mL) of NaH (11.2 g, 280 mmol) was added dropwise. When no gas was generated, diethyl carbonate (20 mL) dissolved in 10 mL of toluene was added dropwise. The mixture was refluxed at 110°C for 24 hours. After the reaction, the reaction solution was cooled and dried to obtain a crude product. The crude product was dissolved in anhydrous toluene (50 mL), and the reaction was refluxed at 110°C for 36 hours. After the reaction solution was cooled, a large amount of solid was obtained. The solid was filtered, washed and dried to obtain 4-hydroxy-7-bromocoumarin (Compound 1b) (16.4 g, yield 73.1%). Compound 1b was used directly in the next step without purification. 1 H NMR (600MHz, DMSO-d6) δ7.74(d,J=8.4Hz,1H),7.70(s,1H),7.54(d,J=8.4Hz,1H),5.60(s,1H); 13 C NMR (150MHz, DMSO-d6) δ165.8,161.8,154.4,127.5,125.9,125.3,119.8,115.7,91.6; ESI-MS[MH - ]:calculated for238.9344,found 238.9346.

[0053] (2) 4-Hydroxy-7-bromocoumarin (Compound 1b) (15.6 g, 64.7 mmol) and K2CO3 (17.0 g, 2 eq) were dissolved in anhydrous acetonitrile (170 mL) respectively, and reacted at 82°C under reflux for 1 hour. Then, benzyl bromide (21.0 g, 123 mmol) dissolved in acetonitrile (13.4 mL) was added dropwise, and the reaction was continued at 82°C under reflux for 6 hours. After the reaction was completed, K2CO3 was filtered out to terminate the reaction. The reaction solution was cooled to -20°C to generate a large amount of solid. The obtained solid was filtered, washed and dried to obtain 4-benzyloxy-7-bromocoumarin (Compound 1c) (7.9 g, yield 36.9%). 1 HNMR(600MHz,DMSO-d6)δ7.76-7.72(m,2H),7.56-7.53(m,3H),7.47-7.41(m,3H)6.10(s,1H),5.35(s,2H); 13C NMR (150MHz, DMSO-d6) δ164.7,161.6,153.6,135.4,129.2,129.0,128.6,127.9,126.1,125.1,119.9,115.1,92.0,71.6.

[0054] (3) Under N2 protection, PdCl2 (107 mg, 0.05 eq), Cs2CO3 (4.7 g, 1.2 eq), 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP, 753 mg, 0.1 eq), thiomorpholine-1,1-dioxide (1.8 g, 1.1 eq) and 4-benzyloxy-7-bromocoumarin (Compound 1c) (4.0 g, 12.1 mmol) were dissolved in anhydrous 1,4-dioxane, and the mixture was stirred for reaction. After the reaction, the reaction solution was cooled to room temperature and filtered. The filter cake was recrystallized from ethyl acetate to obtain Compound 1d (4.23 g, yield: 90.9%). 1 HNMR(400MHz, CDCl3)δ7.76(d,J=9.2Hz,1H),7.43(m,5H),6.80-6.77(m,1H),6.73(d, J=2.4Hz,1H),5.64(s,1H),5.19(s,2H),4.00(t,J=5.2Hz,4H),3.11(t,J=5.2Hz,4H); 13 C NMR (150MHz, CDCl3) δ164.9,162.2,153.6,134.1,129.1,129.0,128.8,127.9,127.4,126.5,124.4,120.0,114.7,91.3,71.40,51.7.

[0055] (4) Compound 1d (2.4 g, 6.23 mmol) was dissolved in anhydrous dichloromethane (20 mL) at 0°C, and then a solution of BBr3 (1.3 mL, 12.5 mmol, 2 eq) in anhydrous dichloromethane (10 mL) was added dropwise. The mixture was reacted overnight at room temperature. After the reaction, the reaction solution was poured into ice water to generate a large amount of solid. The solid was filtered, washed with water, and dried to obtain compound A3 (1.34 g, yield: 72.9%). 1 H NMR (600MHz, DMSO-d6) δ12.20(s,1H),7.64(d,J=9.0Hz,1H),7.04(d,J=9.0Hz,1H),7.00(s,1H),5.38(s,1H),3.95(s,4H),3.14(s,4H); 13CNMR(150MHz,DMSO-d6)δ166.5,163.0,156.2,151.2,124.7,111.4,107.0,101.2,88.2,50.4,46.1; ESI-MS[MH] - :calculated for294.0441,found 294.0446.

[0056] (5) Under the protection of N2, redistilled DMF (2 mL) was added dropwise to POCl3 (2 mL), and the mixture was reacted at 50°C for 30 min to obtain a red solution. Subsequently, a DMF solution (10 mL) of a 7-hydroxycoumarin derivative (A1-A3, 10.0 mmol) was added dropwise, and the reaction was continued at 70°C overnight. After the reaction, the reaction solution was poured into ice water, and the pH was adjusted to 6 with NaOH solution. A large amount of solid precipitated. The solid was filtered, washed with water, dried, and recrystallized from anhydrous ethanol to obtain 3-aldehyde-4-chlorocoumarin derivatives B1-B3.

[0057] B1 (yield 88.2%). 1 H NMR (600MHz, CDCl3) δ10.31 (s, 1H), 7.87 (d, J=

[0058] 9.6Hz,1H),6.72(d,J=9.6Hz,1H),6.46(s,1H),3.51(q,J=6.6,7.2,7.2Hz,4H),1.29(t,J=7.2Hz,6H); 13 C NMR (150MHz, CDCl3) δ187.1,160.0,156.5,154.2,153.7,129.3,111.0,110.6,107.7,96.6,45.4,12.5.

[0059] B2 (yield 83.7%). 1 H NMR (600MHz, CDCl3) δ10.28(s,1H),7.45(s,1H),3.42(q,J=5.4,6.0,4.8Hz,4H),2.89(m,4H),2.01(d,J=6.0Hz,4H); 13 C NMR (150MHz, CDCl3) δ187.3,160.3,153.7,151.4,149.6,124.9,120.4,109.6,107.3,105.6,50.4,50.0,27.5,20.9,20.1,20.0.

[0060] B3 (yield 83.0%). 1H NMR (600MHz, DMSO-d6) δ9.78 (s, 1H), 7.80 (d, J=

[0061] 9.0Hz,1H),7.16(d,J=9.0Hz,1H),7.02(s,1H),4.05(s,4H),3.19(s,4H); 13 C NMR (150MHz, DMSO-d6) δ193.4,174.7,161.4,159.4,154.5,127.1,112.2,104.2,100.5,100.0,50.9,45.8; ESI-MS[M+Na + ]:calculated for 364.0017, found 364.0017.

[0062] (6) Under nitrogen protection, 2,3,3-trimethyl-3H-indole (compound 1, 2.0 mmol), 2-(2-bromoethyl)-1,3-dioxane (compound 2, 3.0 mmol), NaHCO3 (4.0 mmol) and KI (3.0 mmol) were dissolved in anhydrous acetonitrile, and the mixture was stirred at 95°C for 18 hours. After the reaction, the reaction solution was cooled to room temperature, the solvent was dried, and the crude product was separated and purified by column chromatography (developing solvent: dichloromethane / methanol = 9:1, v / v) to obtain compound 3 (0.26 g, yield 50%). 1 HNMR(600MHz,CD3CN)δ7.13(m,2H),6.76(t,J=7.2Hz,1H),6.65(d,J=7.8Hz,1H),4.90(t,J=4.8,4.2Hz,1H ), 4.00 (d, J = 7.8Hz, 3H), 3.91 (s, 1H), 3.83 (s, 2H), 3.66 (t, J = 7.8Hz, 7.2Hz, 2H), 1.93 (m, 2H), 1.32 (s, 6H). 13 C NMR (150MHz, CD3CN) δ161.1,145.7,137.5,127.6,121.8,118.4,105.3,102.3,73.2,64.7,43.9,37.0,29.9,29.3; ESI-MS[M+H] + :calculatedfor260.1645,found 260.1645.

[0063] (7) 3-Formyl-4-chlorocoumarin derivative (compound B1-B3, 1 mmol), compound 3 (1 mmol), triethylamine hydrochloride (1 mmol) and a few drops of piperidine were dissolved in acetonitrile (50 mL) in sequence, and refluxed at 82° C. for 12 hours under vigorous stirring. After the reaction, the reaction solution was cooled to room temperature and the solvent was dried by rotary evaporation to obtain an intermediate. The intermediate was redissolved in a mixed solvent of dichloromethane (50 mL) and concentrated hydrochloric acid (15 mL) and the reaction was continued at 25° C. until the reaction of the raw material was complete as monitored by TLC. After the reaction, the dichloromethane was dried by rotary evaporation, and the remaining acid solution was adjusted to pH 6-7 with a Na2CO3 aqueous solution, extracted and dried to obtain a crude product. The crude product was separated and purified by column chromatography (developing solvent: CH2Cl2:MeOH:TFA=400:10:1, V / V / V) to obtain the target compound RCHC1-3.

[0064] RCHC1: dark green solid (143.0 mg, yield: 30%). 1 H NMR(600MHz, DMSO-d6)δ7.95(s,1H),7.80(q,J=7.2,10.8,7.8Hz,2H),7.70(d,J=7.8Hz,1H ),7.56(m,2H),6.90(d,J=9.0Hz,1H),6.67(s,1H),5.66(q,J=5.4,6.0,4.8Hz,1H),4.72(q, J=4.2,10.2,4.8Hz,1H),4.30(q,J=3.6,12.6,11.4Hz,1H),3.55(d,J=6.6Hz,4H),3.00(t,J =6.6Hz,1H),2.66(q,J=5.4,6.6,5.4Hz,1H),1.73(d,J=12.0Hz,6H),1.17(t,J=7.2Hz,4H); 13 C NMR (150MHz, DMSO-d6)174.2,166.0,159.6,152.6,150.7,143.1,141.3,138.1,129.4,128.7,123.3,122.1,120. 8,114.1,109.2,106.2,100.3,99.4,51.3,50.5,49.9,42.7,27.4,27.2,25.9,25.2,20.8,20.1,19.8; ESI-MS[M] + :calculated441.2173, found 441.2174.

[0065] RCHC2: dark green solid (61.1 mg, yield: 12.2%). 11H NMR (600 MHz, DMSO-d6) δ 7.97 (s, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.60 (s, 1H), 7.55 (d, J = 7.2 Hz, 1H), 7.34 (s, 1H), 5.58 (q, J = 5.4, 6.0, 5.4 Hz, 1H), 4.69 (q, J = 4.8, 9.6, 4.8 Hz, 1H), 4.25 (m, 1H), 3.43 (d, J = 5.4 Hz, 4H), 2.92 (t, J = 6.6, 5.4 Hz, 1H), 2.76 (d, J = 6.0 Hz, 4H), 2.62 (m, 1H), 1.90 (s, 4H), 1.72 (d, J = 12.0 Hz, 6H); 13 13C NMR (150 MHz, DMSO-d6) δ 174.8, 166.6, 159.7, 158.0, 154.7, 143.3, 141.3, 137.9, 129.4, 129.0, 126.7, 123.3, 114.4, 111.2, 109.9, 101.0, 99.8, 97.5, 72.8, 51.5, 45.2, 43.0, 27.3, 25.8, 25.2, 12.9; ESI-MS [M + : calculated for 465.2173, found 465.2173.

[0066] RCHC3: Dark green solid (134.8 mg, yield: 25.0%). 1 1H NMR (600 MHz, CD3CN) δ 8.06 (s, 1H), 7.84 (d, J = 9.0 Hz, 1H), 7.72 (d, J = 6.6 Hz, 1H), 7.62 (s, 1H), 7.07 (d, J = 9.0 Hz, 1H), 6.91 (s, 1H), 5.65 (m, 1H), 4.63 (m, 1H), 4.30 (t, J = 14.4, 12.6 Hz, 1H), 4.09 (s, 4H), 3.14 (s, 4H), 3.04 (s, 1H), 2.66 (m, 1H), 1.78 (d, J = 21.6 Hz, 6H); 13C NMR(150MHz,CD3CN)δ175.8,167.2,159.4,158.3,158.0,157.9,154.4,143.1,140.8,138.6,129.4,129.3,126.5, 122.9,116.2,114.3,113.9,112.1,110.5,101.1,100.4,72.4,51.8,50.8,45.8,42.8,26.5,25.2,24.8; ESI-MS[M + ]: calculated for 503.1636, found 503.1640.

[0067] Example 2

[0068] 1. Test solution preparation

[0069] The dyes RCHC1-3 and the comparative compound CHC1 were prepared into 2 mM stock solutions in DMSO (containing 2% acetic acid) and then diluted to the test concentrations with the solvent to be tested.

[0070] 2. Quantum Yield Determination

[0071] The fluorescence quantum yields of dyes RCHC1-3 and comparative compound CHC1 were measured in representative solvents (CH3CN and PBS), with cresyl violet (Φ=0.58 in ethanol) as the reference compound. The quantum yield was calculated as follows:

[0072]

[0073] Among them, A a and A ref are the absorbance values ​​of the reference and sample solutions at the excitation wavelength, S a and S ref are the fluorescence densities of the reference and sample solutions, n a and n ref are the solvent refractive indices of the reference and sample solutions, respectively. The absorbance of the sample and reference solutions at the excitation wavelength is controlled below 0.05.

[0074] 3. Density functional theory calculations

[0075] Gaussian09 program was used to perform DFT and TDDFT calculations to optimize the molecular structure of the S0 and S1 states of dye RCHC1-3. The b3lyp function method was used in the calculation process, and the 6-31g* basis set was used for all atoms.

[0076] 4. Cell Culture and Fluorescence Imaging

[0077] A549 cells were cultured in DMEM (high glucose) medium containing 10% FBS (fetal bovine serum), 100 U / mL penicillin G sodium, and 100 μg / mL streptomycin at 37°C and 5% carbon dioxide in a humidified environment. Before the cell imaging experiment, the cells were placed on a 30 mm glass-bottomed cell culture dish and allowed to stand for 12 hours until the cells adhered to the wall. The cells were washed three times with phosphate buffered saline (PBS) and then fluorescent imaging was performed using a Ceiss LMS 710 confocal microscope.

[0078] 5. Cytotoxicity

[0079] The cytotoxicity of the dyes RCHC1-3 was studied using the CCK-8 cell proliferation assay. Briefly, adherent HeLa cells were first digested with trypsin to prepare a cell suspension, and then the cells were plated at 8.0 × 10 3 The cells were inoculated in a 96-well plate at a density of 100 μL of culture medium overnight until the cells adhered to the wall. Then, RCHC1-3 in acetonitrile solution was added to the 96-well plate, and the final concentration was kept at 0-5 μM. The control group and the test group were repeated 6 times. After 24 hours of cell culture, the old culture medium was discarded and replaced with fresh culture medium containing 10% CCK-8. After incubation for another 0.5 hours, the absorbance at 450 nm was measured using a SpectraMax 190 plate reader.

[0080] 6. Subcellular localization

[0081] LysoTracker Green DND-26 (LTR, 0.2 μM, 10 min) or MitoTracker Deep Red (MTDR, 0.2 μM, 10 min) was added to A549 cells in advance, and washed with PBS for 3 times. Then, RCHC1 (0.2 μM, 10 min) or RCHC2 (0.2 μM, 10 min) was added, and washed with PBS for 3 times. Fluorescence images were taken at the indicated time points. For RCHC1 / RCHC2, the collection wavelength was 570-630 nm (λ ex =561nm); for LTR and MDR, the collection wavelengths are 490-560nm (λ ex =488nm) and 640-750nm (λ ex =633nm).

[0082] To visualize the autophagy process, LysoTracker Green DND-26 (0.2 μM, 10 min) and RCHC2 (0.2 μM, 10 min) were added to A549 cells in sequence, and the cells were washed twice with D-Hanks buffer. Then, the cells were incubated in D-Hanks buffer for 1.5 h to induce autophagy, and fluorescence images were taken at the indicated time points. For RCHC2, the collection wavelength was 630-740 nm (λ ex =561nm); for LTR, the collection wavelengths are 490-560nm (λ ex =488nm).

[0083] 7. Study on photophysical properties

[0084] First, we studied the photophysical properties of the dyes RCHC1-3 in acetonitrile and PBS, respectively, and compared them with the classic coumarin-hemicyanine dye (CHC1). Fig.12 As shown, the dye RCHC1 exhibits similar absorption and emission wavelengths to CHC1, λ abs / λ em =575nm / 640nm, which suggests that the introduction of a bicyclic system into CHC1 does not change its absorption and emission wavelengths. Compared with RCHC1, the absorption and emission wavelengths of the dye RCHC2 are significantly red-shifted (λ abs / λ em =600nm / 675nm), which may be due to the effective σ-p hyperconjugation between the C-Hσ orbital in julolidine and the p orbital in the adjacent aromatic ring; on the contrary, the absorption and emission wavelengths of the dye RCHC3 are obviously blue-shifted (λ abs / λ em =530nm / 615nm), which is due to the weak electron-donating ability of thiomorpholine-1,1-dioxide, which weakens the ICT effect within the molecule. Density functional theory calculation results show that the HOMO-LUMO energy level difference of the dye RCHC1-3 is consistent with its absorption / emission wavelength trend ( Fig.13). Similar to CHC1, the dyes RCHC1-3 all have large Stokes shifts (60-69nm), which indicates that they can avoid interference from biological autofluorescence and excitation light backscattering during fluorescence imaging. Importantly, compared with CHC1, the fluorescence quantum yields of the dyes RCHC1-3 in acetonitrile and PBS are greatly improved, suggesting that the conformational fixation strategy can effectively inhibit the non-radiative transition process induced by the cis-trans isomerization of the excited state C=C bond. Although the molar absorption coefficients of RCHC1-3 are reduced compared with CHC1, most of the dyes have improved fluorescence brightness due to the high fluorescence quantum yield; although the fluorescence brightness of RCHC2 in PBS is lower, given the huge difference in its fluorescence brightness in acetonitrile and PBS, this dye seems to be more useful in constructing wash-free probes for live cell imaging of intracellular proteins.

[0085] In summary, by introducing a bicyclic system into the skeleton of classical CHCs, the non-radiative transition process induced by the cis-trans isomerization of the excited-state C=C bond can be effectively suppressed. The series of conformationally restricted RCHCs constructed in this way not only have significantly improved fluorescence quantum yields, but also have large Stokes shifts, and therefore have more advantages in biological imaging.

[0086] Table 1 Photophysical properties of dyes RCHC1-3 and comparative compound CHC1 in acetonitrile and PBS

[0087]

[0088] 8. Cell Imaging Studies

[0089] Before performing cell imaging experiments, we first tested the cytotoxicity of the dyes RCHC1-3 using CCK8 assays. Fig.14 As shown, the dyes RCHC1 and RCHC2 have negligible cytotoxicity in the concentration range of 0-2 μM (cell viability is about 85%), and the dye RCHC3 has a cell viability of about 100% in the concentration range of 0-5 μM, indicating that this type of dye has excellent biocompatibility below 2 μM.

[0090] We then used laser confocal microscopy to study the cellular imaging of these dyes. Cellular uptake experiments showed ( Fig.15 ), the dyes CHC1, RCHC1 and RCHC2 can quickly penetrate the cell membrane within 20 minutes. Compared with CHC1, cells loaded with RCHC1 or RCHC2 showed stronger fluorescence signals; however, due to its strong water solubility, RCHC3 cannot penetrate the cell membrane with strong lipid solubility, so cells loaded with RCHC3 showed negligible fluorescence signals, but this does not affect its application in protein labeling.

[0091] Next, we verified the advantages of dyes with large Stokes shifts in cell imaging at the cellular level, and used commercial rhodamine B (Rh B) dye as a comparison. A549 cells were incubated with Rh B, RCHC1, and RCHC2 for 20 minutes and then imaged on a confocal microscope. The excitation wavelength was set to 561 nm, and the collection wavelengths were 570-740 nm, 570-600 nm, 630-660 nm, and 670-700 nm, respectively. Fig.16 As shown, the 570-740nm channel covers the entire emission spectra of Rh B, RCHC1, and RCHC2, and all cells exhibit bright intracellular fluorescence; in the 570-600nm channel, only cells incubated with Rh B exhibit bright intracellular fluorescence, and almost no fluorescence signals are observed in cells incubated with RCHC1 or RCHC2; in the longer 630-660nm or 670-700nm channels, bright fluorescence signals are only observed in cells incubated with RCHC1 or RCHC2, and almost no fluorescence signals are observed in cells incubated with Rh B. The above results indicate that, given the large Stokes shifts of RCHC1 and RCHC2, if RCHC1 or RCHC2 is combined with dyes of appropriate wavelengths, multicolor imaging is expected to be achieved.

[0092] Finally, we evaluated the subcellular localization of RCHC1 and RCHC2. Fig.17 As shown in the figure, although RCHC1 and RCHC2 have similar molecular structures, their localization is completely different. The former is located in lysosomes and the latter is located in mitochondria, which is mainly attributed to their different hydrophilicity and lipophilicity.

[0093] 9. Cell autophagy research

[0094] We combined RCHC2 and the commercial lysosomal dye LTG to verify the process of cell autophagy. A549 cells pre-incubated with RCHC2 and LTG were cultured in D-Hanks buffer solution to induce cell autophagy by starvation, as shown in Figure 2. Fig.18 As shown, initially separated mitochondria and lysosomes can be observed, as shown by the separated green and red fluorescent spots; however, as the starvation time goes on, some mitochondria fuse with lysosomes, suggesting that autophagy is occurring; when the cells have been starved for 80 minutes, the mitochondria are almost completely fused with the lysosomes, and the red and green fluorescent signals are also completely mixed (yellow), and the Pearson's colocalization coefficient gradually increases from 0.16 to 0.76, which also well reflects the fusion process.

[0095] In summary, the present invention introduces a bicyclic system into the skeleton of coumarin hemicyanine dyes (CHCs) to construct a series of conformationally fixed coumarin hemicyanine dyes (RCHC1-3), which not only retain the large Stokes shift of the classic CHCs dyes, but also, more importantly, significantly improve the fluorescence quantum yield. In view of the above advantages, the series of dyes not only show improved fluorescence brightness during cell imaging, but also avoid the interference of biological autofluorescence and excitation light scattering, and therefore have potential application value.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A structurally rigid coumarin hemicyanine dye, characterized in that: Its structural formula is:

2. The method for preparing the structurally rigid coumarin hemicyanine dye according to claim 1, characterized in that: The following steps are involved: (1) Under the protection of N2, redistilled DMF is added dropwise to POCl3 to react to obtain a red solution, and then a DMF solution of 7-hydroxycoumarin derivatives A1-A3 is added dropwise to continue the reaction. After the reaction is completed, the reaction solution is poured into ice water, and the pH is adjusted with NaOH solution until a large amount of solid precipitates. The solid is filtered, washed with water, dried, and recrystallized from anhydrous ethanol to obtain 3-aldehyde-4-chlorocoumarin derivatives B1-B3; (2) Under nitrogen protection, 2,3,3-trimethyl-3H-indole, 2-(2-bromoethyl)-1,3-dioxane, NaHCO3 and KI were dissolved in anhydrous acetonitrile, stirred and reacted. After the reaction, the reaction solution was cooled to room temperature, the solvent was dried, and the crude product was separated and purified by column chromatography to obtain compound 3; (3) dissolving 3-formyl-4-chlorocoumarin derivatives B1-B3, compound 3, triethylamine hydrochloride and piperidine in acetonitrile in turn, and refluxing under vigorous stirring. After the reaction, the reaction solution is cooled to room temperature, and the solvent is dried to obtain an intermediate; the intermediate is redissolved in a mixed solvent of dichloromethane and concentrated hydrochloric acid and the reaction is continued until the reaction of the raw materials is complete as monitored by TLC. After the reaction, the dichloromethane is dried to obtain the remaining acid solution, and the pH is adjusted to 6-7 with a Na2CO3 aqueous solution, and then extracted and dried to obtain a crude product. The crude product is separated and purified by column chromatography to obtain the target compound RCHC1-3; Wherein, the preparation method of 7-hydroxycoumarin derivative A3 is as follows: (1.1) At 0°C, 4-bromo-2-hydroxyacetophenone is dissolved in anhydrous toluene, and then a toluene solution of NaH is added dropwise. When no gas is generated, diethyl carbonate dissolved in toluene is added dropwise, and the reaction is refluxed. After the reaction is completed, the reaction solution is cooled and dried to obtain a crude product. The crude product is dissolved in anhydrous toluene and refluxed to obtain a large amount of solid. The solid is filtered, washed and dried to obtain 4-hydroxy-7-bromocoumarin. (1.2) 4-Hydroxy-7-bromocoumarin and K2CO3 are dissolved in anhydrous acetonitrile respectively, reacted under reflux conditions, and then benzyl bromide dissolved in acetonitrile is added dropwise, and the reaction is continued under reflux conditions. After the reaction is completed, K2CO3 is filtered out to terminate the reaction, and the reaction solution is cooled to -20°C to generate a large amount of solid. The obtained solid is filtered, washed and dried to obtain 4-benzyloxy-7-bromocoumarin; (1.3) Under N2 protection, PdCl2, Cs2CO3, 1,1'-binaphthyl-2,2'-bis(diphenylphosphine), 1,1-thiomorpholine dioxide and 4-benzyloxy-7-bromocoumarin were dissolved in anhydrous 1,4-dioxane, respectively, and stirred for reaction. After the reaction was completed, the reaction solution was cooled to room temperature and filtered. The filter cake was recrystallized from ethyl acetate to obtain compound 1d; (1.4) Compound 1d was dissolved in anhydrous dichloromethane at 0°C, and then a solution of BBr3 in anhydrous dichloromethane was added dropwise. The reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the reaction solution was poured into ice water to generate a large amount of solid. The large amount of solid was filtered, washed with water, and dried to obtain 7-hydroxycoumarin derivative A3; The structural formula of the 7-hydroxycoumarin derivatives A1-A3 is: The structural formula of the 3-formyl-4-chlorocoumarin derivatives B1-B3 is: The structural formula of the compound 3 is: The structural formula of the intermediate is: The structural formula of the compound 1d is: -NR2 in the above structural formula is:

3. The method for preparing the structurally rigid coumarin hemicyanine dye according to claim 2, characterized in that: In the step (1.1), the molar ratio of 4-bromo-2-hydroxyacetophenone to NaH is 1:3; the temperature of the reflux reaction is 110° C. and the time is 24 h; the temperature of the continued reflux reaction is 110° C. and the time is 36 h.

4. The method for preparing the structurally rigid coumarin hemicyanine dye according to claim 2, characterized in that: In the step (1.2), the molar ratio of 4-hydroxy-7-bromocoumarin, K2CO3 and benzyl bromide is 1:2:2; the temperature of the reaction under reflux conditions is 82°C and the time is 1 hour; the temperature of the continued reaction under reflux conditions is 82°C and the time is 6 hours.

5. The method for preparing the structurally rigid coumarin hemicyanine dye according to claim 2, characterized in that: In the step (1.3), the molar ratio of PdCl2, Cs2CO3, 1,1'-binaphthyl-2,2'-bisdiphenylphosphine, thiomorpholine-1,1-dioxide and 4-benzyloxy-7-bromocoumarin is 0.05:1.2:0.1:1.1:1; the temperature of the stirring reaction is 90°C and the time is overnight.

6. The method for preparing the structurally rigid coumarin hemicyanine dye according to claim 2, characterized in that: In the step (1.4), the molar ratio of compound 1d to BBr3 is 1:

2.

7. The method for preparing the structurally rigid coumarin hemicyanine dye according to claim 2, characterized in that: In the step (1), the volume molar ratio of DMF, POCl3 and 7-hydroxycoumarin derivative A1-A3 is 1 mL: 1 mL: 5 mmol; the reaction temperature is 50°C and the reaction time is 30 minutes; the reaction temperature is 70°C and the reaction time is overnight; the pH is adjusted by adjusting the pH to 6 with NaOH solution.

8. The method for preparing the structurally rigid coumarin hemicyanine dye according to claim 2, characterized in that: In the step (2), the molar ratio of 2,3,3-trimethyl-3H-indole, 2-(2-bromoethyl)-1,3-dioxane, NaHCO3 and KI is 2:3:4:3; the temperature of the stirring reaction is 95°C and the time is 18 hours; the developing solvent for the column chromatography separation and purification is CH2Cl2 / MeOH=9:1, v / v.

9. The method for preparing the structurally rigid coumarin hemicyanine dye according to claim 2, characterized in that: In the step (3), the molar ratio of 3-formyl-4-chlorocoumarin derivatives B1-B3, compound 3 and triethylamine hydrochloride is 1:1:1, the volume ratio of dichloromethane and concentrated hydrochloric acid is 50mL:15mL; the temperature of the reflux reaction is 82°C and the time is 12h; the temperature of the continued reaction is 25°C; the developing solvent for the column chromatography separation and purification is CH2Cl2:MeOH:TFA=400:10:1, v / v / v.

10. The use of the rigid coumarin hemicyanine dye according to claim 1, characterized in that: Application in the preparation of products for verifying starvation-induced cell autophagy, and application in the preparation of products for cell imaging.

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