PKa-adjustable aza-boron dipyrromethene fluorescent dye with near-infrared two-region fluorescence emission and preparation method of pKa-adjustable aza-boron dipyrromethene fluorescent dye

Abstract

The invention discloses a pKa-adjustable aza-bodipy fluorescent dye with near-infrared two-region fluorescence emission and a preparation method thereof, in an aza-bodipy framework containing a phenol end group, different halogen atoms are introduced to ortho-position of phenol to obtain a series of fluorescent probes, the series of fluorescent probes are named as AHNU-905 series of probes, including OH-OBDP, FOH-OBDP, ClOH-OBDP, BrOH-OBDP and 2ClOH-OBDP, the pKa is 9.5, 8.5, 8.2, 7.5 and 6.1 respectively, and the pKa is 9.5, 8.5, 8.2, 7.5 and 6.1 respectively. And the 2ClOH-OBDP has very strong near-infrared two-region fluorescence under the physiological condition of pH 7.4, has very good near-infrared two-region fluorescence characteristics, and can be used for high-definition imaging of parts such as blood vessels and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to an aza-fluoro-boron fluorescent dye with adjustable pKa and near-infrared second-region fluorescence emission and a preparation method thereof. Background Art

[0002] Near-infrared II (NIR-II, 900-1700nm) fluorescence imaging can significantly reduce background interference such as tissue autofluorescence and scattering during imaging, and has deeper tissue penetration depth, higher resolution and signal-to-noise ratio (SBR).

[0003] At present, near-infrared region II fluorescent probes are mainly divided into organic small molecule dyes, conjugated polymers, rare earth nanoparticles and single-walled carbon nanotubes. Among them, organic small molecule fluorescent dyes have received more and more attention due to their better biocompatibility and adjustable chemical and physical properties. Currently, the widely reported near-infrared region II organic small molecule dyes mainly include three categories with donor-acceptor-donor (DAD), polymethine (D-π-A) and fluoroborane as the core structure. Among them, azafluoroborane fluorescent dyes have very excellent chemical and photostability, as well as a large molar extinction coefficient, and have always attracted much attention.

[0004] However, the aza-fluoro-boron fluorescent dyes reported so far are mainly of the "always bright" type, which will result in a relatively high background signal of normal tissue during imaging and a decrease in the signal-to-noise ratio of imaging. Therefore, it is crucial to develop a class of activatable fluorescent cores based on aza-fluoro-boron fluorescent dyes with a universal design strategy. Although there are also literature reports on the introduction of phenol as an activatable site in aza-fluoro-boron fluorescent dyes, the pKa of phenol is relatively high at this time, and the fluorescence is very weak under physiological conditions. Therefore, it has been a challenge to develop a class of aza-fluoro-boron fluorescent dye cores with near-infrared second-zone emission that can be activated under physiological conditions. Summary of the invention

[0005] To solve the above technical problems, the present invention provides an aza-fluoro-boron fluorescent dye with adjustable pKa and near-infrared second-zone fluorescence emission and a preparation method thereof. The aza-fluoro-boron fluorescent dye is prepared by introducing different halogen atoms at the ortho position of phenol to obtain a series of dyes, and different halogen atoms will have a significant effect on the deprotonation ability of phenol and thus have different pKa.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The present invention provides an aza-fluoro-boron fluorescent dye with adjustable pKa having near-infrared second-region fluorescence emission, and the structure of the aza-fluoro-boron fluorescent dye is as follows:

[0008]

[0009] Among them, OH-OBDP, F OH-OBDP, Cl OH-OBDP, Br OH-OBDP, 2Cl The pKa of OH-OBDP are 9.5, 8.5, 8.2, 7.5 and 6.1, respectively.

[0010] The pKa-adjustable aza-fluoro-boron fluorescent dye with near-infrared second-zone fluorescence emission is named "AHNU-905" dye. The "AHNU-905" series of dyes have pH-dependent fluorescence emission. As the pH changes from acidic to alkaline, the fluorescence gradually increases, and the maximum emission peak position is 905nm.

[0011] in 2Cl OH-OBDP has excellent near-infrared second-zone luminescence under physiological conditions. Its brightness after 1000nm is OH-OBDP, F OH-OBDP, Cl OH-OBDP, Br 171 times, 129 times, 13.5 times and 10.1 times that of OH-OBDP.

[0012] The present invention also provides a method for preparing the pKa-adjustable aza-fluoro-boron fluorescent dye having near-infrared second-region fluorescence emission, the preparation method comprising the following steps:

[0013] 1) conducting an aldehyde-ketone condensation reaction between p-methoxybenzaldehyde and a vinyl ether-protected acetophenone derivative to obtain an intermediate product A;

[0014] 2) reacting the intermediate product A with nitromethane in an ethanol solvent of sodium hydroxide to obtain an intermediate product B;

[0015] 3) condensing the intermediate product B with ammonium acetate, and then reacting the condensation product with N,N-diisopropylethylamine and boron trifluoride ether to obtain the intermediate product C;

[0016] 4) Deprotecting the intermediate product C from vinyl ether under the action of triphenylphosphine palladium to obtain an aza-fluoro-boron fluorescent dye;

[0017] The structural formula of the vinyl ether protected acetophenone derivative is:

[0018] The structural formula of the intermediate product A is:

[0019] The structural formula of the intermediate product B is:

[0020] The structural formula of the intermediate product C is:

[0021] The step 1) specifically comprises: dissolving p-methoxybenzaldehyde and a vinyl ether protected acetophenone derivative in ethanol, adding sodium hydroxide, reacting at room temperature overnight, filtering and drying to obtain an intermediate product A.

[0022] Furthermore, the molar ratio of p-methoxybenzaldehyde, vinyl ether protected acetophenone derivative and sodium hydroxide is 1:1-1.2:1-1.2; and the concentration of p-methoxybenzaldehyde in ethanol is 0.5-1.5M.

[0023] The step 2) specifically comprises: dissolving the intermediate product A in ethanol, adding nitromethane and sodium hydroxide, reacting at 65-75° C. for 10-12 hours, extracting and purifying by column chromatography to obtain the intermediate product B.

[0024] Furthermore, the molar ratio of the intermediate product A, nitromethane and sodium hydroxide is 1:10-12:1-1.2; and the concentration of the intermediate product A in ethanol is 0.5-1.0M.

[0025] The step 3) specifically comprises: dissolving the intermediate product B in n-butanol, adding ammonium acetate, stirring and reacting at 105-115° C. for 20-24 hours under a nitrogen atmosphere, and then dissolving the obtained product in anhydrous dichloromethane, adding N,N-diisopropylethylamine thereto at -15--25° C. under a N2 atmosphere, and then slowly adding boron trifluoride etherate after 10 minutes, returning to room temperature and stirring and reacting for 10-12 hours, and obtaining the intermediate product C through extraction and column chromatography purification.

[0026] Furthermore, the molar ratio of the intermediate product B, ammonium acetate, N,N-diisopropylethylamine, and boron trifluoride etherate is 1:10-12:3-3.5:5-5.5; and the concentration of the intermediate product B in n-butanol is 0.3-0.5M.

[0027] The step 4) specifically comprises: adding the intermediate product C, 1,3-dimethylbarbituric acid and tetrakistriphenylphosphine palladium to anhydrous DMF under a nitrogen atmosphere, stirring and reacting at room temperature for 1-2 hours, extracting and purifying by column chromatography to obtain an aza-fluoro-boron fluorescent dye.

[0028] Furthermore, the molar ratio of the intermediate product C, 1,3-dimethylbarbituric acid, and tetrakistriphenylphosphine palladium is 1:2.5-3.0:0.4-0.5; and the concentration of the intermediate product C in DMF is 0.1-0.2M.

[0029] The present invention also provides a pKa-adjustable aza-fluorine-boron fluorescent dye with near-infrared second-zone fluorescence emission 2Cl Application of OH-OBDP in fluorescence imaging.

[0030] The pKa-adjustable aza-fluoro-boron fluorescent dye with near-infrared second-region fluorescence emission provided by the present invention can adjust the pKa and luminescence properties of the fluorescent dye by introducing different halogens (F, Cl, Br, I) at the ortho-position of phenol and using them as the end groups of the aza-fluoro-boron fluorescent dye. The optimized and screened aza-fluoro-boron fluorescent dye containing 2,6-dichlorophenol end groups can have very strong near-infrared second-region fluorescence under physiological conditions (pH 7.4), and can achieve high-definition imaging of blood vessels. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The structural formula of the pKa-adjustable aza-fluorine-boron fluorescent dye with near-infrared second-region fluorescence emission;

[0032] Figure 2 This is a general design route for the "AHNU-905" series of fluorescent probes.

[0033] Figure 3 This is the H NMR spectrum of OH-OBDP dye.

[0034] Figure 4 for F H NMR spectrum of OH-OBDP dye.

[0035] Figure 5 for Cl H NMR spectrum of OH-OBDP dye.

[0036] Figure 6 for Br H NMR spectrum of OH-OBDP dye.

[0037] Figure 7 for 2Cl H NMR spectrum of OH-OBDP dye.

[0038] Figure 8 For OH-OBDP, F OH-OBDP, Cl OH-OBDP, Br OH-OBDP, 2Cl Fluorescence emission spectra (excitation at 808 nm) of OH-OBDP micelles (probe concentration was 10 μM) at different pH values.

[0039] Fig. 9 is (a) OH-OBDP, F OH-OBDP, Cl OH-OBDP, Br OH-OBDP, 2Cl(a) Fluorescence emission intensity of the OH-OBDP probe at 905 nm changes with solution pH; (b) Comparison of the fluorescence intensities of the five probes at pH 3.0, pH 7.4, and pH 12.5.

[0040] Fig.10 (a) UV-Vis-NIR changes of OH-OBDP nanoprobe at different pH values; (b) corresponding log(A max -A) / (AA min ) has a linear relationship with pH.

[0041] Fig.11 For (a) F UV-Vis-NIR changes of OH-OBDP nanoprobe at different pH values; (b) corresponding log(A max -A) / (AA min ) has a linear relationship with pH.

[0042] Fig.12 For (a) Cl UV-Vis-NIR changes of OH-OBDP nanoprobe at different pH values; (b) corresponding log(A max -A) / (AA min ) has a linear relationship with pH.

[0043] Fig.13 For (a) Br UV-Vis-NIR changes of OH-OBDP nanoprobe at different pH values; (b) corresponding log(A max -A) / (AA min ) has a linear relationship with pH.

[0044] Fig.14 For (a) 2Cl UV-Vis-NIR changes of OH-OBDP nanoprobe at different pH values; (b) corresponding log(A max -A) / (AA min ) has a linear relationship with pH.

[0045] Fig.15 For OH-OBDP, F OH-OBDP, Cl OH-OBDP, Br OH-OBDP, 2Cl The ratio of the absorption wavelength of OH-OBDP probe at 770nm to 720nm changes with pH.

[0046] Fig.16 For OH-OBDP, FOH-OBDP, Cl OH-OBDP, Br OH-OBDP, 2Cl Brightness of OH-OBDP nanoprobe (10 μM concentration) at pH 11.0, 7.4, 6.5, 5.5 and 3.0 (using 1000 nm

[0047] long-pass filter).

[0048] Fig.17 for 2Cl OH-OBDP micelles were used for near-infrared zone II fluorescence imaging of the back and abdomen of mice. DETAILED DESCRIPTION

[0049] The present invention is described in detail below with reference to the embodiments.

[0050] Example 1

[0051] An aza-fluoro-boron fluorescent dye OH-OBDP, the structural formula of which is:

[0052] Its synthetic route is shown below:

[0053]

[0054] The preparation method is as follows:

[0055] Synthesis of Compound A1: p-Anisaldehyde (2.72 g, 20 mmol, 1.0 equiv.) and 1-(4-(propyleneoxy)phenyl)ethane-1-one (3.52 g, 20 mmol, 1.0 equiv.) were dissolved in 20 ml of ethanol, and sodium hydroxide (800 mg, 20 mmol, 1.0 equiv.) was added thereto. The mixture was reacted at room temperature overnight, and 3.9 g of a solid product was obtained by suction filtration. Yield: 66%. 1 HNMR(400MHz, CDCl3)δ8.16–7.97(m,2H),7.80(d,J=15.6Hz,1H),7.69–7.58(m,2H),7.45(d,J=15.6Hz,1H),7.1 3–6.85(m,4H),6.09(ddt,J=17.3,10.5,5.3Hz,1H),5.60–5.23(m,2H),4.64(dt,J=5.3,1.6Hz,2H),3.87(s,3H). 13C NMR (101MHz, CDCl3) δ188.77,162.28,161.53,143.85,132.58,131.48,131.45,130.69,130 .13,127.83,119.56,118.21,114.51,114.40,113.61,68.92,55.42.ESI-MS(m / z)calc.for Calcd.for[M+H] + ,295.3523,found,295.133.

[0056] Synthesis of compound B1: A1 (3.5 g, 11.9 mmol, 1.0 equiv.) was dissolved in 20 ml of ethanol, and nitromethane (7.26 g, 119 mmol, 10 equiv.) and sodium hydroxide (476 mg, 11.9 mmol, 1.0 equiv.) were added, and then reacted at 70°C for 12 h. The ethanol was spin-dried, and dichloromethane and saturated brine were added for extraction. The organic layer was dried over anhydrous sodium sulfate and then subjected to column chromatography. The column chromatography was performed using an eluent composed of dichloromethane and petroleum ether in a volume ratio of 1:1, and finally 2.5 g of the product was obtained with a yield of 59.2%.

[0057] 1 H NMR (400MHz, CDCl3) δ7.97–7.86(m,2H),7.25–7.15(m,2H),6.97–6.93(m, 2H),6.93–6.81(m,2H),6.06(ddt,J=17.1,10.5,5.3Hz,1H),5.44(dq,J=17 .2,1.6Hz,1H),5.34(dq,J=10.6,1.4Hz,1H),4.82(dd,J=12.3,6.5Hz,1H) ,4.69–4.61(m,3H),4.18(p,J=7.1Hz,1H),3.79(s,3H),3.44–3.29(m,2H). 13 CNMR(101MHz,CDCl3)δ195.45,162.82,159.06,132.38,131.17,130.70,129.61,12 8.52,118.31,114.58,114.52,79.92,68.94,55.26,41.30,38.78.Calcd.for[M+H] + ,356.3924,found,356.150.

[0058] Synthesis of compound C1: Compound B1 (2.2 g, 6.2 mmol, 1.0 equiv.) was dissolved in 20 ml of n-butanol, and ammonium acetate (4.77 g, 62 mmol, 10 equiv.) was added. The mixture was reacted at 110 °C under a nitrogen atmosphere for 24 h and then returned to room temperature. The solvent was dried, saturated brine was added, and the mixture was extracted twice with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated and dried, and then directly proceeded to the next step. The obtained product was dissolved in 50 ml of anhydrous dichloromethane, and N,N-diisopropylethylamine (2.4 g, 18.6 mmol, 3.0 equiv.) was added thereto at -20°C under N2 atmosphere. After 10 minutes, boron trifluoride etherate (4.4 g, 31 mmol, 5.0 equiv.) was slowly added. After returning to room temperature and stirring for 12 hours, saturated brine was slowly added, and the mixture was extracted twice with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated and spin-dried, and then column chromatography was performed using a mixed solution of dichloromethane and petroleum ether in a volume ratio of 1:1 as an eluent, and finally 320 mg of a brown solid product was obtained with a yield of 15.4%. 1 HNMR(400MHz, CDCl3)δ8.33–7.98(m,8H),7.49(s,2H),7.14(ddd,J=13.8,6.8,4.6Hz,8H ),6.20–6.02(m,2H),5.52–5.28(m,4H),4.70(dd,J=19.2,5.2Hz,4H),3.92–3.83(m,6H). 13 CNMR(101MHz,DMSO)δ161.15,160.66,159.58,156.42,149.24,144.77,142.33,134.12,131.98, 130.53,125.03,124.02,118.43,115.44,114.49,68.96,55.90.MALDI-TOF(m / z):Calcd.for[M] + ,669.5235,found,669.4612.

[0059] Synthesis of compound D1: Compound C1 (200 mg, 0.3 mmol, 1.0 equiv.) was added to a dry round-bottom flask, and 1,3-dimethylbarbituric acid (117 mg, 0.75 mmol, 2.5 equiv.) and tetrakistriphenylphosphine palladium (138 mg, 0.12 mmol, 0.4 equiv.) were added. Under a nitrogen atmosphere, anhydrous DMF (2 ml) was added as a solvent. After stirring at room temperature for 1-2 h, dichloromethane was added, and DMF was removed by multiple extractions with saturated brine. The organic phase was dried over anhydrous sodium sulfate, spin-dried, and then column chromatography was performed. The eluent for column chromatography was a mixed solution of DCM:CH3OH in a volume ratio of 95:5 to obtain 120 mg of a brown solid product with a yield of 68%. 1 H NMR (400MHz, DMSO) δ10.43(s,2H),8.15(d,J=8.8Hz,4H),8.06(d,J=8.6Hz,4H),7.43(s,2H),7.17–7.09(m,4H),6.94(d,J=8.5Hz,4H),3.86(s,6H). 13 C NMR(101MHz,DMSO)δ162.78,161.08,,157.44,144.55,141.81,132.32,131.01,1 25.11,122.42,118.22,116.23,114.83,55.86.MALDI-TOF(m / z):Calcd.for[M+H] + ,590.4038,found,590.3479.

[0060] Example 2

[0061] A kind of aza-fluorine-boron fluorescent dye F OH-OBDP, its structural formula is:

[0062] Its synthetic route is shown below:

[0063]

[0064] The preparation method is as follows:

[0065] Synthesis of compound A2: p-Anisaldehyde (2.72 g, 20 mmol, 1.0 equiv.), 1-(4-(propyleneoxy)-3-fluorophenyl)ethane-1-one (3.88 g, 20 mmol, 1.0 equiv.) were dissolved in 20 ml of ethanol, sodium hydroxide (800 mg, 20 mmol, 1.0 equiv.) was added thereto, and then reacted at room temperature overnight, and 4.2 g of solid product was obtained by suction filtration. Yield: 67%.1 H NMR (400MHz, CDCl3) δ7.91–7.73(m,3H),7.69–7.55(m,2H),7.39(d,J=15.5Hz,1H),7.05(t,J=8.2Hz,1H),7.00 –6.88(m,2H),6.10(ddt,J=17.4,10.6,5.3Hz,1H),5.61–5.27(m,2H),4.72(dt,J=5.3,1.5Hz,2H),3.88(s,3H). 13 C NMR (101MHz, CDCl3) δ187.78,161.73,153.49,151.03,144.56,132.08,130.26,127.59,125.44 ,118.85,118.74,116.48,116.29,114.45,113.92,69.97,55.44.ESI-MS(m / z):Calcd.for[M+H] + ,313.3428,found,313.124.

[0066] Synthesis of compound B2: A2 (4.0 g, 12.8 mmol, 1.0 equiv.) was dissolved in 20 ml of ethanol, and nitromethane (7.81 g, 128 mmol, 10 equiv.) and sodium hydroxide (512 mg, 12.8 mmol, 1.0 equiv.) were added, and then reacted at 70°C for 12 h. The ethanol was spin-dried, and dichloromethane and saturated brine were added for extraction. The organic layer was dried over anhydrous sodium sulfate and then subjected to column chromatography. Column chromatography was performed using an eluent composed of dichloromethane and petroleum ether in a volume ratio of 1:1, and finally 2.8 g of the product was obtained with a yield of 58.6%.

[0067] 1 H NMR (400MHz, CDCl3) δ7.76–7.59(m,2H),7.26–7.14(m,2H),6.99(t,J=8.4Hz,1H),6.94–6.81(m,2H),6.07(ddt,J=17.3,10.6,5.4 Hz,1H),5.51–5.34(m,2H),4.80(dd,J=12.3,6.7Hz,1H),4.72–4.60(m,3H),4.17(p,J=7.1Hz,1H),3.79(s,3H),3.43–3.24(m,2H). 13C NMR (101MHz, CDCl3) δ194.69,159.12,153.41,151.26,131.85,130.90,129.85,128.50,125.27,1 18.86,115.98,114.45,113.88,79.83,69.96,55.27,41.30,38.71.ESI-MS(m / z):Calcd.for[M+H] + ,374.3828,found,374.141.

[0068] Synthesis of compound C2: Compound B2 (2.5 g, 6.7 mmol, 1.0 equiv.) was dissolved in 20 ml of n-butanol, and ammonium acetate (5.16 g, 67 mmol, 10 equiv.) was added. The mixture was reacted at 110 °C under a nitrogen atmosphere for 24 h and then returned to room temperature. The solvent was dried, saturated brine was added, and the mixture was extracted twice with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated and dried, and then directly proceeded to the next step. The obtained product was dissolved in 50 ml of anhydrous dichloromethane, and N,N-diisopropylethylamine (2.6 g, 20.1 mmol, 3.0 equiv.) was added thereto at -20°C under N2 atmosphere. After 10 minutes, boron trifluoride etherate (4.75 g, 33.5 mmol, 5.0 equiv.) was slowly added. After returning to room temperature and stirring for 12 hours, saturated brine was slowly added, and the mixture was extracted twice with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated and spin-dried, and then column chromatography was performed using a mixed solution of dichloromethane and petroleum ether in a volume ratio of 1:1 as an eluent, and finally 280 mg of a brown solid product was obtained with a yield of 11.9%. 1 H NMR (400MHz, CDCl3) δ8.06 (d, J=8.6Hz, 4H), 7.96–7.63 (m, 4H), 7.16–6.82 (m, 8H), 6.11 (ddd, J=21.0, 10. 5,5.3Hz,2H),5.59–5.45(m,2H),5.38(dd,J=10.5,1.5Hz,2H),4.72(dt,J=5.4,1.6Hz,4H),3.92(s,6H). 13 C NMR (101MHz, CDCl3) δ160.93,156.83,153.27,148.96,148.89,145.32,143.32,132.30,130.8 8,125.25,118.70,117.16,114.49,114.20,69.98,55.48.MALDI-TOF(m / z):Calcd.for[M+Na] +,706.5124,found,706.4341.

[0069] Synthesis of compound D2: Compound C2 (250 mg, 0.355 mmol, 1.0 equiv.) was added to a dry round-bottom flask, and 1,3-dimethylbarbituric acid (138 mg, 0.887 mmol, 2.5 equiv.) and tetrakistriphenylphosphine palladium (164 mg, 0.142 mmol, 0.4 equiv.) were added. Under a nitrogen atmosphere, anhydrous DMF (2 ml) was added as a solvent. After stirring at room temperature for 1-2 h, dichloromethane was added, and DMF was removed by multiple extractions with saturated brine. The organic phase was dried over anhydrous sodium sulfate, spin-dried, and then column chromatography was performed. The eluent for column chromatography was a mixed solution of DCM:CH3OH in a volume ratio of 95:5 to obtain 160 mg of a brown solid product with a yield of 72%. 1 H NMR (400MHz, DMSO) δ10.96(s,2H),8.26–7.81(m,8H),7.51(s,2H),7.27–7.03(m,6H),3.87(s,6H). 13 C NMR(101MHz,DMSO)δ161.20,156.53,152.27,149.88,148.84,144.79,142.38,131.14,1 27.75,124.93,122.60,118.53,118.38,114.89,55.89.MALDI-TOF(m / z):Calcd.for[M] + ,625.3767,found,625.3702.

[0070] Example 3

[0071] A kind of aza-fluorine-boron fluorescent dye Cl OH-OBDP, its structural formula is:

[0072] Its synthetic route is as follows:

[0073]

[0074] The preparation method is as follows:

[0075] Synthesis of compound A3: p-Anisaldehyde (2.72 g, 20 mmol, 1.0 equiv.), 1-(4-(propyleneoxy)-3-chlorophenyl)ethane-1-one (4.2 g, 20 mmol, 1.0 equiv.) were dissolved in 20 ml of ethanol, sodium hydroxide (800 mg, 20 mmol, 1.0 equiv.) was added thereto, and then reacted at room temperature overnight, and 4.5 g of solid product was obtained by suction filtration. Yield: 68.6%. 1 H NMR (400MHz, CDCl3) δ8.11(d,J=2.1Hz,1H),7.95(dd,J=8.6,2.2Hz,1H),7.81(d,J=15.5Hz,1H),7.66–7.58(m,2H),7.39(d,J=15.5Hz,1H),7.04–6. 93(m,3H),6.10(ddt,J=17.3,10.4,5.1Hz,1H),5.52(dq,J=17.3,1.6Hz,1 H), 5.38 (dq, J=10.5, 1.4Hz, 1H), 4.73 (dt, J=5.1, 1.6Hz, 2H), 3.88 (s, 3H). 13 C NMR (101MHz, CDCl3) δ187.71,161.74,157.54,144.63,132.00,131.91,130.84,130.29,128.67 ,127.58,123.24,118.86,118.44,114.45,112.66,69.75,55.44.ESI-MS(m / z):Calcd.for[M+H] + ,329.7971,found,329.095.

[0076] Synthesis of compound B3: A3 (4.0 g, 12.2 mmol, 1.0 equiv.) was dissolved in 20 ml of ethanol, and nitromethane (7.44 g, 128 mmol, 10 equiv.) and sodium hydroxide (488 mg, 12.2 mmol, 1.0 equiv.) were added, and then reacted at 70°C for 12 h. After the ethanol was dried, dichloromethane and saturated brine were added for extraction. The organic layer was dried over anhydrous sodium sulfate and then subjected to column chromatography. Column chromatography was performed using an eluent composed of dichloromethane and petroleum ether in a volume ratio of 1:1, and finally 2.3 g of the product was obtained with a yield of 48.5%. 1H NMR (400MHz, CDCl3) δ7.96 (d, J=2.2Hz, 1H), 7.81 (dd, J=8.6, 2.3Hz, 1H), 7.24–7.17 (m,2H),6.95(d,J=8.7Hz,1H),6.90–6.82(m,2H),6.07(ddt,J=17.2,10.4,5.0Hz,1 H),5.50(dq,J=17.2,1.6Hz,1H),5.37(dq,J=10.6,1.4Hz,1H),4.80(dd,J=12.4,6. 7Hz,1H),4.72–4.62(m,3H),4.16(p,J=7.1Hz,1H),3.79(s,3H),3.45–3.25(m,2H). 13 CNMR(101MHz, CDCl3)δ194.59,159.11,158.12,131.71,130.92,130.46,130.02,128.51,128.37,1 23.38,118.54,114.44,112.62,79.82,69.76,55.27,41.32,38.68.ESI-MS(m / z):Calcd.for[M+H] + ,390.8371,found,390.111.

[0077] Synthesis of compound C3: Compound B3 (2.0 g, 5.14 mmol, 1.0 equiv.) was dissolved in 20 ml of n-butanol, and ammonium acetate (3.96 g, 51.4 mmol, 10 equiv.) was added. The mixture was reacted at 110 °C under a nitrogen atmosphere for 24 h and then returned to room temperature. The solvent was dried, saturated brine was added, and the mixture was extracted twice with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated and dried, and then directly proceeded to the next step. The obtained product was dissolved in 50 ml of anhydrous dichloromethane, and N,N-diisopropylethylamine (2.0 g, 15.42 mmol, 3.0 equiv.) was added thereto at -20°C under N2 atmosphere. After 10 minutes, boron trifluoride etherate (3.64 g, 25.7 mmol, 5.0 equiv.) was slowly added. After returning to room temperature and stirring for 12 hours, saturated brine was slowly added, and the mixture was extracted twice with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated and spin-dried, and then column chromatography was performed using a mixed solution of dichloromethane and petroleum ether in a volume ratio of 1:1 as an eluent, and finally 220 mg of a brown solid product was obtained with a yield of 11.6%. 1H NMR (400MHz, DMSO) δ8.35–8.04(m,8H),7.60(s,2H),7.37(d,J=9.0Hz,2H),7.23–7.14(m,4H),6.14–6.06(m,2H),5. 54–5.47(m,2H),5.34(dd,J=10.6,1.7Hz,2H),4.83(d,J=5.0Hz,4H),3.89(s,6H).MALDI-TOF(m / z):Calcd.for[M+H] + ,739.4210found,739.3973.

[0078] Synthesis of compound D3: Compound C3 (200 mg, 0.27 mmol, 1.0 equiv.) was added to a dry round-bottom flask, and 1,3-dimethylbarbituric acid (106 mg, 0.678 mmol, 2.5 equiv.) and tetrakistriphenylphosphine palladium (125 mg, 0.108 mmol, 0.4 equiv.) were added. Under a nitrogen atmosphere, anhydrous DMF (2 ml) was added as a solvent. After stirring at room temperature for 1-2 h, dichloromethane was added, and DMF was removed by multiple extractions with saturated brine. The organic phase was dried over anhydrous sodium sulfate, spin-dried, and then column chromatography was performed. The eluent for column chromatography was a mixed solution of DCM:CH3OH in a volume ratio of 95:5 to obtain 150 mg of a brown solid product with a yield of 84.5%. 1 H NMR (400MHz, DMSO-D6) δ8.34–8.05(m,6H),7.96(dd,J=9.0,2.5Hz,2H),7.52–7.39(m,2H),7.16–7.02(m,4H),6.87(t,J=5.6Hz,2H),3.85(s,6H). 13 CNMR(101MHz,DMSO)δ160.75,144.61,133.94,132.56,132.53,132.01,131.91,131.00,1 30.83,129.43,125.32,121.67,118.04,114.74,55.84.MALDI-TOF(m / z):Calcd.for[M+H] + ,659.2933,found,659.3292.

[0079] Embodiment 4:

[0080] A kind of aza-fluorine-boron fluorescent dye Br OH-OBDP, its structural formula is:

[0081] Its synthetic route is as follows:

[0082]

[0083] The preparation method is as follows:

[0084] Synthesis of compound A4: p-Anisaldehyde (2.72 g, 20 mmol, 1.0 equiv.), 1-(4-(propyleneoxy)-3-bromophenyl)ethane-1-one (5.08 g, 20 mmol, 1.0 equiv.) were dissolved in 20 ml of ethanol, sodium hydroxide (800 mg, 20 mmol, 1.0 equiv.) was added thereto, and then reacted at room temperature overnight, and 4.1 g of solid product was obtained by suction filtration. Yield: 55.1%. 1 H NMR (400MHz, CDCl3) δ8.28(d,J=2.1Hz,1H),8.00(dd,J=8.6,2.1Hz,1H),7.81(d,J=15.5Hz,1H),7.69–7.58(m,2H),7.39(d,J=1 5.5Hz,1H),6.97(dd,J=8.6,5.8Hz,3H),6.10(ddt,J=17.6,10.2,4.9Hz,1H),5.58–5.34(m,2H),4.77–4.66(m,2H),3.88(s,3H). 13 C NMR (101MHz, CDCl3) δ187.59,161.74,158.37,144.65,134.00,132.45,131.87,130.30,129 .42,127.59,118.87,118.32,114.45,112.48,69.78,55.45.ESI-MS(m / z):Calcd.for[M+H] + ,374.2484,found,373.045and 375.043

[0085] Synthesis of compound B4: A4 (4.0 g, 12.2 mmol, 1.0 equiv.) was dissolved in 20 ml of ethanol, and nitromethane (6.25 g, 107.5 mmol, 10 equiv.) and sodium hydroxide (430 mg, 10.75 mmol, 1.0 equiv.) were added, and then reacted at 70°C for 12 h. After the ethanol was dried, dichloromethane and saturated brine were added for extraction. The organic layer was dried over anhydrous sodium sulfate and then subjected to column chromatography. Column chromatography was performed using an eluent composed of dichloromethane and petroleum ether in a volume ratio of 1:1, and finally 2.8 g of the product was obtained with a yield of 60.1%. 1H NMR (400MHz, CDCl3) δ8.14(d,J=2.1Hz,1H),7.87(dd,J=8.7,2.2Hz,1H),7.27–7.13(m,2H),7.02–6.82(m,3H),6.07(ddt ,J=17.4,10.3,5.0Hz,1H),5.58–5.32(m,2H),4.85–4.60(m,4H),4.17(p,J=7.1Hz,1H),3.80(s,3H),3.44–3.27(m,2H). 13 C NMR (101MHz, CDCl3) δ194.45,159.12,158.97,133.68,131.65,130.89,130.48,129.09,128.5 1,118.44,114.46,112.48,79.80,69.80,55.28,41.32,38.68.ESI-MS(m / z):Calcd.for[M+H] + ,435.2884,found,437.195.

[0086] Synthesis of compound C4: Compound B4 (2.5 g, 5.77 mmol, 1.0 equiv.) was dissolved in 20 ml of n-butanol, and ammonium acetate (4.45 g, 57.7 mmol, 10 equiv.) was added. The mixture was reacted at 110 °C under a nitrogen atmosphere for 24 h and then returned to room temperature. The solvent was dried, saturated brine was added, and the mixture was extracted twice with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated and dried, and then directly proceeded to the next step. The obtained product was dissolved in 50 ml of anhydrous dichloromethane, and N,N-diisopropylethylamine (2.25 g, 17.31 mmol, 3.0 equiv.) was added thereto at -20°C under N2 atmosphere. After 10 min, boron trifluoride etherate (4.09 g, 28.85 mmol, 5.0 equiv.) was slowly added. After returning to room temperature and stirring for 12 h, saturated brine was slowly added, and the mixture was extracted twice with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated and spin-dried, and column chromatography was performed using a mixed solution of dichloromethane and petroleum ether in a volume ratio of 1:1 as an eluent to finally obtain 300 mg of a brown solid product with a yield of 12.6%. 1H NMR (400MHz, DMSO) δ8.54–7.83(m,6H),7.65–6.85(m,10H),6.17–5.98(m,2H),5.51(d,J=17.5Hz,2H) ,5.34(d,J=13.7Hz,2H),4.91–4.59(m,4H),3.81(d,J=57.6Hz,6H).MALDI-TOF(m / z):Calcd.for[M+H] + ,828.3236,found,828.3050.

[0087] Synthesis of compound D4: Compound C4 (200 mg, 0.24 mmol, 1.0 equiv.) was added to a dry round-bottom flask, and 1,3-dimethylbarbituric acid (94.5 mg, 0.606 mmol, 2.5 equiv.) and tetrakistriphenylphosphine palladium (111 mg, 0.096 mmol, 0.4 equiv.) were added. Under a nitrogen atmosphere, anhydrous DMF (2 ml) was added as a solvent. After stirring at room temperature for 1-2 h, dichloromethane was added, and DMF was removed by multiple extractions with saturated brine. The organic phase was dried over anhydrous sodium sulfate, spin-dried, and then column chromatography was performed. The eluent for column chromatography was a mixed solution of DCM:CH3OH in a volume ratio of 95:5 to obtain 140 mg of a brown solid product with a yield of 78.4%. 1 H NMR (400MHz, DMSO) δ8.41(d,J=2.2Hz,2H),8.22–8.11(m,4H),8.03(dd,J=8.7,2.3Hz,2H),7.53(s,2H),7.18–7.06(m,6H),3.88(s,6H). 13 C NMR(101MHz,DMSO)δ161.15,157.83,155.91,144.76,142.13,131.34,131.12,125.0 0,123.56,118.43,116.88,114.87,110.58,55.89.MALDI-TOF(m / z):Calcd.for[M+H] + ,748.1959,found,748.2293.

[0088] Example 5

[0089] A kind of aza-fluorine-boron fluorescent dye 2Cl OH-OBDP, its structural formula is:

[0090] The synthetic route is as follows:

[0091]

[0092] The preparation method is as follows:

[0093] Synthesis of compound A5: p-Anisaldehyde (2.72 g, 20 mmol, 1.0 equiv.), 1-(4-(propyleneoxy)-3,5-dichlorophenyl)ethane-1-one (4.88 g, 20 mmol, 1.0 equiv.) were dissolved in 20 ml of ethanol, sodium hydroxide (800 mg, 20 mmol, 1.0 equiv.) was added thereto, and then reacted at room temperature overnight, and 4.9 g of solid product was obtained by suction filtration. Yield: 67.7%. 1 H NMR(500MHz, CDCl3)δ7.96(s,2H),7.82(d,J=15.5Hz,1H),7.69–7.55(m,2H),7.30(d,J=15.5Hz,1H),7.03–6.79(m,2H),6.16(dd t,J=16.6,10.3,6.0Hz,1H),5.45(dq,J=17.1,1.5Hz,1H),5.32(dq,J=10.3,1.2Hz,1H),4.66(dt,J=6.0,1.3Hz,2H),3.87(s,3H). 13 C NMR (126MHz, CDCl3) δ187.33,162.44,154.95,146.31,135.75,132.93,130.89,130.45,129.42,127.59,119.71,118.57,114.89,74.99,55.83.

[0094] Synthesis of compound B5: A5 (4.5 g, 12.4 mmol, 1.0 equiv.) was dissolved in 20 ml of ethanol, and nitromethane (7.56 g, 124 mmol, 10 equiv.) and sodium hydroxide (496 mg, 12.4 mmol, 1.0 equiv.) were added, and then reacted at 70°C for 12 h. After the ethanol was dried, dichloromethane and saturated brine were added for extraction. The organic layer was dried over anhydrous sodium sulfate and then subjected to column chromatography. Column chromatography was performed using an eluent composed of dichloromethane and petroleum ether in a volume ratio of 1:1, and finally 3.5 g of the product was obtained with a yield of 66.8%.

[0095] 1H NMR(500MHz, CDCl3)δ7.86(s,2H),7.28–7.15(m,2H),6.95–6.80(m,2H),6.15(ddt,J=17.1,10.3,6.0Hz,1H),5.53–5.25(m,2H),4.7 8(dd,J=12.4,6.9Hz,1H),4.68(d,J=7.7Hz,1H),4.65(dt,J=6.0,1.1Hz,2H),4.16(p,J=7.1Hz,1H),3.80(s,3H),3.47–3.32(m,2H). 13 C NMR (126MHz, CDCl3) δ194.28,159.54,155.69,133.53,132.84,132.74,130.91,130.65,129. 41,129.12,128.85,119.90,114.85,79.99,77.32,75.05,55.63,41.93,38.85,30.07,24.67.

[0096] Synthesis of compound C5: Compound B5 (3.0 g, 7.1 mmol, 1.0 equiv.) was dissolved in 20 ml of n-butanol, and ammonium acetate (5.47 g, 71 mmol, 10 equiv.) was added. The mixture was reacted at 110 °C under a nitrogen atmosphere for 24 h and then returned to room temperature. The solvent was dried, saturated brine was added, and the mixture was extracted twice with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated and dried, and then directly proceeded to the next step. The obtained product was dissolved in 50 ml of anhydrous dichloromethane, and N,N-diisopropylethylamine (2.75 g, 21.3 mmol, 3.0 equiv.) was added thereto at -20°C under N2 atmosphere. After 10 minutes, boron trifluoride etherate (5.0 g, 35.5 mmol, 5.0 equiv.) was slowly added. After returning to room temperature and stirring for 12 hours, saturated brine was slowly added, and the mixture was extracted twice with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated and spin-dried, and then column chromatography was performed using a mixed solution of dichloromethane and petroleum ether in a volume ratio of 1:1 as an eluent, and finally 240 mg of a brown solid product was obtained with a yield of 8.4%. 1 H NMR(400MHz, CDCl3)δ8.04(d,J=7.4Hz,8H),7.04–6.88(m,6H),6.20(ddt,J=16.5,10.3,5.9H z,2H),5.50(dq,J=17.1,1.7Hz,2H),5.35(d,J=10.3Hz,2H),4.73–4.62(m,4H),3.92(s,6H). 13C NMR (101MHz, CDCl3) δ161.32,155.75,153.10,145.65,144.35,132.83,131 .07,129.91,128.69,124.89,119.03,117.20,114.33,77.25,74.66,55.50.

[0097] Synthesis of compound D5: Compound C5 (200 mg, 0.248 mmol, 1.0 equiv.) was added to a dry round-bottom flask, and 1,3-dimethylbarbituric acid (97 mg, 0.62 mmol, 2.5 equiv.) and tetrakistriphenylphosphine palladium (115 mg, 0.10 mmol, 0.4 equiv.) were added. Under a nitrogen atmosphere, anhydrous DMF (2 ml) was added as a solvent. After stirring at room temperature for 1-2 h, dichloromethane was added, and DMF was removed by multiple extractions with saturated brine. The organic phase was dried over anhydrous sodium sulfate, spin-dried, and then column chromatography was performed. The eluent for column chromatography was a mixed solution of DCM:CH3OH in a volume ratio of 95:5 to obtain 150 mg of a brown solid product with a yield of 83.3%. 1 H NMR (400MHz, DMSO) δ8.30–8.12(m,8H),7.62(s,2H),7.12(d,J=8.7Hz,4H),3.87(s,6H). 13 C NMR (101MHz, DMSO) δ161.24,132.01,131.91,131.17,130.46,129.31,129.19,124.95,123.12,114.89,55.90. 13 C NMR (101MHz, DMSO) δ161.24,132.55,132.01,131.91,131.17,130.46,129.31,129.19,124.95,123.12,114.89,55.90.

[0098] Test Example 1

[0099] Determination of the pKa of the probe

[0100] Take a certain amount of OH-OBDP prepared in the above examples, F OH-OBDP, Cl OH-OBDP, Br OH-OBDP and 2Cl OH-OBDP was dissolved in DMSO to prepare a 2 mM stock solution. 10 mg of DSPE-PEG 2kDissolve in 9.8 mL of deionized water, and under high-speed stirring, take 200 μL of the above dye mother solution and quickly add it to the water, and stir for 10 minutes to obtain a micellar solution. Then take 1.5 mL of the micellar solution and add a pH buffer solution (pH from 3.0 to 11.0), and then test the ultraviolet absorption spectrum. Then calculate the pKa according to the Henderson-Hasselbalch formula, the formula is:

[0101] Log[(A max -A) / (AA min )]=pKa-pH

[0102] Among them A max and A min Respectively represent the maximum and minimum absorbance at the measurement wavelength. A represents the absorbance value at different pH. With pKa as the horizontal axis, log[(A max -A) / (AA min )] as the ordinate to plot a linear relationship, and the intercept is pKa.

[0103] OH-OBDP, F OH-OBDP, Cl OH-OBDP, Br OH-OBDP, 2Cl The pKa of OH-OBDP are 9.5, 8.5, 8.2, 7.5 and 6.1 respectively.

[0104] Test Example 2

[0105] Fluorescence emission spectra of the probe at different pH

[0106] The OH-OBDP prepared in each example, F OH-OBDP, Cl OH-OBDP, Br OH-OBDP and 2Cl OH-OBDP was dissolved in DMSO to prepare a 2 mM stock solution, and then 10 mg of DSPE-PEG 2000 Dissolve in 9.9 mL of deionized water, and under high-speed stirring, take 0.1 mL of the DMSO mother solution of the above dye and quickly add it to the above 9.9 mL of water to prepare micellar solutions of the above dyes. The concentration of the dye is 20 μM. Take 1.0 mL of the above micellar solution and mix it with 1.0 mL of buffer solution of different pH values, and then measure the fluorescence. The excitation wavelength of the fluorescence is 808 nm. The results are as follows Figure 8 shown.

[0107] The fluorescence emission intensity of each probe at 905 nm changes with the solution pH as shown in Fig. 9As shown in Figure a), the comparison of the fluorescence intensity of the probe at pH 3.0, pH 7.4 and pH 12.5 is shown in Figure 2. Fig. 9 As shown in Figure b). It can be seen from the figure that the fluorescence intensity of each probe gradually increases with the increase of pH; 2Cl OH-OBDP has very strong near-infrared second-zone fluorescence under physiological conditions (pH 7.4), and its brightness (wavelength above 1000nm) is 171 times that of OH-OBDP. F 129 times of OH-OBDP, Cl OH-OBDP is 13.5 times that of Br 10.1 times that of OH-OBDP.

[0108] Test Example 3

[0109] UV-visible spectroscopic determination of the probe at different pH

[0110] The OH-OBDP prepared in each example, F OH-OBDP, Cl OH-OBDP, Br OH-OBDP and 2Cl OH-OBDP was dissolved in DMSO to prepare a 2 mM probe stock solution. 10 mg of DSPE-PEG2000 was dissolved in 9.8 mL of deionized water. Under high-speed stirring, 0.2 mL of the above probe stock solution was taken and quickly added to the above DSPE-PEG 2000 1.5 mL of the above micelle solution was added to 1.5 mL of buffer solution of different pH values, and then the UV-visible-near infrared absorption spectra were tested. The results are as follows: Figure 10-Figure 14 As shown in the figure, it can be seen that as the pH of the buffer solution increases, the peak around 720nm gradually decreases, and the absorption peaks around 770nm and 820nm gradually increase.

[0111] The ratio of the absorption wavelengths of each probe at 770 nm to 720 nm changes with pH. Fig.15 As shown in the figure, it can be seen that 2Cl The OH-OBDP probe is sensitive to changes in solution pH. The ratio of the peaks at 770 nm to 720 nm gradually increases from pH 5.5. F OH-OBDP, Cl OH-OBDP and Br OH-OBDP starts to change at around pH 7.4, while OH-OBDP starts to change at around pH 9.0.

[0112] Test Example 4

[0113] Brightness of the probe at different pH values

[0114] 50 μL of the micelle solution with a final concentration of 20 μM prepared in Test Example 2 was taken into small centrifuge tubes, and then 50 μL of buffer solutions of different pH values ​​were added thereto. The images were taken using a near-infrared zone 2 imager, using an 808 nm laser for excitation and a 1000 nm long pass filter for collection. The test results are shown in FIG. Fig.16 As shown in the figure, at pH 11.0, except for OH-OBDP, the other probes all have high brightness. At pH 7.4, only 2Cl OH-OBDP has strong brightness. 2Cl OH-OBDP has very strong near-infrared second region fluorescence under physiological conditions (pH 7.4).

[0115] Application Examples

[0116] Near-infrared fluorescence imaging of the back and abdomen of mice

[0117] Healthy Balb / c mice of 5-8 weeks old were injected into the tail vein 2Cl Nano-micelles of OH-OBDP (DSPE-PEG 2k Stable, concentration is 0.2g / L, 200μL), near-infrared second-zone fluorescence imaging of the abdomen and back of mice was performed at different time points (3min, 15min, 30min, 45min, 60min, 90min), using 808nm laser excitation and 1000nm long-pass filter for collection.

[0118] The test results are as follows Fig.12 As shown in the figure, it can be seen that 3 minutes after the probe was injected, the abdominal and back blood vessels were clearly visible, and the abdominal blood vessels were still very clear until 90 minutes. From the imaging results, the probe was mainly enriched in the liver, and a small part was metabolized from the bladder through the kidneys. 2Cl OH-OBDP has excellent near-infrared second-zone fluorescence characteristics and can be used for high-definition imaging of blood vessels and other parts.

[0119] The detailed description of the pKa-adjustable nitrogen-fluoroboron fluorescent dyes with near-infrared second-zone fluorescence emission and their preparation methods with reference to the above-mentioned examples is illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. A pKa-adjustable aza-fluoro-boron fluorescent dye with near-infrared second-region fluorescence emission, characterized in that: The structure of aza-fluoro-boron fluorescent dyes is as follows: Among them, OH-OBDP, F OH-OBDP, Cl OH-OBDP, Br OH-OBDP, 2Cl The pKa of OH-OBDP are 9.5, 8.5, 8.2, 7.5 and 6.1, respectively.

2. The method for preparing the pKa-adjustable aza-fluoro-boron fluorescent dye with near-infrared second-region fluorescence emission according to claim 1, characterized in that: The preparation method comprises the following steps: 1) conducting an aldehyde-ketone condensation reaction between p-methoxybenzaldehyde and a vinyl ether-protected acetophenone derivative to obtain an intermediate product A; 2) reacting the intermediate product A with nitromethane in an ethanol solvent of sodium hydroxide to obtain an intermediate product B; 3) condensing the intermediate product B with ammonium acetate, and then reacting the condensation product with N,N-diisopropylethylamine and boron trifluoride ether to obtain the intermediate product C; 4) Deprotecting the intermediate product C from vinyl ether under the action of triphenylphosphine palladium to obtain an aza-fluoro-boron fluorescent dye; The structural formula of the vinyl ether protected acetophenone derivative is: The structural formula of the intermediate product A is: The structural formula of the intermediate product B is: The structural formula of the intermediate product C is:

3. The preparation method according to claim 2, characterized in that: The step 1) specifically comprises: dissolving p-methoxybenzaldehyde and a vinyl ether protected acetophenone derivative in ethanol, adding sodium hydroxide, reacting at room temperature overnight, filtering and drying to obtain an intermediate product A.

4. The preparation method according to claim 2, characterized in that: The step 2) specifically comprises: dissolving the intermediate product A in ethanol, adding nitromethane and sodium hydroxide, reacting at 65-75° C. for 10-12 hours, extracting and purifying by column chromatography to obtain the intermediate product B.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the intermediate product A, nitromethane and sodium hydroxide is 1:10-12:1-1.2; the concentration of the intermediate product A in ethanol is 0.5-1.0M.

6. The preparation method according to claim 2, characterized in that: The step 3) specifically comprises: dissolving the intermediate product B in n-butanol, adding ammonium acetate, stirring and reacting at 105-115° C. for 20-24 hours under a nitrogen atmosphere, and then dissolving the obtained product in anhydrous dichloromethane, adding N,N-diisopropylethylamine thereto at -15--25° C. under a N2 atmosphere, and then slowly adding boron trifluoride etherate after 10 minutes, returning to room temperature and stirring and reacting for 10-12 hours, and obtaining the intermediate product C through extraction and column chromatography purification.

7. The preparation method according to claim 6, characterized in that: The molar ratio of the intermediate product B, ammonium acetate, N,N-diisopropylethylamine and boron trifluoride ether is 1:10-12:3-3.5:5-5.5; the concentration of the intermediate product B in n-butanol is 0.3-0.5M.

8. The preparation method according to claim 2, characterized in that: The step 4) specifically comprises: adding the intermediate product C, 1,3-dimethylbarbituric acid and tetrakistriphenylphosphine palladium to anhydrous DMF under a nitrogen atmosphere, stirring and reacting at room temperature for 1-2 hours, extracting and purifying by column chromatography to obtain an aza-fluoro-boron fluorescent dye.

9. The preparation method according to claim 8, characterized in that: The molar ratio of the intermediate product C, 1,3-dimethylbarbituric acid and tetrakistriphenylphosphine palladium is 1:2.5-3.0:0.4-0.5; the concentration of the intermediate product C in DMF is 0.1-0.2M.

10. The pKa-adjustable aza-fluoro-boron fluorescent dye with near-infrared second-region fluorescence emission as claimed in claim 1 2Cl Application of OH-OBDP in fluorescence imaging.

Images