Synthesis method and application of boron dipyrromethene framework near-infrared two-region chemiluminescence probe based on X-ray activation
By synthesizing an X-ray activated fluoroboron fluorescent framework near-infrared II chemiluminescent probe, the energy loss and molecular accessibility issues of NIR-II CL nanoprobes during energy conversion were resolved, enabling efficient tumor imaging and deep tissue penetration, especially showing excellent performance in liver imaging.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-25
- Publication Date
- 2026-03-27
AI Technical Summary
Existing NIR-II CL nanoprobes suffer from energy loss and low intermolecular energy conversion efficiency during energy conversion, resulting in reduced probe strength. Furthermore, the embedded nanoparticles have difficulty accessing the target molecules, limiting their depth and resolution for in vivo imaging.
A near-infrared II chemiluminescent probe based on an X-ray activated fluoroboron fluorescent framework was developed. By synthesizing stable electron-donating compounds A and C-4, and combining them with a click reaction and a trifluoromethanesulfonate group that responds to superoxide anion radicals, compound CL-1050 was prepared, achieving high energy conversion and long-wavelength emission.
This probe can emit chemiluminescence exceeding 970nm when activated by X-rays, significantly improving tumor imaging resolution and tissue penetration depth, enabling real-time liver imaging, and overcoming the energy loss and molecular accessibility problems of existing technologies.
Smart Images
Figure CN121735983A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of chemiluminescence probe technology, specifically to a method for synthesizing and applying a near-infrared II chemiluminescence probe based on an X-ray activated fluorine-boron-fluorescent framework. Background Technology
[0002] Activity-based optical sensing has become an indispensable technology for disease diagnosis and bioanalysis. Chemiluminescence (CL) analysis relies on emission excited states formed by chemical reactions, rather than external light excitation, thus reducing the autofluorescence and scattering caused by external light excitation. Therefore, CL exhibits excellent sensitivity, micron-level resolution at millimeter-scale depth, and high signal-to-noise ratio (SNR). However, currently developed CL probes typically emit light in the visible light (400–650 nm) region, and their low resolution and penetration limit their further application in vivo. On the other hand, optical imaging in the near-infrared (NIR) region (typically defined as NIR-I (650–900 nm) and NIR-II (900–1,700 nm) shows reduced photon absorption and scattering, high spatial resolution, and deep penetration. Although some NIR CL nanoprobes have been reported, most are based on the intermolecular chemiluminescence resonance energy transfer (CRET) effect between the CL donor and the fluorophore acceptor. CRET refers to the nonradiative energy transfer from the CL donor to the fluorophore acceptor candidate gene. Compared to Förster resonance energy transfer (FRET), CRET without excitation light input has relatively higher resolution and deeper penetration. For example, Zhang et al. reported an NIR-II CL nanoprobe by assembling a chemical donor and two alternative dye acceptors into nanoparticles, and then using sequential CRET and FRET to achieve chemical energy transfer to NIR-II CL. The conversion of emission. Due to the multi-step process and low intermolecular energy conversion efficiency, this modification suffers from energy loss and additional energy relay processes, leading to a decrease in NIR-II CL intensity. Furthermore, probes embedded in nanoparticles may also have difficulty accessing the target molecule. To overcome these shortcomings, Song et al. developed a single-molecule NIR CL probe excited by H2S by covalently linking a high-energy CL donor and a candidate NIR fluorophore acceptor, achieving great success. However, this single-molecule probe only achieved CL imaging at 970 nm, and the development of NIR-II CL single-molecule probes is almost non-existent. Therefore, the development of this type of probe is of great significance, not only overcoming almost all the shortcomings of previous probes but also having extraordinary clinical implications. Thus, in-depth exploration of this type of probe is both necessary and urgent. Compared to light, X-rays have deep tissue penetration, allowing the delivery of high-energy photons into the human body for diagnostic imaging and cancer radiotherapy. Inspired by the mechanism of radioluminescence, X-rays have recently been used to replace light in inducing afterglow imaging.Similarly, X-rays can be used as an energy source to initiate the photodynamic process of photoagents to achieve radiodynamic therapy (RDT). Due to the local generation of cytotoxic reactive oxygen species (ROS) by the photoagents, RDT can minimize radiation damage to normal tissues and exert tumor ablation effects in deep tissues.
[0003] Currently, most NIR-II CL nanoprobes employ sequential CRET and FRET to convert chemical energy into NIR-II CL emission. Due to the multi-step process and low intermolecular energy conversion efficiency, this modification suffers from energy loss and additional energy relay processes, leading to a decrease in NIR-II CL intensity. Furthermore, probes embedded in nanoparticles may have difficulty accessing the target molecule. Moreover, the wavelength of even high-energy-conversion-efficiency single-molecule probes is limited to around 970 nm, making the development of NIR-II CL single-molecule probes virtually nonexistent.
[0004] Therefore, it is necessary to develop an X-ray activated NIR-II CL single-molecule probe with a longer emission wavelength, which can achieve better tumor imaging results. Summary of the Invention
[0005] To address the aforementioned technical problems, the main objective of this invention is to provide an X-ray activated fluorine-boron-fluorescent framework near-infrared II chemiluminescent probe and its preparation method.
[0006] To achieve the above objectives, on the one hand, the present invention provides an X-ray activated fluorine-boron-fluorescent framework near-infrared II chemiluminescent probe, the chemical structure of which is shown in formula CL-1050.
[0007] .
[0008] According to a second aspect of the present invention, the present invention also provides a method for preparing an X-ray activated fluorine-boron-fluorescent framework near-infrared II chemiluminescent probe, comprising the following steps:
[0009] 1. Synthesize stable electron-donating group compounds A and C-4 with a high degree of overlap with the incident wavelength of electron-withdrawing groups.
[0010] 2. Synthesize novel BODIPY compounds 14 and 15 with strong or narrow emission bands.
[0011] 3. The two molecules were synthesized by click reaction to obtain compound CL-950.
[0012] 4. Finally, connect the response superoxide anion radical (O2) - The responsive group trifluoromethanesulfonate group (·) forms the final compound CL-1050.
[0013] The specific preparation method of compound CL-1050 is as follows:
[0014] S1, Synthesis of Compound A
[0015] S1-1, 2-bromo-5-hydroxybenzaldehyde, trimethyl orthoformate and tetrabutylammonium bromide were dissolved in anhydrous methanol and reacted at room temperature under nitrogen protection. After the reaction was completed, the mixture was purified to obtain compound 1.
[0016]
[0017] S1-2, Compound 1, imidazole and tert-butyldimethylchlorosilane were dissolved in anhydrous dichloromethane and reacted at room temperature under nitrogen protection. After the reaction was completed, the mixture was purified to obtain Compound 2.
[0018]
[0019] S1-3, Compound 2 and trimethyl phosphite were dissolved in anhydrous dichloromethane and stirred for 10-15 minutes under anhydrous and oxygen-free conditions at -10-0 °C. Then titanium tetrachloride was added dropwise and the reaction was stirred continuously. After the reaction was completed, the reaction was quenched with saturated sodium bicarbonate solution and purified to obtain compound 3.
[0020]
[0021] S1-4, Compound 3 was dissolved in anhydrous THF, and diisopropylaminolithium was added dropwise under anhydrous and oxygen-free conditions at -70-90 °C. Then, 2-adamantanone was added and stirred at room temperature. After the reaction was completed, the mixture was purified to obtain Compound 4.
[0022]
[0023] S1-5, Compound 4 was dissolved in anhydrous THF, and after cooling to -70-90 °C, n-butyllithium and DMF were added dropwise under anhydrous and oxygen-free conditions. The reaction was stirred at room temperature, and after the reaction was completed, the reaction was quenched with saturated ammonium chloride solution and purified to obtain Compound 5.
[0024]
[0025] S1-6, Compound 5 was dissolved in a mixture of anhydrous THF and methanol, cooled to -10-0 °C, and NaBH4 was added dropwise. The mixture was stirred at room temperature and the reaction was completed. After purification, Compound 6 was obtained.
[0026]
[0027] S1-7, Compound 6 and NaOH were dissolved in anhydrous methanol, Icl was added dropwise and stirred at room temperature. After the reaction was completed, the reaction was quenched with saturated ammonium chloride solution and purified to obtain Compound 7;
[0028]
[0029] S1-8, Compound 7, PDC, silica gel, and 5-10 molecular sieves were injected into a mixture of anhydrous pyridine and anhydrous DCM and stirred at room temperature. After the reaction was completed, the reaction was quenched with saturated ammonium chloride solution and purified to obtain Compound A.
[0030]
[0031] Synthesis of S2 and compound C-4
[0032] S2-1, p-methoxythiophenol was dissolved in polyphosphoric acid, heated under reflux and stirred at 80-90 °C, then ethyl acetoacetate was added and the reaction was continued with stirring. The reaction was quenched with ice water and purified to obtain compound C.
[0033]
[0034] S2-2, compound C was dissolved in anhydrous DCM, boron tribromide was added and stirred under N2 protection at -10-0 ℃, and the reaction was continued at room temperature. The solution turned red and the reaction was terminated by adding water. The solution was then purified to obtain compound C-1.
[0035]
[0036] S2-3, both compound C-1 and malononitrile were dissolved in acetic anhydride and reacted with stirring at 130-150 °C. After the reaction was completed, compound C-2 was obtained by purification.
[0037]
[0038] S2-4, Compound C-2 and 2-azidoethyl 4-toluenesulfonic acid were stirred and reacted. After the reaction was completed, the mixture was purified to obtain compound C-3.
[0039]
[0040] S2-5, Compound A, Compound C-3, acetonitrile and piperidine are mixed and stirred at 80-90 °C to react. After the reaction is completed, the mixture is purified to obtain Compound C-4.
[0041]
[0042] Synthesis of S3 and compound CL-1050
[0043] S3-1, compound julonidine was added to the liquid resulting from the reaction of phosphorus oxychloride and anhydrous DMF at -10-0 °C, and then stirred at room temperature. After the reaction was completed, the mixture was purified to obtain compound 9.
[0044]
[0045] S3-2, p-methoxyacetophenone and NaOH were added to compound 9, and the mixture was stirred at room temperature. After the reaction was completed, the mixture was purified to obtain compound 10.
[0046]
[0047] S3-3, Nitromethane and KOH were added to compound 10, and the mixture was stirred at room temperature. After the reaction was completed, the mixture was purified to obtain compound 11.
[0048]
[0049] S3-4, Compound 11 was dissolved in n-butanol and ammonium acetate was added and stirred at room temperature. After the reaction was completed, the mixture was purified to obtain Compound 12.
[0050]
[0051] S3-5, compound 12 dissolved in anhydrous DCM was added with DIPEA and boron trifluoride diethyl ether and stirred at room temperature. After the reaction was completed, the mixture was purified to obtain compound 13.
[0052]
[0053] S3-6, Compound 13 was dissolved in anhydrous DCM, and Br2 dissolved in anhydrous DCM was added. The mixture was stirred at room temperature and the reaction was completed. After the reaction was completed, the mixture was purified to obtain Compound 14.
[0054]
[0055] S3-7, Compound 14 was dissolved in anhydrous THF, and ethynyltri-n-butyltin and tetra(triphenylphosphine)palladium dissolved in anhydrous THF were added. The mixture was stirred at room temperature and the reaction was completed. After the reaction was completed, the mixture was purified to obtain Compound 15.
[0056]
[0057] S3-8, compound 15 and compound C-4 were dissolved in anhydrous DMSO, and NaAsc and CuSO4 dissolved in deionized water were added. The mixture was stirred at room temperature and the reaction was completed. After purification, compound CL-950 was obtained.
[0058]
[0059] In S3-9, under nitrogen protection, compound CL-950 was dissolved in anhydrous DCM. Anhydrous pyridine and trifluoromethanesulfonic anhydride were added at -70-90 °C and reacted for 10-60 min. The reaction was then carried out at room temperature for 6-12 hours. After the reaction was complete, the compound CL-1050 was purified to obtain the X-ray activated fluoroboron-fluorescent framework near-infrared II chemiluminescent probe.
[0060] .
[0061] According to a third aspect of the invention, the invention also provides the application of the X-ray activated fluorine-boron-fluorescent framework near-infrared II chemiluminescent probe of the first aspect as a chemiluminescent probe.
[0062] Compared with the prior art, the present invention can achieve the following beneficial effects:
[0063] From the perspective of preparation and material properties: Compound 15 is a novel BODIPY complex with high quantum yield and strong or narrow emission bands. It is simple to prepare, has high yield, and is easy to prepare. As an energy acceptor for CRET, it has longer incident and emission wavelengths. Compound C-4 is a chemiluminescent substrate with a dicyanomethyldioxane structure. Its preparation materials are simple and readily available, with almost no precursors for toxic or explosive purposes, making it easy to prepare. The process from the trifluoromethanesulfonate ester responder group on CL-950 to the CL-1050 probe also has a corresponding reference reaction, making it easy to prepare.
[0064] From an application perspective: this probe features a dual-locking mechanism and, upon excitation, emits chemiluminescence at a wavelength exceeding 970 nm (1050 nm). X-ray activation will significantly improve the imaging resolution and tissue penetration depth of tumors, and X-ray activation can enable real-time imaging, such as of the liver. Attached Figure Description
[0065] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.
[0066] Figure 1 The diagram shows the synthesis circuit of the X-ray activated fluorine-boron-fluorescent framework near-infrared II chemiluminescent probe of Example 1.
[0067] Figure 2 The 1H NMR spectrum of compound A ( 1 (H NMR) image.
[0068] Figure 3 The 1H NMR spectrum of compound 13 ( 1 (H NMR) image.
[0069] Figure 4The 1H NMR spectrum of compound 14 ( 1 (H NMR) image.
[0070] Figure 5 The 1H NMR spectrum of compound 15 ( 1 (H NMR) image.
[0071] Figure 6 The 1H NMR spectrum of compound C-3 ( 1 (H NMR) image.
[0072] Figure 7 The 1H NMR spectrum of compound C-4 ( 1 (H NMR) image.
[0073] Figure 8 The 1H NMR spectrum of compound CL-950 ( 1 (H NMR) image.
[0074] Figure 9 The 1H NMR spectrum of compound CL-1050 ( 1 (H NMR) image.
[0075] Figure 10 The carbon NMR spectrum of compound C-4 ( 13 (C NMR) plot.
[0076] Figure 11 The carbon NMR spectrum of compound 15 ( 13 (C NMR) plot.
[0077] Figure 12 The carbon NMR spectrum of compound CL-950 ( 13 (C NMR) plot.
[0078] Figure 13 The carbon NMR spectrum of compound CL-1050 ( 13 (C NMR) plot.
[0079] Figure 14 This is the mass spectrum (MS) of compound 15.
[0080] Figure 15 This is the mass spectrum (MS) of compound C-4.
[0081] Figure 16 This is the mass spectrum (MS) of compound CL-950.
[0082] Figure 17 This is the mass spectrum (MS) of compound CL-1050.
[0083] Figure 18 The image shows the spectral characterization of CL-1050.
[0084] Figure 19 This is a characterization diagram of the simple double-locking mechanism of the CL-1050.
[0085] Figure 20 For selective characterization and chemiluminescence duration characterization.
[0086] Figure 21 The graph shows the relationship between fluorescence and chemiluminescence intensity and KO2 concentration.
[0087] Figure 22 Characterization maps of singlet oxygen and tissue penetration depth were generated using the ESR method for CL-950 material.
[0088] The objectives, features, and advantages of this invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation
[0089] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.
[0090] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.
[0091] Example 1
[0092] like Figure 1 The synthetic circuit shown in this embodiment provides a method for preparing an X-ray activated fluoroboron fluorescent framework near-infrared II chemiluminescent probe, as detailed below:
[0093] S1, Synthesis of Compound A
[0094] S1-1, Synthesis steps of compound 1
[0095] 1. Dissolve 2-bromo-5-hydroxybenzaldehyde (10 g, 49.7 mmol), trimethyl orthoformate (5.8 g, 54.7 mmol), and tetrabutylammonium bromide (TBAB, 239.6 mg, 0.497 mmol) in anhydrous methanol (100 mL).
[0096] 2. Stir the reaction at room temperature under nitrogen protection for 12-24 hours (overnight).
[0097] 3. After the reaction is complete, concentrate under reduced pressure, wash with water and extract with ethyl acetate.
[0098] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 1.
[0099] S1-2, Synthesis steps of compound 2
[0100] 1. Compound 1 (5 g, 20.2 mmol), imidazole (4.1 g, 60.7 mmol) and tert-butyldimethylchlorosilane (6.1 g, 40.4 mmol) were dissolved in anhydrous dichloromethane (50 mL).
[0101] 2. Stir the reaction at room temperature under nitrogen protection for 0.5-2 hours, and monitor the reaction progress by TLC.
[0102] 3. After the reaction is complete, wash with water and extract with dichloromethane.
[0103] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 2.
[0104] S1-3, Synthesis steps of compound 3
[0105] 1. Compound 2 (3 g, 8.3 mmol) and trimethyl phosphite (1.5 g, 12.4 mmol) were dissolved in anhydrous dichloromethane (50 mL).
[0106] 2. Stir the reaction for 10-15 minutes under anhydrous and oxygen-free conditions at 0°C.
[0107] 3. Slowly add titanium tetrachloride (2.3 g, 12.4 mmol) dropwise while stirring the reaction.
[0108] 4. After the reaction is complete, quench the reaction with saturated sodium bicarbonate solution and extract with dichloromethane.
[0109] 5. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 3.
[0110] S1-4, Synthesis steps of compound 4
[0111] 1. Compound 3 (2 g, 4.6 mmol) was dissolved in anhydrous THF (15 mL), and lithium diisopropylamino (3.4 mL) was added dropwise under anhydrous and oxygen-free conditions at -78°C.
[0112] 2. After stirring for 20-30 minutes, add dropwise 2-adamantanone (1.4 g, 9.2 mmol, dissolved in anhydrous THF).
[0113] 3. Stir the reaction at room temperature for 0.5-2 hours and monitor the reaction progress by TLC.
[0114] 4. After the reaction is complete, wash with water and extract with ethyl acetate.
[0115] 5. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 4.
[0116] S1-5, Synthesis steps of compound 5
[0117] 1. Compound 4 (1.5 g, 3.2 mmol) was dissolved in anhydrous THF (20 mL), cooled at -78°C for 10 min, and then n-butyllithium (2.6 mL, 2.5 M) and DMF (1.0 mL) were added dropwise under anhydrous and oxygen-free conditions.
[0118] 2. Stir the reaction at room temperature for 0.5-2 hours and monitor the reaction progress by TLC.
[0119] 3. After the reaction is complete, quench the reaction with saturated ammonium chloride solution and extract with ethyl acetate.
[0120] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 5.
[0121] S1-6, Synthesis steps of compound 6
[0122] 1. Compound 5 (0.5 g, 1.68 mmol) was dissolved in anhydrous THF and MeOH (5 mL: 3 mL), and after cooling at 0°C for 10 min, NaBH4 (0.07 g, 1.85 mmol) was added dropwise.
[0123] 2. Stir the reaction at room temperature for 0.5-2 hours and monitor the reaction progress by TLC.
[0124] 3. After the reaction is complete, extract with ethyl acetate.
[0125] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 6.
[0126] S1-7, Compound 7 Synthesis Steps
[0127] 1. Compound 6 (0.52 g, 1.73 mmol) and NaOH (0.104 g, 2.6 mmol) were dissolved in anhydrous MeOH (10 mL), and ICl (0.28 g, 1.72 mmol) was added dropwise at 0°C.
[0128] 2. Stir the reaction at room temperature for 0.5-2 hours and monitor the reaction progress by TLC.
[0129] 3. After the reaction is complete, quench the reaction with saturated ammonium chloride solution and extract with ethyl acetate.
[0130] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 7.
[0131] S1-8, Synthesis steps of compound A
[0132] 1. Compound 7 (1.5 g, 3.52 mmol), PDC (2.12 g, 9.8 mmol), silica gel (2 g, 33.3 mmol), 5 molecular sieves, anhydrous pyridine (732 mL) and anhydrous DCM (10 mL) were injected and reacted at 0°C for 5–10 min.
[0133] 2. Transfer to room temperature and stir for 1-2 hours, monitoring the reaction progress by TLC.
[0134] 3. After the reaction is complete, quench the reaction with saturated ammonium chloride solution and extract with ethyl acetate.
[0135] 4. Combine the organic layers, dry and concentrate, then purify by silica gel column chromatography to obtain compound A, whose 1H NMR spectrum is shown below. Figure 2 As shown.
[0136] Synthesis of S2, compound C-4
[0137] S2-1, Synthesis of Compound C
[0138] 1. Dissolve p-methoxythiophenol (200 mg, 1.426 mmol) in 2.5 mL of polyphosphoric acid, heat under reflux at 90 °C with stirring, then add ethyl acetoacetate (220 mg, 1.690 mmol) and continue stirring for 1 h.
[0139] 2. Quench the reaction with ice water, then extract with EA.
[0140] 3. Dry by rotary evaporation. Column chromatography: PE:EA = 5:1, yielding yellow crystalline compound C.
[0141] Synthesis of S2-2, compound C-1
[0142] 1. Compound C (200 mg, 1 mmol) was dissolved in 15 mL of anhydrous DCM. 10 mL of boron tribromide was added dropwise under N2 protection at 0 °C with stirring. Stirring was continued for 4 h at room temperature. The reaction was monitored by TLC, and the solution turned red.
[0143] 2. The reaction is terminated by adding water. The solution color lightens and yellow-brown flocculent matter is formed. Extract with EA.
[0144] 3. The solution was dried over anhydrous magnesium sulfate, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography to obtain compound C-1.
[0145] Synthesis of S2-3, compound C-2
[0146] 1. Dissolve compound C-1 (150 mg, 0.78 mmol) and malononitrile (77.32 mg, 1.17 mmol) in 2.5 mL of acetic anhydride.
[0147] 2. Stir at 140℃ for 3.5 h-8 h.
[0148] 3. During the reaction, TLC monitoring was performed, water was removed and the mixture was concentrated, and compound C-2 was obtained by column chromatography.
[0149] Synthesis of S2-4, compound C-3
[0150] 1. Mix compound C-2 and 2-azidoethyl 4-toluenesulfonic acid together for 1 h to 8 h.
[0151] 2. After the reaction is complete, extract with EA.
[0152] 3. After water removal and concentration, compound C-3 was obtained by column chromatography, and its 1H NMR spectrum is shown below. Figure 6 As shown.
[0153] Synthesis of S2-5, compound C-4
[0154] 1. Acetonitrile (157 mmol) and piperidine were injected into a double-necked round-bottom flask containing compound A (100 mg, 235.7 mmol) and compound C-3 (48.6 mg, 157 mmol). The mixture was then refluxed in an oil bath at 85°C without a condenser and stirred for 8 hours.
[0155] 2. After the reaction, the powder was spotted onto a TLC plate. The yellow and white powder turned into a red powder. The solution was collected with 0.5M hydrochloric acid and DCM.
[0156] 3. After water removal and concentration, column chromatography was performed initially with PE:EA = 50:1, followed by the addition of DCM to increase polarity, ultimately yielding compound C-4. Its 1H NMR spectrum is shown below. Figure 7 As shown, the carbon NMR spectrum is as follows: Figure 10 As shown, the mass spectrometry is as follows Figure 15 As shown.
[0157] S3, Synthesis of compound CL-950
[0158] S3-1, Synthesis steps of compound 9
[0159] 1. Add the compound julonidine (2 g, 11.5 mmol) to the liquid after the reaction of phosphorus oxychloride (3.6 mL) and anhydrous DMF (7 mL) at 0°C.
[0160] 2. Stir the reaction at room temperature for 0.5-2 hours and monitor the reaction progress by TLC.
[0161] 3. After the reaction is complete, extract with ethyl acetate.
[0162] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 9.
[0163] S3-2, Synthesis steps of compound 10
[0164] 1. Add p-methoxyacetophenone (1.55 g, 10.3 mmol) and NaOH (6 mL) to compound 9 (1.265 g, 6.29 mmol).
[0165] 2. Stir the reaction at room temperature for 12-24 hours and monitor the reaction progress by TLC.
[0166] 3. After the reaction is complete, extract with ethyl acetate.
[0167] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 10.
[0168] S3-3, Synthesis steps of compound 11
[0169] 1. Nitromethane (13 mL) and KOH (1.04 g, 18.5 mmol) were added to compound 10 (4.15 g, 12.4 mmol).
[0170] 2. Stir the reaction at room temperature for 12-24 hours and monitor the reaction progress by TLC.
[0171] 3. After the reaction is complete, extract with ethyl acetate.
[0172] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 11.
[0173] S3-4, Synthesis steps of compound 12
[0174] 1. Add ammonium acetate (13.79 g, 178.9 mmol) to compound 11 (4.7 g, 11.9 mmol) dissolved in n-butanol (25 mL).
[0175] 2. Stir the reaction at room temperature for 12-24 hours and monitor the reaction progress by TLC.
[0176] 3. After the reaction is complete, extract with ethyl acetate.
[0177] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 12.
[0178] S3-5, Synthesis steps of compound 13
[0179] 1. Add DIPEA (20.86 mL) and boron trifluoride-ethyl ether (22 mL) to compound 12 (8.34 g, 11.9 mmol) dissolved in anhydrous DCM (25 mL).
[0180] 2. Stir the reaction at room temperature for 12-24 hours and monitor the reaction progress by TLC.
[0181] 3. After the reaction is complete, extract with DCM.
[0182] 4. The organic layers were combined, dried, concentrated, and purified by silica gel column chromatography to obtain compound 13, whose 1H NMR spectrum is shown below. Figure 3 As shown.
[0183] S3-6, Synthesis steps of compound 14
[0184] 1. Add Br2 dissolved in anhydrous DCM to compound 13 (0.03 g, 4 μL) dissolved in anhydrous DCM (8 mL).
[0185] 2. Stir the reaction at room temperature for 10-30 minutes and monitor the reaction progress by TLC.
[0186] 3. After the reaction is complete, extract with DCM.
[0187] 4. The organic layers were combined, dried, concentrated, and purified by silica gel column chromatography to obtain compound 14, whose 1H NMR spectrum is shown below. Figure 4 As shown.
[0188] S3-7, Synthesis steps of compound 15
[0189] 1. Add 0.186 g, 0.6 mmol of ethynyltri-n-butyltin (THF) and 0.0137 g, 11.84 mmol of tetrakis(triphenylphosphine)palladium (THF) dissolved in anhydrous THF (2 mL) to compound 14 (0.055 g, 0.06 mmol).
[0190] 2. Stir the reaction at room temperature for 6-10 hours and monitor the reaction progress by TLC.
[0191] 3. After the reaction is complete, extract with DCM.
[0192] 4. The organic layers were combined, dried, concentrated, and purified by silica gel column chromatography to obtain compound 15, whose 1H NMR spectrum is shown below. Figure 5 As shown, the carbon NMR spectrum is as follows: Figure 11 As shown, the mass spectrometry is as follows Figure 14 .
[0193] S3-8, Synthesis steps of compound CL-950
[0194] 1. Dissolve compound 15 and compound C-4 in anhydrous DMSO (8 mL) in a double-fisted flask.
[0195] 2. Inject NaASC and CuSO4 dissolved in deionized water. 4。
[0196] 3. Stir the reaction at room temperature for 6-10 hours and monitor the reaction progress by TLC.
[0197] 4. After the reaction is complete, extract with DCM.
[0198] 5. The organic layers were combined, dried, concentrated, and purified by silica gel column chromatography to obtain compound CL-950, whose 1H NMR spectrum is shown below. Figure 8 As shown, the carbon NMR spectrum is as follows: Figure 12 As shown, the mass spectrometry is as follows Figure 16 As shown.
[0199] S3-9, Synthetic steps of compound CL-1050
[0200] 1. Dissolve compound CL-950 in anhydrous DCM and evacuate under nitrogen protection.
[0201] 2. Inject anhydrous pyridine and trifluoromethanesulfonic anhydride (Tf2O) into the well at -80℃.
[0202] 3. React at -80℃ for half an hour in a cold well, then react at room temperature for 8-12 hours, and monitor the reaction progress using TLC.
[0203] 4. After the reaction is complete, DCM extraction is performed.
[0204] 5. The organic layers were combined, dried, concentrated, and purified by silica gel column chromatography to obtain compound CL-1050, whose 1H NMR spectrum is shown below. Figure 9 As shown, the carbon NMR spectrum is as follows: Figure 13 As shown, the mass spectrometry is as follows Figure 17 As shown.
[0205] The compound CL-1050 prepared in Example 1 was characterized in vitro, and the results are as follows:
[0206] Figure 18 The spectral characterization diagrams are: (a) ultraviolet absorption spectrum and (b) chemiluminescence spectrum. It can be seen that the absorption wavelength of CL-1050 is 680 nm and the chemiluminescence emission wavelength is 1050 nm.
[0207] Figure 19 Characterization diagrams for a simple double-locking mechanism: (a) Chemiluminescence intensity of CL-1050 probe under X-ray irradiation in the presence of KO2 (b) Chemiluminescence intensity of CL-1050 probe under X-ray irradiation with and without KO2. From (a) and (b), it can be seen that CL-1050 material requires both X-ray irradiation and KO2 conditions to produce chemiluminescence, while CL-950 requires X-ray activation to produce chemiluminescence.
[0208] Figure 20 For selective characterization and chemiluminescence duration characterization: (a) The enhancement of CL after X-ray irradiation with different types of ions in PBS shows that CL-1050 material has high selectivity for KO2; (b) The CL kinetic curve after activation of CL-1050 in PBS in the presence of KO2 shows that the chemiluminescence duration of CL-1050 material is about 4 min.
[0209] Figure 21 The following graphs characterize the relationship between fluorescence and chemiluminescence intensities and KO2 concentration: (a) the relationship between CL-1050 intensity and concentration in the range of 0.1 to 2 mM, and (b) the linear relationship between CL-1050 and KO2 concentration in the range of 0 to 200 μM. The graphs show a certain linear relationship between (a) FL intensity and (b) CL intensity and KO2 concentration.
[0210] Figure 22 Characterization of singlet oxygen and tissue penetration depth for CL-950 material using ESR method: (a) ESR spectrum 1 O2 generation depends on whether X-ray irradiation is involved (2 Gy, 100 mGy / s). -1 Three groups were identified: one group irradiated for 20 seconds and another quenched with NaN3. (b) Chemiluminescence and fluorescence imaging of chicken breast tissue induced by X-ray and without chicken breast tissue were detected. (a) Fluorescence and chemiluminescence imaging of CL-950 activated in PBS (0.01 M, pH 7.4, 50% DMSO). It was observed that CL-950 produced singlet oxygen under X-ray irradiation, and the tissue penetration depth of CL-950 material was approximately 1.0 cm.
[0211] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. The scope of protection of the present invention is defined only by the appended claims.
Claims
1. A near-infrared II chemiluminescent probe based on an X-ray activated fluorine-boron-fluorescent framework, characterized in that, Its chemical structure is shown in formula CL-1050: 。 2. A method for preparing the X-ray activated fluorine-boron-fluorescent framework near-infrared II chemiluminescent probe as described in claim 1, characterized in that, Includes the following steps: S1, Synthesis of Compound A S1-1, 2-bromo-5-hydroxybenzaldehyde, trimethyl orthoformate and tetrabutylammonium bromide were dissolved in anhydrous methanol and reacted at room temperature under nitrogen protection. After the reaction was completed, the mixture was purified to obtain compound 1. ； S1-2, Compound 1, imidazole and tert-butyldimethylchlorosilane were dissolved in anhydrous dichloromethane and reacted at room temperature under nitrogen protection. After the reaction was completed, the mixture was purified to obtain Compound 2. ； S1-3, Compound 2 and trimethyl phosphite were dissolved in anhydrous dichloromethane and stirred for 10-15 minutes under anhydrous and oxygen-free conditions at -10-0 °C. Then titanium tetrachloride was added dropwise and the reaction was stirred continuously. After the reaction was completed, the reaction was quenched with saturated sodium bicarbonate solution and purified to obtain compound 3. ； S1-4, Compound 3 was dissolved in anhydrous THF, and diisopropylaminolithium was added dropwise under anhydrous and oxygen-free conditions at -70-90 °C. Then, 2-adamantanone was added and stirred at room temperature. After the reaction was completed, the mixture was purified to obtain Compound 4. ； S1-5, Compound 4 was dissolved in anhydrous THF, and after cooling to -70-90 °C, n-butyllithium and DMF were added dropwise under anhydrous and oxygen-free conditions. The reaction was stirred at room temperature, and after the reaction was completed, the reaction was quenched with saturated ammonium chloride solution and purified to obtain Compound 5. ； S1-6, Compound 5 was dissolved in a mixture of anhydrous THF and methanol, cooled to -10-0 °C, and NaBH4 was added dropwise. The mixture was stirred at room temperature and the reaction was completed. After purification, Compound 6 was obtained. ； S1-7, Compound 6 and NaOH were dissolved in anhydrous methanol, Icl was added dropwise and stirred at room temperature. After the reaction was completed, the reaction was quenched with saturated ammonium chloride solution and purified to obtain Compound 7; ； S1-8, Compound 7, PDC, silica gel, and 5-10 molecular sieves were injected into a mixture of anhydrous pyridine and anhydrous DCM and stirred at room temperature. After the reaction was completed, the reaction was quenched with saturated ammonium chloride solution and purified to obtain Compound A. ； Synthesis of S2 and compound C-4 S2-1, p-methoxythiophenol was dissolved in polyphosphoric acid, heated under reflux and stirred at 80-90 °C, then ethyl acetoacetate was added and the reaction was continued with stirring. The reaction was quenched with ice water and purified to obtain compound C. ； S2-2, compound C was dissolved in anhydrous DCM, boron tribromide was added and stirred under N2 protection at -10-0 ℃, and the reaction was continued at room temperature. The solution turned red and the reaction was terminated by adding water. The solution was then purified to obtain compound C-1. ； S2-3, both compound C-1 and malononitrile were dissolved in acetic anhydride and reacted with stirring at 130-150 °C. After the reaction was completed, compound C-2 was obtained by purification. ； S2-4, Compound C-2 and 2-azidoethyl 4-toluenesulfonic acid were stirred and reacted. After the reaction was completed, the mixture was purified to obtain compound C-3. ； S2-5, Compound A, Compound C-3, acetonitrile and piperidine are mixed and stirred at 80-90 °C to react. After the reaction is completed, the mixture is purified to obtain Compound C-4. ； Synthesis of S3 and compound CL-1050 S3-1, compound julonidine was added to the liquid resulting from the reaction of phosphorus oxychloride and anhydrous DMF at -10-0 °C, and then stirred at room temperature. After the reaction was completed, the mixture was purified to obtain compound 9. ； S3-2, p-methoxyacetophenone and NaOH were added to compound 9, and the mixture was stirred at room temperature. After the reaction was completed, the mixture was purified to obtain compound 10. ； S3-3, Nitromethane and KOH were added to compound 10, and the mixture was stirred at room temperature. After the reaction was completed, the mixture was purified to obtain compound 11. ； S3-4, Compound 11 was dissolved in n-butanol and ammonium acetate was added and stirred at room temperature. After the reaction was completed, the mixture was purified to obtain Compound 12. ； S3-5, compound 12 dissolved in anhydrous DCM was added with DIPEA and boron trifluoride diethyl ether and stirred at room temperature. After the reaction was completed, the mixture was purified to obtain compound 13. ； S3-6, Compound 13 was dissolved in anhydrous DCM, and Br2 dissolved in anhydrous DCM was added. The mixture was stirred at room temperature and the reaction was completed. After the reaction was completed, the mixture was purified to obtain Compound 14. ； S3-7, Compound 14 was dissolved in anhydrous THF, and ethynyltri-n-butyltin and tetra(triphenylphosphine)palladium dissolved in anhydrous THF were added. The mixture was stirred at room temperature and the reaction was completed. After the reaction was completed, the mixture was purified to obtain Compound 15. ； S3-8, compound 15 and compound C-4 were dissolved in anhydrous DMSO, and NaAsc and CuSO4 dissolved in deionized water were added. The mixture was stirred at room temperature and the reaction was completed. After purification, compound CL-950 was obtained. ； In S3-9, under nitrogen protection, compound CL-950 was dissolved in anhydrous DCM. Anhydrous pyridine and trifluoromethanesulfonic anhydride were added at -70-90 °C and reacted for 10-60 min. The reaction was then carried out at room temperature for 6-12 hours. After the reaction was complete, the compound CL-1050 was purified to obtain the X-ray activated fluoroboron-fluorescent framework near-infrared II chemiluminescent probe. 。 3. The application of the X-ray activated fluorine-boron-fluorescent framework near-infrared II chemiluminescent probe of claim 1 as a chemiluminescent probe.