BODIPY derivatives with dual-lock cascade activation and their application in brain Abeta and ClO － detection

Abstract

The application discloses a kind of double lock cascade activated BODIPY derivatives and its preparation method and application.The BODIPY derivative has formula I structure, wherein R is selected from hydrogen or ethyl.The derivative introduces pyrrole PET unit in the meso position of BODIPY mother nucleus, introduces 4-dimethylaminostyryl TICT unit in 3-position and 5-position, and constructs "double lock" fluorescent probe.The derivative only in Alzheimer's disease (AD) related pathological marker Aβ aggregate and ClO － 50-100 times cascade amplification fluorescence enhancement is generated when existing simultaneously.The probe can penetrate blood-brain barrier, realizes Aβ aggregate and ClO － Real-time bimodal imaging in AD transgenic mouse brain, fluorescence intensity is positively correlated with the severity of pathology, and can be down-regulated by antioxidant therapy.The application provides an effective molecular imaging tool for early diagnosis, pathological research and efficacy evaluation of AD.(formula I)

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a double-locked cascade activated BODIPY derivative, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a progressive, irreversible neurodegenerative disease characterized by cognitive decline and memory loss, and has become a major public health problem posing a serious threat to the health of the elderly. The pathological mechanisms of AD are very complex, involving multiple factors such as abnormal aggregation of β-amyloid protein (Aβ) to form senile plaques, hyperphosphorylation of tau protein, oxidative stress, and chronic neuroinflammation.

[0003] Recent studies have shown that Aβ aggregates are not only a core pathological marker of Alzheimer's disease (AD), but can also activate microglia, induce the overexpression of myeloperoxidase (MPO), and subsequently catalyze the production of excessive hypochlorous acid / hypochlorite (HClO / ClO) ions. － Notably, MPO expression is significantly upregulated in AD brains and exhibits high spatial colocalization with Aβ aggregates (J. Biol. Chem. 2009, 284, 3158). This Aβ aggregation is associated with ClO₂. － The synergistic effect of mediated oxidative damage is considered a key factor driving the occurrence and development of Alzheimer's disease (AD). Therefore, developing a method to simultaneously detect Aβ aggregates and ClO2 is crucial. － Molecular imaging tools are of great significance for elucidating the pathogenesis of Alzheimer's disease and achieving early and accurate imaging.

[0004] However, due to the presence of the blood-brain barrier (BBB), effective methods for real-time monitoring of dual biomarkers in the in vivo brain remain very limited. The BBB blocks almost 100% of macromolecules and 98% of small molecules (Neurobiol. Dis. 2010, 37, 48–57). Furthermore, small-molecule probes used for in vivo brain imaging require excitation and emission wavelengths in the near-infrared region (650–900 nm) to achieve deep tissue penetration and avoid autofluorescence interference, and the probes need to exhibit sensitive and specific responses to the target analyte. These issues place extremely stringent requirements on the design of fluorescent probes suitable for detecting dual biomarkers in the brain of Alzheimer's disease (AD).

[0005] Botryolean dipyrrole (BODIPY) dyes are classic fluorescent labeling reagents, possessing high molar extinction coefficients, high fluorescence quantum yields, and excellent photostability. In recent years, structural modifications to the BODIPY parent compound, such as extending the conjugated system and introducing strong electron donors, have yielded BODIPY derivatives with emission wavelengths in the near-infrared region. However, to our knowledge, there are currently no derivatives that simultaneously possess BBB permeability, near-infrared emission, and the ability to achieve both Aβ and ClO₂ emission. － A report on the BODIPY probe with a dual-lock cascade activation response.

[0006] In the existing technology, although there are a few dual-response probes targeting Aβ and reactive oxygen species, such as BTNPO reported by Xie et al. (Anal. Chem. 2021, 93, 15088) which can simultaneously image Aβ and peroxynitrite, these probes have the following shortcomings: (1) The response mechanism is based on "OR" logic, with the two stimuli generating signal changes independently, lacking a synergistic amplification effect, and showing obvious fluorescence response even in the presence of a single marker, easily leading to false positives; (2) The affinity for Aβ aggregates is low (dissociation constants are mostly in the μM range), making it difficult to detect early micro-aggregates; (3) The emission wavelength is relatively short (mostly <650 nm), limiting the tissue penetration depth; (4) The BBB penetration ability is poor, limiting its direct application in the living brain. In particular, there is currently no probe that can simultaneously image Aβ and ClO2. － Single-molecule probes that produce significant fluorescence enhancement only when both pathological factors are present simultaneously result in insufficient imaging contrast between the coexisting and non-coexisting regions.

[0007] Therefore, if a method is developed that can efficiently penetrate the blood-brain barrier, has near-infrared emission characteristics, and emits only in Aβ and ClO2, it would be beneficial. － The "double-lock" fluorescent probe, which generates a cascaded amplified fluorescence signal when two AD-related biomarkers are present simultaneously, will have significant research value and clinical application prospects for accurate imaging of AD. Summary of the Invention

[0008] To address the problems existing in the prior art, the purpose of this invention is to provide a class of double-locked cascade activated near-infrared fluorescent probes that can cross the blood-brain barrier and sensitively respond to Aβ aggregates and ClO in vitro and in vivo. － Ultimately, precise AD imaging is achieved through cascaded amplification changes in fluorescence intensity.

[0009] To achieve the objectives of this invention, the technical solution is as follows: In a first aspect, the present invention provides a dual-locked cascade activated near-infrared fluorescent BODIPY derivative, characterized in that it has the following general formula (Formula I): (Formula I) R is selected from hydrogen or ethyl.

[0010] When R is hydrogen, the derivative is named D-BDY; When R is ethyl, the derivative is named T-BDY.

[0011] The design concept of the BODIPY derivative described in this invention is as follows: a pyrrole derivative is introduced as a photoinduced electron transfer (PET) unit at the meso site of the BODIPY core (i.e., the central carbon atom connecting the two pyrrole rings, the C-8 position). This unit can be activated by ClO － Oxidation shuts down the PET effect; a 4-dimethylaminostyrene group is attached to the 3- or 5-position of the BODIPY core via Knoevenagel condensation, acting as a molecular rotor and generating a twisted intramolecular charge transfer (TICT) effect. In an aqueous environment, both PET and TICT fluorescence quenching mechanisms work together to completely quench the probe's fluorescence. When ClO2 is present in the environment... － When pyrrole units are oxidized, the PET effect is suppressed, but the TICT effect still exists, and fluorescence is only partially recovered (approximately 2.5–3 times enhanced). When Aβ aggregates are present in the environment, the probe binds to the hydrophobic cavity of the Aβ aggregates, restricting molecular rotor rotation and suppressing the TICT effect, but the PET effect still exists, and fluorescence recovers to a certain degree. Only when ClO － When coexisting with Aβ aggregates, ClO － First, the pyrrole oxidase unit shuts down PET, and then the probe binds to Aβ to shut down TICT. The two quenching mechanisms are released in sequence, resulting in a cascade amplification effect and achieving significant fluorescence enhancement.

[0012] Furthermore, the BODIPY derivative of the present invention has a high affinity for Aβ aggregates.

[0013] Preferably, the BODIPY derivative is effective against Aβ. 1-42 The dissociation constant Kd of the aggregate is ≤ 50 nM. More preferably, the BODIPY derivative is effective against Aβ. 1-42 The dissociation constant of the aggregates ranges from 26.91 to 39.91 nM. This nanomolar level affinity far exceeds that of most existing probes, which are at the micromolar level. This enables the probe of this invention to detect small, early-stage Aβ aggregates, providing a powerful tool for the early diagnosis of Alzheimer's disease.

[0014] Furthermore, the BODIPY derivative of the present invention has a fluorescence response characteristic of dual-lock cascade activation.

[0015] Preferably, the BODIPY derivative is in Aβ 1-42 Aggregates and ClO －When both are present, the fluorescence enhancement factor is ≥50-fold. More preferably, when R is hydrogen (D-BDY), the fluorescence enhancement factor can reach 100-fold; when R is ethyl (T-BDY), the fluorescence enhancement factor can reach 50-fold. In comparison, when only ClO is present... － When present, the fluorescence enhancement factor is approximately 2.5 to 3 times; when only Aβ aggregates are present, the fluorescence enhancement factor is approximately 12.5 to 20 times. This indicates that the BODIPY derivative described in this invention achieves a true "AND gate" logic response, producing a significant cascaded amplification of fluorescence signal only when both stimuli are present simultaneously, effectively avoiding false positives caused by a single marker.

[0016] Furthermore, the BODIPY derivative of the present invention possesses near-infrared emission characteristics. Preferably, the maximum emission wavelength of the BODIPY derivative is 715–725 nm. Near-infrared light has advantages such as large tissue penetration depth and low interference from biological autofluorescence, making it particularly suitable for imaging deep tissues in vivo.

[0017] Furthermore, the BODIPY derivative of this invention can penetrate the blood-brain barrier. Preferably, the uptake rate of the BODIPY derivative in the mouse brain is ≥5% ID / g, more preferably 6.73 ± 0.71% ID / g. This characteristic allows the probe to be used directly for in vivo brain imaging without invasive drug delivery, showing good prospects for clinical translation.

[0018] Furthermore, the BODIPY derivative of this invention exhibits good biocompatibility. Testing showed that the BODIPY derivative achieved a cell viability of >90% at a concentration of 20 μM and a hemolysis rate of <2% at a concentration of 150 μM, demonstrating low cytotoxicity and low hemolytic activity.

[0019] Secondly, the present invention provides a method for preparing the above-mentioned BODIPY derivative, comprising the following steps.

[0020] (1) Synthesis of intermediate BDP: Cl-BDP, pyrrole derivatives, and a catalytic amount of triethylamine were mixed and dissolved in anhydrous toluene at a molar ratio of 0.05~0.1:0.1~0.2:0.003~0.01 to achieve a molar concentration of Cl-BDP of 5 mM~10 mM. The mixture was heated to 80~100℃ under a nitrogen atmosphere and reacted for 6~12 h. After cooling to room temperature, the solvent was removed, and the residue was dissolved in dichloromethane and washed 2~3 times with saturated brine. Finally, the mixture was purified by column chromatography to obtain the orange solid intermediate BDP.

[0021] Further, the pyrrole derivative is 2,4-dimethylpyrrole or 2,4-dimethyl-3-ethylpyrrole. When 2,4-dimethylpyrrole is used, an intermediate in which R is hydrogen (D-BDP) is prepared accordingly; when 2,4-dimethyl-3-ethylpyrrole is used, an intermediate in which R is ethyl is prepared accordingly (T-BDP).

[0022] Preferably, the reaction temperature in step (1) is 90°C and the reaction time is 8 h.

[0023] (2) Synthesis of the target product BODIPY derivative: The intermediate BDP obtained in step (1) and 4-dimethylaminobenzaldehyde were added to anhydrous toluene at a molar ratio of 1:1.5~2. Piperidine and acetic acid were added under stirring, with the volume of piperidine and acetic acid being 1 / 100~1 / 50 of the volume of toluene, respectively. The mixture was refluxed under a nitrogen atmosphere for 20~28 h. After the reaction was completed, the mixture was cooled to room temperature, the solvent was removed, the residue was dissolved in dichloromethane, the solution was washed with brine and water, the organic layer was separated and dried, and the BODIPY derivative (blue-black solid) was obtained by column chromatography and vacuum distillation to remove the solvent.

[0024] Preferably, the reflux reaction time in step (2) is 24 hours.

[0025] The above preparation method uses commercially available or synthesized Cl-BDP by known methods as the starting material. The target product can be obtained through two steps. The synthetic route is short, the operation is simple, and the yield can reach 54.6%–58.9%, making it suitable for large-scale preparation. The solvent can be removed by vacuum distillation or other conventional methods in the field.

[0026] Thirdly, the present invention provides a method for simultaneously detecting Aβ 1-42 Aggregates and ClO － Fluorescent probes or fluorescent imaging reagents.

[0027] The fluorescent probe or fluorescent imaging reagent is a BODIPY derivative as described above, or contains a BODIPY derivative as described above. Preferably, the fluorescent probe or fluorescent imaging reagent is composed of a BODIPY derivative as described above.

[0028] Furthermore, the fluorescent probe or fluorescent imaging reagent can penetrate the blood-brain barrier and is used to detect Aβ aggregates and ClO2 in the brains of Alzheimer's disease animals. － Imaging or monitoring.

[0029] In this invention, the "imaging" includes static imaging after a single drug administration, used to determine Aβ aggregates and ClO in the brain. －The presence and relative content of the fluorescent probe; the "monitoring" includes dynamic tracking imaging after multiple administrations to assess disease progression or treatment efficacy. For example, in the APP / PS1 transgenic AD mouse model, after tail vein injection of the fluorescent probe or fluorescent imaging reagent, the peak fluorescence signal in the brain was reached in about 25 minutes, and the fluorescence intensity in the brain of AD mice was about twice that of wild-type mice; after 3 days of treatment with the antioxidant N-acetylcysteine ​​(NAC), the fluorescence intensity decreased significantly, indicating that the probe can be used for dynamic monitoring of drug efficacy.

[0030] Furthermore, the fluorescent probe or fluorescent imaging reagent exhibits high selectivity. Testing revealed that the fluorescent probe or fluorescent imaging reagent is highly selective for ClO₂. － It exhibits high selectivity for other reactive oxygen species (including H2O2, ·OH, ONOO). － (NO) hardly produces a fluorescence response; at the same time, it has high selectivity for Aβ aggregates and for common brain metal ions (including Na+, NO) ＋ K ＋ Ca 2＋ Cu 2＋ Fe 3＋ Fe 2＋ Zn 2＋ Co 2＋ ) and amino acids (including GSH, Cys, Leu, Phe, Val) hardly produce a fluorescent response.

[0031] Fourthly, this invention provides the aforementioned BODIPY derivatives for the preparation of Aβ aggregates and ClO2 in the brain. － Applications in testing reagents.

[0032] Furthermore, based on the relationship between Aβ aggregates and ClO in AD pathology － The importance and high relevance of oxidative stress to Aβ and ClO － The combined detection of these components can be used for precise imaging of Alzheimer's disease (AD) and evaluation of drug efficacy. Therefore, this invention also provides the application of the aforementioned BODIPY derivatives in fluorescence imaging of the brain before and after treatment in AD model mice, and further, the use of the BODIPY derivatives described in this invention in the preparation of diagnostic reagents or efficacy assessment reagents for Alzheimer's disease.

[0033] In specific embodiments, this invention uses D-BDY as an example to illustrate its use in in vitro and in vivo environments for Aβ aggregates and ClO. －The detection showed that D-BDY could be effectively enriched in the brain at the same time interval (BBB uptake rate of approximately 6.73% ID / g). Simultaneously, this invention experimentally verified that T-BDY, due to having the same BODIPY fluorescent nucleus and the same dual-lock response mechanism (PET+TICT), also possesses Aβ and ClO2 in vitro and in vivo. － The detection capability and ability to cross the blood-brain barrier. Changes in the substituents on the pyrrole ring (hydrogen group → ethyl group) affect the strength of PET efficiency, but do not change the basic mechanism of the double-lock cascade response. In in vitro AD mouse brain slice experiments, D-BDY can specifically label Aβ aggregates, and the Pearson correlation coefficient with ThT co-localization is 0.31; when the probe is pre-treated with ClO － After the reaction, the co-location coefficient significantly increased to 0.79, indicating that ClO － Activation significantly enhances the probe's ability to stain the entire region of Aβ aggregates. In in vivo experiments, D-BDY was effectively enriched in the brain at the same time interval (BBB uptake rate approximately 6.73% ID / g). In APP / PS1 transgenic AD mice (10 months old, naturally occurring Aβ aggregates and high levels of ClO2) after tail vein injection, this significantly improved the staining performance. － The near-infrared fluorescence intensity of the brain in T-BDY mice was approximately twice that of wild-type mice. After 3 days of treatment with the antioxidant NAC, the fluorescence intensity significantly decreased but remained higher than that of wild-type mice (approximately 1.7 times), demonstrating its potential for drug efficacy evaluation. Furthermore, this invention experimentally verified that T-BDY, possessing the same BODIPY fluorescent nucleus and the same dual-lock response mechanism (PET+TICT), also exhibits similar characteristics in in vitro AD brain slice Aβ aggregate staining and in vivo Aβ and ClO2 staining. － The ability to detect and cross the blood-brain barrier. Changes in substituents on the pyrrole ring (from hydrogen to ethyl) affect the strength of PET efficiency, but do not change the basic mechanism of the double-lock cascade response.

[0034] Therefore, the BODIPY derivative developed in this invention can be used in an older transgenic AD mouse model (APP / PS1, which develops Aβ aggregates and higher levels of ClO2 in the brain with age). － ), to achieve the targeting of Aβ aggregates and ClO in the brain － Endogenous dual-analyte cascade fluorescence imaging. The BODIPY derivatives described in this invention can effectively penetrate the blood-brain barrier and target endogenous Aβ aggregates and ClO2 in the animal brain. － Real-time near-infrared fluorescence imaging was performed to reflect the coexistence and synergistic changes of two biomarkers in the pathological state of Alzheimer's disease (AD).

[0035] The raw materials or reagents involved in this invention are all commercially available products, and the operations involved are all routine operations in the field unless otherwise specified.

[0036] The beneficial effects of this invention are at least as follows: This invention develops and synthesizes a novel class of BODIPY derivatives that can serve as near-infrared fluorescent probes for dual-lock cascade activation, effectively enabling the in vitro and in vivo reaction of Aβ aggregates with ClO₂. － This probe enables simultaneous detection and real-time monitoring. It possesses advantages such as AND gate cascade amplification, high affinity, near-infrared emission, and efficient blood-brain barrier penetration. Aβ aggregates and ClO2 were successfully detected in the brains and brain slices of AD transgenic mice. － Its highly specific imaging capabilities, and the signals that can reflect the severity of the pathology and the effectiveness of drug treatment, provide a valuable research tool for the pathological study, precise imaging, and efficacy evaluation of Alzheimer's disease. Attached Figure Description

[0037] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 This is the synthetic route for the BODIPY derivative described in this invention.

[0040] Figure 2 The spectral properties of the BODIPY derivatives described in this invention are shown in different solvent environments; wherein, (A): UV absorption intensity curves of D-BDY and T-BDY, and (B): fluorescence intensity curves of D-BDY and T-BDY.

[0041] Figure 3 The Aβ detection performance of BODIPY derivatives in this embodiment of the invention; wherein, (A): D-BDY and T-BDY for Aβ 1-42 Fluorescence response of aggregates (final concentration 7 μM) and bovine serum albumin (BSA, 200 μM), (B): D-BDY and T-BDY with Aβ 1-42 Saturation binding curves of aggregates (7 μM) at different probe concentrations, (C): D-BDY and T-BDY for common brain metal ions (Na+). ＋ K ＋ Ca 2＋ Cu 2＋ Fe 3＋ Fe 2＋ Zn 2＋ Co2＋ ), amino acids (GSH, Cys, Leu, Phe, Val) and Aβ 1-42 Selectivity of aggregates.

[0042] Figure 4 The BODIPY derivative in this embodiment of the invention is used to counteract ClO － The fluorescence response; where, (A): added ClO － Before and after, normalized UV-Vis absorption spectra of D-BDY and T-BDY in ethanol, (B): after titration of ClO － Fluorescence intensity changes of D-BDY and T-BDY (1 μM dissolved in ethanol) at (0~100 μM), (C): D-BDY and T-BDY with ClO － The high-resolution mass spectrometry (HRMS) spectra of the reaction products confirmed the formation of D-BDY-OH and T-BDY-OH, respectively.

[0043] Figure 5 The BODIPY derivative in this embodiment of the invention is used to counteract ClO － The response kinetics and selectivity; where, (A): D-BDY (1 μM) and T-BDY (1 μM) for ClO － Response kinetics (30–90 μM), (B): Selectivity of D-BDY and T-BDY for common reactive oxygen species (ClO-, H2O2, ·OH, ONOO-, NO).

[0044] Figure 6 The fluorescence responses of BODIPY derivatives under different activation conditions are shown in the embodiments of the present invention; wherein, (A) represents the fluorescence responses of D-BDY and T-BDY under ClO₂ conditions. － Aβ 1-42 Fluorescence spectral changes in the presence of aggregates, (B) shows the quantitative analysis of fluorescence intensity of D1-D4 and T1-T4 samples in the well plate using the IVIS in vivo imaging system.

[0045] Figure 7 In this embodiment of the invention, the BODIPY derivative is used to stain Aβ plaques in brain slices from AD mice in vitro; wherein, (A): without ClO － Under conditions of (60 µM), Aβ in mouse brain slices co-stained with ThT (10 µM, green) and D-BDY (2 µM, red) 1-42 Confocal laser scanning microscopy (CLSM) image of the plaque, (B): in the presence of ClO － Under conditions of (60 µM), Aβ in mouse brain slices co-stained with ThT (10 µM, green) and D-BDY (2 µM, red) 1-42CLSM image of the plaque, (C): without ClO － Under conditions of (60 µM), Aβ in mouse brain slices co-stained with ThT (10 µM, green) and T-BDY (2 µM, red) 1-42 CLSM image of the plaque, (D): Presence of ClO － Under conditions of (60 µM), Aβ in mouse brain slices co-stained with ThT (10 µM, green) and T-BDY (2 µM, red) 1-42 CLSM image of the plaque.

[0046] Figure 8 The results of the determination of BBB uptake rate of BODIPY derivative D-BDY in the embodiments of the present invention are shown. Among them, (A) is the HPLC chromatogram and peak area of ​​D-BDY at different concentrations, which is used for the determination of the standard curve, (B) is the corresponding standard curve, and (C) is the HPLC chromatogram and peak area of ​​D-BDY in mouse brain tissue.

[0047] Figure 9 Cell viability and hemolysis rate of BODIPY derivatives in embodiments of the present invention. Wherein, (A): cytotoxicity of DBDY and T-BDY, (B): hemolysis rate of D-BDY and T-BDY.

[0048] Figure 10 This invention provides in vivo fluorescence imaging of the brain regions of the BODIPY derivative D-BDY in wild-type mice (WT, C57BL / 6J, 10 months, female), transgenic mice (APP / PS1, 10 months, female), and APP / PS1 NAC mice (APP / PS1, 10 months, female). (A): D-BDY (1 mg / kg) was intravenously injected into mice, and fluorescence signals were obtained via IVIS. (B): Clearance curves of D-BDY in WT, APP / PS1, and APP / PS1 NAC mice. (C): Schematic diagram of the experimental design, showing the pretreatment and imaging procedures for AD mice, as well as the treatment groups, including the dosages of D-BDY and NAC.

[0049] Figure 11 The above are the proton NMR spectra of the BODIPY derivative intermediates D-BDP and T-BDP in this embodiment of the invention; wherein, (A) is the NMR spectrum of D-BDP. 1 H NMR spectrum, (B) is T-BDP. 1 H NMR spectrum.

[0050] Figure 12 These are the proton NMR spectra of BODIPY derivatives D-BDY and T-BDY in embodiments of the present invention; wherein, (A) is the NMR spectrum of D-BDY.1 H NMR spectrum (CDCl3, 400 MHz), (B) is T-BDY. 1 H NMR spectrum (CDCl3, 400 MHz).

[0051] Figure 13 The images show high-resolution mass spectra of the BODIPY derivative intermediates D-BDP and T-BDP in this embodiment of the invention; wherein, (A) is the HR-MS (ESI) spectrum of D-BDP and (B) is the HR-MS (ESI) spectrum of T-BDP.

[0052] Figure 14 The images show high-resolution mass spectra of BODIPY derivatives D-BDY and T-BDY in this embodiment of the invention; wherein, (A) is the HR-MS (ESI) spectrum of D-BDY and (B) is the HR-MS (ESI) spectrum of T-BDY. Detailed Implementation

[0053] To fully demonstrate the technical solution and advantages of this invention, the implementation of this invention will be further described below. It should be noted in advance that, without creating contradictions, the various specific solutions and technical features within these solutions can be combined and used in combination.

[0054] In the following description, several specific details will be set forth to provide a thorough understanding of the invention. However, the invention can be practiced in many other ways than those described herein; it should be understood that the specific examples listed herein are only a part, not all, of the possible embodiments of the invention.

[0055] The preferred embodiments of the present invention will be described in detail below with reference to several examples. These examples are intended only to illustrate the present invention and not to limit the scope of protection of the present invention in any way. All equivalent substitutions or modifications made without departing from the core concept and spirit of the present invention should fall within the scope of protection of the present invention.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0057] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0058] In the examples described below, Cl-BDP is a compound described in the reference (Organic Letters, 2012, 14, 24, 6150-6153, doi:10.1021 / ol3028225), and can be synthesized according to the synthesis method described in the reference.

[0059] Example 1: Preparation method and product verification of BODIPY derivatives according to Figure 1 The synthetic route shown is used to prepare BODIPY derivatives.

[0060] 1. Synthesis of intermediate BDP 1.1 Synthesis of D-BDP Under N2 protection, Cl-BDP (56.4 mg, 0.2 mmol) was dissolved in 15 mL of anhydrous toluene, and a catalytic amount of triethylamine (2.8 μL, 0.02 mmol) was added. The mixture was stirred at room temperature for 10 minutes. 2,4-Dimethylpyrrole (28.5 mg, 0.3 mmol) was added, and the mixture was heated to 90 °C and reacted for 8 hours. The solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 1, v / v) to give an orange solid.

[0061] Characterization of D-BDP: 1 H NMR (400 MHz, CDCl3) δ 7.51 (s, 1H), 6.02 (s, 1H), 5.81 (s, 1H), 2.56 (s, 6H), 2.30 (s, 3H), 1.91 (s, 3H), 1.64 (s, 6H). HR-MS (ESI,positive) calcd for C 19 H 23 BF2N3 [M+H] ＋ 342.195107, found 342.19509.

[0062] The proton NMR spectrum and high-resolution mass spectra are shown below. Figure 11 and Figure 13 .

[0063] 1.2 Synthesis of T-BDP Under N2 protection, Cl-BDP (56.4 mg, 0.2 mmol) was dissolved in 15 mL of anhydrous toluene, and a catalytic amount of triethylamine (2.8 μL, 0.02 mmol) was added. The mixture was stirred at room temperature for 10 minutes. 3-Ethyl-2,4-dimethylpyrrole (36.9 mg, 0.3 mmol) was added, and the mixture was heated to 90 °C and reacted for 8 hours. The solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 1, v / v) to give an orange solid, T-BDP.

[0064] Characterization of T-BDP: 1H NMR (400 MHz, CDCl3) δ 7.35 (s, 1H), 6.01 (s, 1H), 2.56 (s, 6H), 2.42 (q, J = 7.5 Hz, 2H), 2.25 (s, 3H), 1.87 (s, 3H), 1.62 (s, 6H), 1.07 (t, J = 7.5 Hz, 3H). HR-MS (ESI, positive) calcd for C 21 H 27 BF2N3[M+H] ＋ 370.226442, found 370.22653.

[0065] The proton NMR spectrum and high-resolution mass spectra are shown below. Figure 11 and Figure 13 .

[0066] 2. Synthesis of the target compound BDY 2.1 Synthesis of D-BDY D-BDP (68.2 mg, 0.2 mmol) and 4-dimethylaminobenzaldehyde (36.9 mg, 0.3 mmol) were added to a 100 mL round-bottom double-necked flask and dissolved in 15 mL of anhydrous toluene. Then, acetic acid (0.2 mL) and piperidine (0.19 mL) were added with stirring. After reflux under nitrogen atmosphere for 24 h, the mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane, and the solution was washed with brine and water. The organic layer was separated and dried over anhydrous magnesium sulfate. After evaporating the solvent, petroleum ether:dichloromethane (3:7) was used as the eluent. The mixture was purified by column chromatography and the solvent was removed by reduced pressure distillation to obtain a deep blue solid, D-BDY (56.1 mg, 58.9% yield).

[0067] Characterization of D-BDY: 1 H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 8.6 Hz, 4H), 7.26(s, 1H), 6.76 (s, 1H), 6.64 (s, 1H), 6.01 (s, 1H), 5.82 (s, 1H), 3.06 (s,6H), 2.60 (s, 3H), 2.32 (s, 3H), 1.92 (s, 3H), 1.69 (s, 3H), 1.64 (s, 3H). HR-MS (ESI, positive) calcd for C 28 H31 BF2N4[M+H] ＋ 473.26093, found 473.26044.

[0068] The proton NMR spectrum and high-resolution mass spectra are shown below. Figure 12 and Figure 14 .

[0069] 2.2 Synthesis of T-BDY T-BDP (79.2 mg, 0.2 mmol) and 4-dimethylaminobenzaldehyde (36.9 mg, 0.3 mmol) were added to a 100 mL round-bottom double-necked flask and dissolved in 15 mL anhydrous toluene. Then, acetic acid (0.2 mL) and piperidine (0.19 mL) were added with stirring. After reflux under nitrogen atmosphere for 24 h, the mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane, and the solution was washed with brine and water. The organic layer was separated and dried over anhydrous magnesium sulfate. After evaporating the solvent, petroleum ether:dichloromethane (3:7) was used as the eluent. The mixture was purified by column chromatography and the solvent was removed by reduced pressure distillation to obtain a deep blue solid, T-BDY (54.6 mg, yield 54.6%).

[0070] Characterization of T-BDY: 1 H NMR (400 MHz, CDCl3) δ 7.57 – 7.51 (m, 3H), 7.36 (s,1H), 7.23 (d, J = 16.3 Hz, 1H), 6.78 (s, 1H), 6.64 (s, 1H), 6.01 (s, 1H), 3.06 (s, 6H), 2.60 (s, 3H), 2.42 (q, J = 7.4 Hz, 2H), 2.25 (s, 3H), 1.88 (s,3H), 1.66 (s, 3H), 1.62 (s, 3H), 1.07 (t, J = 7.5 Hz, 3H). HR-MS (ESI,positive) calcd for C 30 H 35 BF2N4 [M+H] ＋ 500.29226, found 500.29171.

[0071] The proton NMR spectrum and high-resolution mass spectra are shown below. Figure 12 and Figure 14 .

[0072] Example 2: Spectral properties of probes D-BDY and T-BDY D-BDY and T-BDY were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions (1 mM), and then dissolved in PBS buffer (pH 7.4, with 1% ethanol as a solubilizer) to prepare working solutions (2 μM). Their UV-Vis and fluorescence spectra were then measured. UV spectroscopy analysis showed that the maximum absorption wavelengths of D-BDY and T-BDY in PBS were 648 nm and 644 nm, respectively. Figure 2 A). Fluorescence spectroscopy showed that the maximum emission wavelengths of D-BDY and T-BDY were 725 nm and 715 nm, respectively. Figure 2 B). As solvent polarity increases, the emission wavelengths of both molecules redshift, indicating the presence of a distorted intramolecular charge transfer (TICT) effect.

[0073] Example 3: Probes D-BDY and T-BDY on Aβ 1-42 fluorescence response of aggregates D-BDY and T-BDY (2 μM) were respectively reacted with Aβ 1-42 After incubating the aggregates (7 μM) in PBS for 30 minutes, the fluorescence spectra were measured. The results showed that the fluorescence enhancement of D-BDY was approximately 18.90-fold, and that of T-BDY was approximately 12.50-fold, with both exhibiting a blue shift to 659 nm. Figure 3 A). The dissociation constant (Kd) was determined using a saturated binding experiment. The Kd for D-BDY was 39.91 ± 7.28 nM, and the Kd for T-BDY was 26.91 ± 1.72 nM. Figure 3 B), indicating that both have a high affinity for Aβ aggregates. Selectivity experiments showed that both have a high affinity for common brain metal ions (Na+). ＋ K ＋ Ca 2＋ Cu 2＋ Fe 3＋ Fe 2＋ Zn 2＋ Co 2＋ ) and amino acids (GSH, Cys, Leu, Phe, Val) showed almost no fluorescence response ( Figure 3 C).

[0074] Example 4: Probes D-BDY and T-BDY on ClO － fluorescence response Dissolve D-BDY and T-BDY (1 μM) in ethanol, and gradually add ClO － (0–100 μM). For example... Figure 4As shown in A and 4B, the fluorescence intensity of D-BDY and T-BDY increased by approximately 2.50 times and 2.70 times, respectively, and the maximum emission wavelength redshifted from 660 nm to 680 nm. Response kinetics indicate that both exhibit rapid responses within 600 seconds and tend to stabilize after 3000 seconds. Figure 5 A). High-resolution mass spectrometry confirmed that the oxidation products were D-BDY-OH (m / z = 488.2574) and T-BDY-OH (m / z = 516.2899). Figure 4 C). Selectivity experiments show that both have an effect on ClO － It exhibits high selectivity and is effective against other reactive oxygen species (H2O2, ·OH, ONOO). － NO) hardly causes any change in fluorescence. Figure 5 B).

[0075] Example 5: Probes D-BDY and T-BDY for Aβ and ClO － Cascaded Amplification Response D-BDY and T-BDY (2 μM) were placed under the following four conditions: PBS control, +ClO － (60 μM), +Aβ 1-42 Aggregates (7 μM), +Aβ+ClO － .like Figure 6 As shown in Figure A, D-BDY in only ClO － The fluorescence enhancement was approximately 2.5-fold when present, approximately 18.9-fold when Aβ alone was present, and approximately 100-fold when both coexisted; T-BDY showed an approximately 50-fold enhancement when both coexisted. IVIS imaging also showed a similar trend. Figure 6 B). This result indicates that the probe exhibits a double-locked cascade amplification effect.

[0076] Example 6: Co-localization imaging of probes D-BDY and T-BDY in AD brain slices Brain tissue was collected from APP / PS1 transgenic AD mice (10 months old) and prepared into 5 μm thick paraffin sections. After dewaxing and rehydration, the sections were stained with ThT (10 μM) and D-BDY (2 μM), respectively, and observed under a laser confocal microscope. Figure 7 As shown in Figure A, the Pearson correlation coefficient between D-BDY and ThT is 0.31, indicating that the unactivated probe mainly binds to the plaque core region. When the probe is pre-treated with ClO... － After reacting with (60 μM) and then staining, the Pearson correlation coefficient increased to 0.79, and the red and green fluorescence highly overlapped. Figure 7 B). Under the same conditions, the Pearson coefficient for T-BDY increased from 0.49 to 0.69 (B). Figure 7 C-7D). The results show that ClO －Activation significantly enhanced the probe's staining ability on the entire Aβ plaque.

[0077] Example 7: Biocompatibility and Blood-Brain Barrier Penetration of Probes D-BDY and T-BDY (1) Cytotoxicity: PC12 cells were incubated with different concentrations (0, 2, 5, 10, 15, 20 μM) of D-BDY or T-BDY for 24 hours, and cell viability was determined by the MTT assay. The results showed that the cell viability at the 20 μM concentration was >90%. Figure 9 A).

[0078] (2) Hemolysis test: Red blood cells were collected and incubated for 3 hours with PBS (negative control), water (positive control), and different concentrations (10, 20, 30, 50, 100, 150 μM) of probes D-BDY and T-BDY. The absorbance of the supernatant at 490 nm was measured. The results showed that the hemolysis rate at a concentration of 150 μM was <2%. Figure 9 B).

[0079] (3) BBB uptake rate: D-BDY (1.42 mg / kg) was injected into KM mice (n=3) via the tail vein. The mice were sacrificed 25 minutes later, and the brain tissue was homogenized, extracted with acetonitrile, and analyzed by HPLC.

[0080] Based on the standard curve, the BBB uptake rate was calculated to be 6.73 ± 0.71% ID / g ( Figure 8 ).

[0081] Example 8: Near-infrared fluorescence imaging of probe D-BDY in the brain of live AD mice like Figure 10 As shown in Figure C, 10-month-old female C57BL / 6J wild-type mice (WT), APP / PS1 transgenic AD mice, and APP / PS1 mice pretreated with N-acetylcysteine ​​(NAC, 20 mg / kg, intraperitoneal injection, for 3 consecutive days) were selected. D-BDY (1 mg / kg) was injected via the tail vein, and brain fluorescence signals were continuously acquired at 0, 5, 15, 25, 35, 45, and 60 minutes post-injection using an IVIS Lumina III imaging system (excitation 640 nm, emission 700 nm). Figure 10 As shown in Figures A and 10B, the fluorescence intensity in the brains of AD mice peaked at 25 minutes, approximately twice that of WT mice; the fluorescence intensity in the NAC pretreatment group was between the two (approximately 1.7 times). The signal attenuation curves indicate that the probe clearance rate in the brains of AD mice was slower than that in WT mice, which is related to the retention of the probe after binding to Aβ plaques. These results suggest that D-BDY can effectively penetrate the blood-brain barrier, achieving effective clearance of Aβ plaques and ClO2 in the brains of AD mice. － It provides real-time imaging, and the signals can reflect the effects of antioxidant treatment.

[0082] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A near-infrared fluorescent BODIPY derivative with dual-lock cascade activation, characterized in that, It has the following general formula (Formula I) structure: (Equation I) R is selected from hydrogen or ethyl.

2. The BODIPY derivative according to claim 1, characterized in that, The BODIPY derivative affects Aβ 1-42 The dissociation constant Kd of the aggregate is ≤ 50 nM.

3. The BODIPY derivative according to claim 1, characterized in that, The BODIPY derivative in Aβ 1-42 Aggregates and ClO － When both are present, the fluorescence enhancement factor is ≥50 times.

4. The method for preparing the BODIPY derivative according to claim 1, characterized in that, The steps include the following: (1) Synthesis of intermediate BDP: Cl-BDP, pyrrole derivatives, and a catalytic amount of triethylamine were mixed and dissolved in anhydrous toluene at a molar ratio of 0.05–0.1: 0.1–0.2: 0.003–0.01, so that the molar concentration of Cl-BDP was 5 mM–10 mM. The mixture was heated to 80–100 °C under a nitrogen atmosphere and reacted for 6–12 h. After cooling to room temperature, the solvent was removed, the residue was dissolved in dichloromethane, and washed 2–3 times with saturated brine. Finally, the intermediate BDP was purified by column chromatography. (2) Synthesis of the target product BODIPY derivative: The intermediate BDP obtained in step (1) and 4-dimethylaminobenzaldehyde were added to anhydrous toluene at a molar ratio of 1:1.5 to 2. Piperidine and acetic acid were added under stirring, with the volume of piperidine and acetic acid being 1 / 100 to 1 / 50 of the volume of toluene, respectively. The mixture was refluxed under a nitrogen atmosphere for 20 to 28 h. After the reaction was completed, the mixture was cooled to room temperature, the solvent was removed, the residue was dissolved in dichloromethane, the solution was washed with brine and water, the organic layer was separated and dried, and the BODIPY derivative was obtained by column chromatography and vacuum distillation to remove the solvent.

5. The preparation method according to claim 4, characterized in that, The pyrrole derivative mentioned in step (1) is 2,4-dimethylpyrrole or 2,4-dimethyl-3-ethylpyrrole.

6. The preparation method according to claim 4, characterized in that, In step (1), the reaction temperature is 90℃ and the reaction time is 8 h; in step (2), the reflux reaction time is 24 h.

7. A method for detecting Aβ 1-42 Aggregates and ClO － A fluorescent probe or fluorescent imaging reagent, characterized in that, The fluorescent probe or fluorescent imaging reagent is a BODIPY derivative as described in any one of claims 1 to 3.

8. The fluorescent probe or fluorescent imaging reagent according to claim 7, characterized in that, The fluorescent probe or fluorescent imaging reagent can penetrate the blood-brain barrier and is used to detect Aβ aggregates and ClO2 in the brains of Alzheimer's disease animals. － Imaging or monitoring.

9. The BODIPY derivative according to any one of claims 1 to 3 in the preparation of Aβ aggregates and ClO for the brain － Applications in testing reagents.

10. The use of the BODIPY derivative according to any one of claims 1 to 3 in the preparation of diagnostic reagents or efficacy assessment reagents for Alzheimer's disease.

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