Alkaline phosphatase response type near-infrared fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses an alkaline phosphatase response type near-infrared fluorescent probe and a preparation method and application thereof, the fluorescent probe is a compound XDM-P, the molecular structure of the fluorescent probe comprises a donor-pi-conjugate-receptor type near-infrared fluorophore constructed by taking xanthene and cyanoisophorone as parent nucleuses, and a recognition group linked by a phosphate bond; the preparation method comprises Knoevenagel condensation, phosphorylation and selective dealkylation reaction. The probe is subjected to specific hydrolysis under the action of alkaline phosphatase (ALP), the intramolecular charge transfer effect is recovered, and near-infrared fluorescence 'OFF-ON' response at 774 nm is realized. The probe provided by the invention has high sensitivity, good linear response and deep tissue penetrating ability, and can be used for intracellular ALP detection and non-invasive, real-time and semi-quantitative in-vivo fluorescence imaging of liver cancer tumor-bearing mouse and drug-induced liver injury mouse models.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to an alkaline phosphatase-responsive near-infrared fluorescent probe, its preparation method, and its application. Background Technology

[0002] Alkaline phosphatase (ALP) is a hydrolytic enzyme widely found in various human tissues, including the liver, bones, kidneys, and placenta. It catalyzes the hydrolysis and transphosphorylation of phosphate monoesters. Under physiological conditions, ALP participates in regulating key processes such as intracellular phosphate metabolism and signal transduction. However, abnormal ALP activity levels are often closely related to various pathological conditions, particularly in liver pathologies such as hepatocellular carcinoma (HCC) and drug-induced liver injury (DILI), where serum and tissue ALP levels are significantly elevated. Therefore, ALP is clinically recognized as a key biomarker for assessing liver function and diagnosing related diseases. Developing highly sensitive and specific methods for detecting ALP activity is of great clinical significance for the early diagnosis, efficacy evaluation, and pathological mechanism research of liver cancer and drug-induced liver injury.

[0003] Currently, conventional methods for ALP detection mainly include radioisotope methods, chromatography, electrochemical methods, and the traditional colorimetric method based on p-nitrophenyl phosphate (pNPP). Although these methods are relatively mature, they also have many limitations: for example, radioactive methods pose radiation safety risks; while colorimetric methods are simple to operate, their sensitivity is relatively limited and they are easily affected by factors such as turbidity and endogenous light-absorbing substances in biological samples, leading to decreased detection accuracy; the aforementioned traditional detection methods usually require lysis of cell or tissue samples, which is cumbersome and makes it difficult to achieve in situ, non-invasive, and visualized dynamic monitoring of ALP activity in living organisms in real time. In recent years, small molecule fluorescent probe technology has provided a powerful tool for real-time detection of biological enzyme activity in vivo due to its advantages such as high sensitivity, simple operation, good spatiotemporal resolution, and minimal damage to biological samples. In particular, near-infrared (NIR, wavelength typically 650-900 nm) fluorescent probes, due to their emission wavelength being located within the "optical window" of biological tissues, have stronger tissue penetration, lower biological autofluorescence background interference, and less phototoxicity, making them an ideal choice for the field of in vivo imaging.

[0004] Although some fluorescent probes for ALP detection have been reported, existing probes still have significant shortcomings in practical applications, mainly in the following aspects: Limited emission wavelength: Many existing ALP probes have short emission wavelengths (e.g., in the visible light region or less than 700 nm), making them susceptible to absorption and scattering by biological tissues (e.g., skin, blood, fat) during in vivo imaging. This results in shallow tissue penetration, low signal-to-noise ratio, and difficulty in meeting the imaging needs of deep tissues or solid tumors; Poor response performance: Some probes have slow enzyme response kinetics, requiring long incubation times to reach the fluorescence signal plateau, failing to capture rapidly changing enzyme activity fluctuations in vivo; or the probes have poor water solubility and biocompatibility, easily accumulating or precipitating under physiological conditions; Limited application scenarios: Most existing probes have only been validated for feasibility in single cell lines or simple animal models, lacking the ability to perform specific recognition, high-contrast imaging, and simultaneous detection in complex pathological environments (e.g., involving the complex processes of liver cancer development and drug-induced liver injury simultaneously).

[0005] In summary, there is an urgent need to develop a novel near-infrared fluorescent probe. This probe should possess excellent sensitivity, specificity, good water solubility and biocompatibility, and its emission wavelength should be located in the near-infrared region to meet the needs of in vivo imaging. In particular, it should be able to achieve accurate and simultaneous visualization detection of ALP activity in two key liver pathological states: liver cancer and drug-induced liver injury. This would overcome the bottlenecks of existing technologies and provide more effective technical means for the clinical diagnosis and basic medical research of related diseases. Summary of the Invention

[0006] One of the technical problems to be solved by the present invention is to provide an alkaline phosphatase-responsive near-infrared fluorescent probe to solve the problems of short emission wavelength, insufficient tissue penetration, slow enzyme response, poor anti-interference ability, and inability to achieve high-contrast imaging under complex pathological conditions in the prior art.

[0007] To overcome the shortcomings of the prior art, the present invention provides an alkaline phosphatase-responsive near-infrared fluorescent probe, wherein the fluorescent probe is a compound XDM-P, the molecular structure of which comprises: a donor-π-conjugated-acceptor type near-infrared fluorophore constructed with oxanthracene and cyanoisophorone as the parent nucleus; and a phosphate ester recognition group connected to the phenolic hydroxyl group of the fluorophore via a phosphate ester bond.

[0008] As a preferred embodiment, the fluorescent probe has the structure shown in formula (I): Formula (I).

[0009] Compared with existing technologies (related technologies), the alkaline phosphatase-responsive near-infrared fluorescent probe XDM-P provided in this application has the following advantages: The alkaline phosphatase-responsive near-infrared fluorescent probe XDM-P provided by this invention has achieved comprehensive breakthroughs in molecular design strategy, response mechanism, optical performance, and bioimaging capabilities. By constructing a D–π–A type fluorescent core with significant push-pull electron interaction using xanthracene and cyanoisophorone, and introducing a phosphate ester recognition group that can be specifically hydrolyzed by ALP onto the donor phenolic hydroxyl group, the probe is initially in an "OFF" state with weak near-infrared fluorescence due to the quenching effect of the phosphate ester on the ICT process. When encountering ALP, the phosphate ester bond is efficiently broken, exposing the electron donor hydroxyl group, thereby restoring the intramolecular charge transfer (ICT) process and causing the probe to rapidly transition to an "ON" state with strong near-infrared emission. The structural features and mechanism of action of this invention are closely related, resulting in rapid response, high selectivity, a large Stokes shift (up to 194 nm), and 774 nm emission. The core optical advantages, such as nm near-infrared emission, significantly enhance its imaging performance in complex biological systems. Furthermore, the fluorescent probe of this invention significantly optimizes the probe's solubility and cell compatibility in aqueous phase, exhibiting excellent linear detection performance in the 0–100 U / L ALP concentration range, and possessing low detection limit, high catalytic efficiency, and high affinity. It also shows high selectivity for various endogenous interfering substances such as metal ions, reactive oxygen species, thiols, and amino acids. Based on the above synergistic design, the probe of this invention can not only achieve high-contrast imaging of endogenous ALP in HepG2 cells, but also accurately reflect the changes in ALP elevation during APAP-induced drug-induced liver injury. It can also achieve time-dependent deep tissue near-infrared imaging in tumor models and animal models of liver injury, overcoming the technical difficulties of existing probes such as weak penetration, low imaging contrast, and inability to adapt to multiple pathological states simultaneously. Thus, it realizes synchronous, non-invasive, and real-time visualization analysis of liver cancer and drug-induced liver injury.

[0010] Another technical problem to be solved by the present invention is to provide a method for preparing an alkaline phosphatase-responsive near-infrared fluorescent probe, so as to solve the problems of cumbersome steps, poor site selectivity and low yield of conventional preparation methods in the prior art.

[0011] To overcome the shortcomings of the prior art, the present invention provides a method for preparing the alkaline phosphatase-responsive near-infrared fluorescent probe, comprising the following steps: S1: Using 6-hydroxy-2,3-dihydro-1H-xan-4-carboxaldehyde and dicyanoisophorone compounds as raw materials, a Knauvengel condensation reaction is carried out to obtain the intermediate XDM-OH. S2: Phosphorylation of the phenolic hydroxyl group of intermediate XDM-OH with dimethyl chlorophosphate yields intermediate XDM-PMe2; S3: The intermediate XDM-PMe2 was dealkylated using trimethylbromosilane to obtain the probe XDM-P; The structural formulas of the intermediates XDM-OH and XDM-PMe2 are as follows: .

[0012] Compared with existing technologies, the preparation method of the alkaline phosphatase-responsive near-infrared fluorescent probe disclosed in this application has the following advantages: The preparation method of this invention has significant advantages in reaction route design, step control, and intermediate controllability. By optimizing the traditional complex process into a three-step efficient tandem route: First, in step S1, 6-hydroxy-2,3-dihydro-1H-xan-4-carboxaldehyde undergoes a Knevengel condensation reaction with dicyanoisophorone compounds, allowing the active methylene group and aldehyde to efficiently form a conjugated C=C structure, directly constructing the probe's D-... A π–A fluorescent framework is formed; subsequently, in step S2, dimethyl chlorophosphate is used to selectively phosphorylate the phenolic hydroxyl group, achieving efficient introduction of an ALP-specific recognition group through nucleophilic substitution; finally, in step S3, trimethyl bromide is used for mild dealkylation, achieving highly selective removal of the methyl group of the phosphate ester, yielding the target product XDM-P with a well-defined structure and high purity. The reaction conditions of the above three steps are mild and regioselective, the intermediate structure is stable, and there is no need for complex introduction and removal of protecting groups, making the overall synthetic route simple, highly reproducible, and with a higher overall yield than traditional routes. By combining the steps of "condensation framework formation—phosphorylation introduction—selective dealkylation," the preparation method of this invention effectively solves the problems of cumbersome steps, poor site selectivity, and low yield in the prior art, and can stably obtain target probes with precise structures, providing a reliable chemical basis for subsequent bioimaging and in vivo applications.

[0013] In one possible implementation, in step S1, the molar ratio of 6-hydroxy-2,3-dihydro-1H-xan-4-carboxaldehyde to dicyanoisophorone compounds is 1:(1±0.2), the reaction solvent is a polar solvent, and the polar solvent includes ethanol, acetonitrile, methanol, tetrahydrofuran, the catalyst is piperidine, and the reaction temperature is 60-80℃.

[0014] Compared with existing technologies, the above-mentioned technical solution enables the Knauwengel condensation reaction to proceed efficiently under mild conditions through precise control of the raw material molar ratio, solvent polarity, and base catalyst. The use of a polar solvent is beneficial for the deprotonation of the active methylene group, while piperidine, as an organic base, can promote the activation of the aldehyde group and the formation of carbanion, making the conjugation of the 6-hydroxycoumarin precursor and dicyanoisophorone compounds more complete, thereby obtaining the D–π–A type intermediate XDM-OH with good regioselectivity and stable double bond configuration. Furthermore, by limiting the molar ratio to a narrow range of 1:(1±0.2), the generation of by-products can be significantly reduced, and the conversion rate and yield of the condensation reaction can be improved. The intermediate obtained by this embodiment has high structural purity, good batch reproducibility, and is easier to perform in subsequent phosphorylation steps, ultimately ensuring the integrity of the probe backbone and the stability of its photophysical properties, effectively overcoming the technical problems of side reactions and unstable yields in the backbone construction steps of existing ALP probe synthesis.

[0015] In one possible implementation, in step S2, the molar ratio of the intermediate XDM-OH to dimethyl chlorophosphate is 1:(3.6±0.5), the phosphorylation reaction is carried out in an anhydrous solvent, and the anhydrous solvent includes anhydrous dichloromethane, anhydrous acetonitrile, and anhydrous trichloromethane, the catalyst is a nucleophilic base, and the nucleophilic base includes triethylamine, 4-dimethylaminopyridine, and 1,8-diazacyclo[5.4.0]undec-7-ene, the reaction temperature is 0°C, and then the mixture is stirred at room temperature.

[0016] Compared with existing technologies, the above-mentioned technical solution enables highly selective phosphorylation of the phenolic hydroxyl group of XDM-OH by dimethyl chlorophosphate under anhydrous conditions and nucleophilic base promotion. The anhydrous solvent avoids side reactions of phosphoryl chloride in the presence of water, while nucleophilic bases such as triethylamine or 4-dimethylaminopyridine can simultaneously act as deacidifying agents and catalysts, improving the nucleophilicity of phenoxy anions and making the reaction with the P=O center more rapid and effective. Furthermore, controlling the molar ratio at 1:(3.6±0.5) ensures that the phosphorylating reagent is not excessive and that the phenolic hydroxyl group is completely converted, avoiding the formation of polyphosphorylation byproducts. This embodiment improves the regioselectivity and chemoselectivity of phosphate ester bond formation, further making the chemical structure of intermediate XDM-PMe2 highly stable, significantly reducing side reactions, facilitating subsequent dealkylation reactions, promoting the precise introduction of phosphate recognition groups, and improving the optical stability and biological response consistency of the finished probe.

[0017] In one possible implementation, in step S3, the molar ratio of the bromotrimethylsilane to the intermediate XDM-PMe2 is (4.0-6.0):1, the reaction solvent is anhydrous dichloromethane, the reaction is carried out by adding bromotrimethylsilane at 0°C, and then stirring the reaction at room temperature.

[0018] Compared with existing technologies, the above-mentioned technical solution can achieve selective dealkylation of methyl groups in phosphate esters of XDM-PMe2 under mild conditions. Trimethylbromosilane has strong electrophilicity and can undergo silanization and cleavage with methoxy groups in phosphate esters to form intermediate silicides, which are then quenched with methanol to generate the target phosphate group. Furthermore, by controlling the molar ratio of trimethylbromosilane to the intermediate to (4.0-6.0):1, the demethylation reaction is ensured to be sufficient without destroying the D–π–A fluorescent core structure. The dropwise addition at 0°C and the stirring process at room temperature avoid phosphate ester migration or structural rearrangement that may occur at high temperatures. Through the synergistic effect of the above conditions, this embodiment achieves high selectivity of the dealkylation reaction and does not damage the probe backbone, ultimately obtaining a target probe XDM-P with stable structure and high purity.

[0019] This invention provides an application of the fluorescent probe described above in detecting alkaline phosphatase at different concentrations. When the fluorescent probe reacts with different concentrations of ALP, a concentration-dependent fluorescence enhancement phenomenon occurs: under the action of ALP, the phosphate ester bonds in the probe undergo specific hydrolysis, releasing a strongly fluorescent dephosphorylated product, restoring the intramolecular charge transfer effect, and realizing an "OFF-ON" fluorescence response; and within the ALP concentration range of 0-100 U / L, the fluorescence intensity shows a linear relationship with the ALP concentration.

[0020] Compared with existing technologies, this invention utilizes the cleavable properties of phosphate ester bonds in the probe molecule to directly convert the enzymatic hydrolysis of ALP into a quantifiable change in near-infrared fluorescence signal. ALP has a highly specific dephosphorylation ability for phosphate esters, which can rapidly break the phosphate ester bonds of the probe under aqueous conditions, exposing the phenolic hydroxyl groups of strong electron donors and restoring the D–π–A structure through the ICT process, thereby significantly enhancing the near-infrared emission intensity at 774 nm. This mechanism makes the probe fluorescence change in a concentration-dependent manner with ALP concentration, forming an excellent linear relationship in the range of 0–100 U / L, achieving accurate quantification. Furthermore, the above-mentioned applications provided by this invention have high detection sensitivity, fast dynamic response, and strong anti-interference ability, enabling accurate quantification of ALP activity in complex biological samples. This overcomes the problems of low sensitivity, strong susceptibility to interference, and narrow linear range of traditional fluorescent probes in existing colorimetric methods, thus achieving rapid, stable, and high signal-to-noise ratio analysis and detection of ALP activity.

[0021] This invention provides an application of the fluorescent probe described above in near-infrared fluorescence imaging for detecting intracellular alkaline phosphatase, the application comprising the following steps: S1: Co-incubate the fluorescent probe with the cell to allow the fluorescent probe to enter the cell; S2: Under conditions of stimulation by ALP donors at different concentrations or drug-induced injury, fluorescence signals were detected in the near-infrared channel, and the intensity of the fluorescence signal increased with the increase of ALP concentration or degree of cell damage. S3: The fluorescent probe is used to achieve semi-quantitative visualization analysis of intracellular alkaline phosphatase concentration.

[0022] Compared with existing technologies, the probe of this invention, using the above technical solution, can achieve specific activation and near-infrared fluorescence enhancement in the cellular environment. XDM-P has good cell permeability and can smoothly enter the cytoplasm region of the cell. Under cell damage conditions induced by different concentrations of exogenous ALP stimulation or drugs, the intracellular ALP level is upregulated, which promotes the accelerated hydrolysis of the probe's phosphate ester bond and releases highly fluorescent XDM-OH, thereby generating a significant and uniformly enhanced fluorescence signal in the near-infrared channel. The fluorescence imaging process has a high signal-to-noise ratio and low cytotoxicity, enabling semi-quantitative analysis of the dynamic changes of intracellular ALP and distinguishing the differences in ALP levels caused by different degrees of damage. It overcomes the limitations of existing probes, such as insufficient cell permeability, poor anti-interference ability, or inability to reflect the degree of cell damage, and enables the real-time and quantitative imaging capture of minute changes in ALP during cellular pathology.

[0023] This invention provides an application of the fluorescent probe described above in alkaline phosphatase fluorescence imaging in an animal model of liver cancer, the application comprising the following steps: S1: Establish a tumor-bearing mouse model and introduce the fluorescent probe into the mouse body by injection; S2: At different time points after the probe enters the tumor-bearing mouse, the fluorescence signal in the near-infrared region of the tumor site is detected by an in vivo imaging system; S3: Based on the fluorescence signal that increases over time at the tumor site, non-invasive, real-time visual monitoring of liver cancer lesions and high ALP expression within the tumor can be achieved.

[0024] Compared with existing technologies, the above-mentioned application of the present invention, using the above-mentioned technical solution, can perform non-invasive, real-time imaging of ALP activity in tumor tissue in liver cancer-bearing mice. Tumor tissue usually has high ALP expression and higher microenvironment permeability, causing the probe XDM-P to preferentially accumulate in the tumor area after entering the body and be locally catalyzed and hydrolyzed by ALP, generating a strong near-infrared fluorescence signal of 774 nm. As the reaction time increases, the fluorescence in the tumor area continues to accumulate and increases in a time-dependent manner. The above-mentioned application of the present invention has strong imaging signal penetration and low background interference, which can clearly distinguish tumor tissue from normal tissue and achieve high-contrast visualization and localization of the tumor area. It overcomes the problems of limited penetration depth, unclear identification of tumor sites, and inability to reflect changes in enzyme activity of traditional probes in vivo. This allows liver cancer lesions and their biochemical characteristics (high ALP expression) to be accurately and in real-time presented, providing an effective tool for tumor diagnosis and treatment monitoring.

[0025] This invention provides an application of the fluorescent probe described above in alkaline phosphatase fluorescence imaging in a drug-induced liver injury animal model, the application comprising the following steps: S1: Establish a mouse model of drug-induced liver injury by introducing the fluorescent probe into the mouse body via injection; S2: Detecting fluorescence signals in the near-infrared region of the liver using a live in vivo imaging system; S3: Based on the positive correlation between fluorescence signal intensity and inducing drug dosage, the degree of drug-induced liver injury can be assessed and the efficacy of hepatoprotective drugs can be monitored.

[0026] Compared with existing technologies, the above-mentioned technical solution can directly convert the increase in alkaline phosphatase (ALP) activity during drug-induced liver injury into a visual signal of near-infrared fluorescence. Drug-induced hepatocyte injury causes cell membrane rupture or upregulation of enzyme expression, leading to the accumulation or leakage of ALP in liver tissue. This accelerates the hydrolysis of the probe's phosphate ester bond, generating a strongly fluorescent product XDM-OH, which forms significantly enhanced near-infrared fluorescence in the liver region. The fluorescence signal intensity is positively correlated with the degree of injury and can be significantly inhibited by hepatoprotective agents, further demonstrating that fluorescence changes are highly correlated with ALP activity. This enables real-time and quantitative monitoring of the severity of liver injury at the in vivo level. Attached Figure Description

[0027] Figure 1 The 1H NMR spectrum (500 MHz, DMSO-d6) of the intermediate XDM-OH prepared in Example 1 of this invention. Figure 2 The carbon NMR spectrum (126 MHz, DMSO-d6) of the intermediate XDM-OH prepared in Example 1 of this invention. Figure 3High-resolution mass spectrum (ESI-HRMS) of intermediate XDM-OH prepared in Example 1 of this invention. Figure 4 The nuclear magnetic resonance hydrogen spectrum (500 MHz, MeOD-d4) of the probe XDM-P prepared in Example 1 of this invention. Figure 5 The nuclear magnetic resonance carbon spectrum (126 MHz, MeOD-d4) of the probe XDM-P prepared in Example 1 of this invention. Figure 6 The phosphorus NMR spectrum of the probe XDM-P prepared in Example 1 of this invention (202 MHz, MeOD-d4). Figure 7 This is a high-resolution mass spectrum (ESI-HRMS) of the probe XDM-P prepared in Example 1 of the present invention. Figure 8 This is a comparison of the UV-Vis absorption spectra of probe XDM-P, intermediate XDM-OH, and probe XDM-P after reaction with alkaline phosphatase (ALP) in Example 2 of the present invention. Figure 9 The following are fluorescence titration curves and low-concentration linear fitting graphs of the reaction between probe XDM-P and different concentrations of ALP (0–600 U / L) in Example 2 of this invention. Figure 10 The Michaelis-Menten kinetic curve and double reciprocal fitting plot of the reaction between probe XDM-P and ALP in Example 2 of this invention are shown. Figure 11 This is a bar chart showing the selectivity test of probe XDM-P for ALP and various potential biological interference substances in Example 2 of the present invention. Figure 12 The figure shows the cytotoxicity test results of probe XDM-P and intermediate XDM-OH on human liver cancer cells HepG2 (A) and human umbilical vein endothelial cells HUVEC (B) in Example 3 of the present invention. Figure 13 The image shows a confocal fluorescence imaging (A) and a corresponding semi-quantitative fluorescence intensity analysis (B) of the probe XDM-P used in Example 3 of this invention to monitor the endogenous ALP activity of human hepatocellular carcinoma cells HepG2. Figure 14 The image shows a fluorescence image (A) and a corresponding semi-quantitative fluorescence intensity analysis image (B) of the cell model of drug-induced liver injury induced by acetaminophen (APAP) induced by probe XDM-P in Example 3 of the present invention. Figure 15 The images show the in vivo fluorescence imaging (A) of the probe XDM-P in the 4T1 tumor-bearing mouse model in Example 4 of this invention and the statistical graph of the change in fluorescence intensity over time at the tumor site (B). Figure 16 The images show (A) of the in vivo fluorescence imaging of probe XDM-P in a mouse model of drug-induced liver injury and (B) of the statistical analysis of fluorescence intensity in the liver region in Example 4 of this invention. Figure 17 This is the chemical structural formula of the alkaline phosphatase-responsive near-infrared fluorescent probe XDM-P described in this invention. Detailed Implementation

[0028] First, those skilled in the art should understand that these embodiments are merely used to explain the technical principles of the embodiments of this application and are not intended to limit the scope of protection of the embodiments of this application. Those skilled in the art can make adjustments as needed to adapt to specific application scenarios.

[0029] This invention provides an alkaline phosphatase-responsive near-infrared fluorescent probe, the structure of which is as follows: Figure 17 As shown, the fluorescent probe is compound XDM-P, whose molecular structure includes: a donor-π-conjugated-acceptor type near-infrared fluorophore constructed with oxanthracene and cyanoisophorone as the parent nucleus; and a phosphate ester recognition group linked to the phenolic hydroxyl group of the fluorophore via a phosphate ester bond.

[0030] As a preferred embodiment, the fluorescent probe has the structure shown in formula (I): Formula (I).

[0031] The molecular formula of the fluorescent probe described in this invention is C 26 H 25 N3O5P, with a relative molecular mass of 476.46, has a core push-pull electron system in its molecular structure. It contains a hydroxycoumarin derivative as an electron donor (D) and a dicyanoisophorone unit as an electron acceptor (A), which are connected by an ethylene bridge (π-conjugated bridge) to form an efficient intramolecular charge transfer (ICT) mechanism. On the phenolic hydroxyl group of the electron donor (hydroxycoumarin) in this ICT framework, a phosphate group is connected by a phosphate ester bond as a specific recognition and reaction site for ALP. This phosphate group also acts as an effective fluorescence quencher, making the probe initially weakly fluorescent ("OFF" state). Under the action of ALP, the phosphate ester bond undergoes specific hydrolysis, releasing the strongly fluorescent dephosphorylated product XDM-OH (with the structure shown in the intermediate XDM-OH), restoring the ICT effect and realizing an "OFF-ON" fluorescence response. Its maximum emission wavelength is located in the near-infrared region of 774 nm.

[0032] This invention provides a method for preparing the alkaline phosphatase-responsive near-infrared fluorescent probe, comprising the following steps: S1: Using 6-hydroxy-2,3-dihydro-1H-xan-4-carboxaldehyde and dicyanoisophorone compounds as raw materials, a Knauvengel condensation reaction is carried out to obtain the intermediate XDM-OH. S2: Phosphorylation of the phenolic hydroxyl group of intermediate XDM-OH with dimethyl chlorophosphate yields intermediate XDM-PMe2; S3: The intermediate XDM-PMe2 was dealkylated using trimethylbromosilane to obtain the probe XDM-P; The structural formulas of the intermediates XDM-OH and XDM-PMe2 are as follows: .

[0033] As a preferred embodiment, in step S1, the molar ratio of 6-hydroxy-2,3-dihydro-1H-xan-4-carboxaldehyde to dicyanoisophorone compounds is 1:(1±0.2), the reaction solvent is a polar solvent, and the polar solvent includes ethanol, acetonitrile, methanol, tetrahydrofuran, the catalyst is piperidine, and the reaction temperature is 60-80℃.

[0034] As a preferred embodiment, in step S2, the molar ratio of the intermediate XDM-OH to dimethyl chlorophosphate is 1:(3.6±0.5), the phosphorylation reaction is carried out in an anhydrous solvent, and the anhydrous solvent includes anhydrous dichloromethane, anhydrous acetonitrile, and anhydrous trichloromethane, the catalyst is a nucleophilic base, and the nucleophilic base includes triethylamine, 4-dimethylaminopyridine, and 1,8-diazacyclic[5.4.0]undec-7-ene, the reaction temperature is 0°C, and then the mixture is stirred at room temperature.

[0035] As a preferred embodiment, in step S3, the molar ratio of the bromotrimethylsilane to the intermediate XDM-PMe2 is (4.0-6.0):1, the reaction solvent is anhydrous dichloromethane, the reaction is carried out by adding bromotrimethylsilane at 0°C, and then stirring the reaction at room temperature.

[0036] This invention provides an application of the fluorescent probe described above in detecting alkaline phosphatase at different concentrations. When the fluorescent probe reacts with different concentrations of ALP, a concentration-dependent fluorescence enhancement phenomenon occurs: under the action of ALP, the phosphate ester bonds in the probe undergo specific hydrolysis, releasing a strongly fluorescent dephosphorylated product, restoring the intramolecular charge transfer effect, and realizing an "OFF-ON" fluorescence response; and within the ALP concentration range of 0-100 U / L, the fluorescence intensity shows a linear relationship with the ALP concentration.

[0037] This invention provides an application of the fluorescent probe described above in near-infrared fluorescence imaging for detecting intracellular alkaline phosphatase, the application comprising the following steps: S1: Co-incubate the fluorescent probe with the cell to allow the fluorescent probe to enter the cell; S2: Under conditions of stimulation by ALP donors at different concentrations or drug-induced injury, fluorescence signals were detected in the near-infrared channel, and the intensity of the fluorescence signal increased with the increase of ALP concentration or degree of cell damage. S3: The fluorescent probe is used to achieve semi-quantitative visualization analysis of intracellular alkaline phosphatase concentration.

[0038] This invention provides an application of the fluorescent probe described above in alkaline phosphatase fluorescence imaging in an animal model of liver cancer, the application comprising the following steps: S1: Establish a tumor-bearing mouse model and introduce the fluorescent probe into the mouse body by injection; S2: At different time points after the probe enters the tumor-bearing mouse, the fluorescence signal in the near-infrared region of the tumor site is detected by an in vivo imaging system; S3: Based on the fluorescence signal that increases over time at the tumor site, non-invasive, real-time visual monitoring of liver cancer lesions and high ALP expression within the tumor can be achieved.

[0039] This invention provides an application of the fluorescent probe described above in alkaline phosphatase fluorescence imaging in a drug-induced liver injury animal model, the application comprising the following steps: S1: Establish a mouse model of drug-induced liver injury by introducing the fluorescent probe into the mouse body via injection; S2: Detecting fluorescence signals in the near-infrared region of the liver using a live in vivo imaging system; S3: Based on the positive correlation between fluorescence signal intensity and inducing drug dosage, the degree of drug-induced liver injury can be assessed and the efficacy of hepatoprotective drugs can be monitored.

[0040] The following are specific embodiments, incorporating concrete data, preparation methods, and applications, to further elaborate on the technical solution of the present invention: Example 1: This embodiment provides an alkaline phosphatase-responsive near-infrared fluorescent probe and its preparation method. The synthetic route of the preparation method is as follows: ; Wherein: the bold numbers at the bottom of the above structural formula represent the serial numbers of the intermediate items, and for ease of description thereafter, these serial numbers will be used directly to replace the structural formula.

[0041] The synthesis method of XDM-P in this embodiment specifically includes the following steps: S1: Synthesis of intermediate XDM-OH: 6-hydroxy-2,3-dihydro-1H-xan-4-carboxaldehyde (200 mg, 0.88 mmol) and 2-(3,5,5-trimethylcyclohexyl-2-eneyl)malononitrile (164 mg, 0.88 mmol) were dissolved in ethanol (20 mL) and added to a 100 mL round-bottom flask. Piperidine (0.1 mL) was then added, and the mixture was refluxed at 80°C for 16 hours. After the reaction was completed, the crude product was purified by rapid column chromatography (eluent: petroleum ether / ethyl acetate mixture) to give a blue-black solid XDM-OH (292 mg, yield 84%). 1 H NMR (500 MHz, DMSO- d6 ) δ 10.00 (s,1H), 7.63 (d, J = 15.5 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 6.70 (s, 1H), 6.66(d, J = 2.4 Hz, 2H), 6.60 – 6.51 (m, 2H), 2.58 – 2.43 (m, 12H), 1.70 (p, J =6.0 Hz, 2H), 1.03 (s, 6H). 13 C NMR (126 MHz, DMSO- d6 ) δ 169.7, 159.7, 157.6,153.9, 151.0, 133.2, 127.9, 126.7, 125.8, 124.8, 120.9, 115.3, 114.4, 114.2,111.9, 111.8, 102.4, 72.5, 42.8, 38.7, 32.1, 29.3, 27.9, 24.6, 21.0. HR-MS(ESI): C 26 H 24 N₂O₂, [M+H] + m / z calcd for 397.1911, found 397.1901. (As shown in Figure 1-3); S2: Synthesis of intermediate XDM-PMe2: Intermediate XDM-OH (210 mg, 0.53 mmol) was dissolved in anhydrous dichloromethane (20 mL) containing triethylamine (161 mg, 1.59 mmol). After cooling the solution to 0°C, dimethyl chlorophosphate (276 mg, 1.91 mmol) was slowly added dropwise. The resulting mixture was stirred at room temperature for 6 hours, followed by washing with 1 M HCl and saturated brine sequentially. The organic phase was dried over anhydrous sodium sulfate and concentrated to give a purple solid, which was intermediate XDM-PMe2, and could be used directly in the next reaction without further purification. S3: Synthesis of probe XDM-P: The purple solid was dissolved in anhydrous dichloromethane (15 mL), and trimethylbromosilane (5.0 equivalents) was added at 0°C. The reaction mixture was stirred at room temperature for 12 hours, then quenched with methanol and concentrated. Finally, it was purified by preparative high-performance liquid chromatography (C18 column, water / acetonitrile gradient elution) to obtain 199 mg of the blue-black powder target product XDM-P, with a two-step yield of 79%. The structures of the intermediate and the final product were confirmed by 1H NMR spectroscopy (1H NMR spectroscopy). 1 H NMR, carbon spectrum ( 13 C NMR, phosphorus spectrum ( 31 Confirmed by pNMR and high-resolution mass spectrometry (HRMS). 1 H NMR (500MHz, Methanol- d4 ) δ 1 H NMR (500 MHz, Methanol- d 4) δ 7.64 (dd, J = 15.8, 5.3 Hz, 1H), 7.08 (dd, J = 8.3, 2.1 Hz, 1H), 7.01 (s, 1H), 6.90 – 6.84 (m, 1H), 6.68(d, J = 3.0 Hz, 1H), 6.53 – 6.42 (m, 2H), 2.58 – 2.46 (m, 8H), 1.79 (t, J = 6.3Hz, 2H), 1.09 – 1.06 (m, 6H). 13 C NMR (126 MHz, Methanol- d4) δ 169.3, 156.5,153.3, 150.9, 132.1, 129.4, 126.6, 125.7, 122.6, 121.0, 118.4, 115.3, 112.1,106.8, 74.0, 42.6, 38.5, 31.5, 29.4, 26.7, 24.2, 20.6. 31 P NMR (202 MHz, Methanol-) d 4) δ -5.08. HR-MS (ESI): m / z C 26 H 25 N₂O₅P, calcd for [M+H] + 477.1574, found 477.1567. (e.g.) Figure 4-7 (As shown).

[0042] Example 2: This embodiment provides an application of the fluorescent probe XDM-P in detecting different concentrations of alkaline phosphatase (ALP) to verify the spectral properties and reaction kinetics characteristics of the probe: To characterize the photophysical properties of the probe, the probe XDM-P and the intermediate XDM-OH were prepared into a 10 μmol / L (10 M) 20% DMSO / Tris-HCl buffer solution, and UV-Vis absorption and fluorescence spectroscopy were performed. The UV-Vis absorption spectra showed that XDM-P, the solution of XDM-P co-incubated with ALP, and the control compound XDM-OH all exhibited a maximum absorption peak at approximately 580 nm. (e.g.) Figure 8 (As shown).

[0043] The analytical performance of the probe was then quantitatively evaluated using fluorescence titration: within the ALP concentration range of 0 to 600 U / L, the fluorescence intensity of the probe emission spectrum at 774 nm gradually increased with increasing ALP concentration; in the low concentration range of 0–100 U / L, the fluorescence intensity showed a good linear relationship with the ALP concentration (linear equation: y = 34.94938x + 1166.62822, coefficient of determination R0). 2 = 0.99204 (e.g.) Figure 9 (As shown). Based on the calibration curve, the detection limit of the probe for ALP is calculated to be 0.17 U / L (signal-to-noise ratio S / N = 3), indicating that the probe of the present invention has excellent sensitivity, which is superior to existing ALP detection methods.

[0044] To further investigate the interaction between the enzyme and the substrate, the reaction kinetics of the probe were verified: when different alkaline phosphatases (ALPs) were added, the fluorescence intensity of the probe emission spectrum at 774 nm increased linearly with increasing probe concentration (R0). 2 = 0.98863). By calculating the initial reaction rate (V0) at different probe concentrations and fitting the Michaelis equation, the maximum reaction rate (Vmax) was found to be 1.76 μM / min, and the Michaelis constant (Km) was 8.05 μM (e.g., 0.98863). Figure 10 As shown in the figure, this result indicates that the probe XDM-P has good binding affinity and high catalytic efficiency with ALP.

[0045] In a physiological environment, various potential interfering substances can affect the accuracy of probes. This embodiment investigated the XDM-P probe in the presence of various interfering substances to further evaluate its selectivity for ALP. The fluorescence intensity of XDM-P did not change significantly in the presence of various potential interfering substances (including biologically related enzymes, active species, metal ions, thiols, and amino acids). Figure 11 As shown in the figure, this further confirms that the probe has excellent anti-interference ability when detecting ALP.

[0046] Example 3: This embodiment provides an application of the fluorescent probe XDM-P in ratiometric fluorescence imaging for detecting intracellular alkaline phosphatase, to verify the probe's response performance and biosafety at the live cell level: Cytotoxicity assay: Before application in bioimaging, the cytotoxicity of XDM-P and XDM-OH against human hepatocellular carcinoma cells (HepG2, cancer cells) and human umbilical vein endothelial cells (HUVECs, normal cells) was evaluated using the CCK-8 assay. Different concentrations (0-100 μM) of the compound were co-incubated with HepG2 and HUVECs for 24 hours, respectively, and the survival rate of both cell lines remained above 90%. This indicates that the probes have good biocompatibility, low toxicity, and are suitable for biological applications at the live cell level (e.g., Figure 12 (As shown).

[0047] Imaging of endogenous ALP in liver cancer cells: HepG2 cells with high ALP expression were selected as a model. The probe XDM-P (10 μM) was co-incubated with the cells, and changes in intracellular fluorescence signals were monitored using laser confocal microscopy. At the initial time (0 min), only weak background fluorescence was observed in the cells; with prolonged incubation, the red fluorescence signal in the cytoplasm increased in a time-dependent manner, reaching a relatively strong level at approximately 2 hours. This indicates that the probe can effectively penetrate the cell membrane and is specifically hydrolyzed and activated by the highly expressed endogenous ALP, releasing a strong fluorescence signal (e.g., ...). Figure 13 (As shown).

[0048] Imaging of a Drug-Induced Liver Injury (DILI) Cell Model: An in vitro drug-induced liver injury model was established using acetaminophen (APAP)-induced HepG2 cells. Cells were exposed to different concentrations (0.1 mM and 0.2 mM) of APAP for 12 hours, followed by imaging with the probe XDM-P. Compared with the untreated control group, the fluorescence intensity in the APAP-injured group was significantly enhanced, and the enhancement was positively correlated with the APAP concentration, suggesting that drug injury led to abnormal upregulation of intracellular ALP activity. Furthermore, pretreatment of cells with the hepatoprotective drug N-acetylcysteine ​​(NAC) significantly inhibited the APAP-induced fluorescence enhancement (e.g., ...). Figure 14 (As shown in the figure). This result demonstrates that the probe XDM-P can be used to assess drug hepatotoxicity and screen hepatoprotective drugs at the cellular level.

[0049] Example 4: This embodiment provides an application of the fluorescent probe XDM-P in alkaline phosphatase ratio fluorescence imaging in animal models of diseases, to verify the dynamic response performance and specific imaging effect of the probe in live animals, specifically including the application of the fluorescent probe in alkaline phosphatase fluorescence imaging in animal models of liver cancer and in alkaline phosphatase fluorescence imaging in animal models of drug-induced liver injury. In vivo imaging of tumor-bearing mice: A 4T1 tumor-bearing mouse model was established. The XDM-P probe was injected via the tail vein, and near-infrared fluorescence images were acquired at different time points using a small animal in vivo imaging system. The results are as follows: Figure 15 As shown, after probe injection, the fluorescence signal at the tumor site gradually increased over time. The tumor outline could be clearly distinguished 30 minutes after injection, and the signal continued to accumulate during the 90-minute monitoring period. This gradual fluorescence enhancement indicates that the probe can accumulate in tumor tissue through the EPR effect or active uptake, and be activated in situ by the highly expressed ALP within the tumor, thereby releasing the fluorescent product XDM-OH, achieving high-contrast visual monitoring of tumor lesions. Imaging of a mouse model of drug-induced liver injury (DILI): Acute liver injury mouse models induced by different doses of APAP (0-300 mg / kg) were established. Abdominal imaging was performed after tail vein injection of probe XDM-P. The results are as follows: Figure 16As shown, compared with the saline control group, the near-infrared fluorescence signal in the liver region of mice treated with APAP was significantly enhanced; the fluorescence intensity showed a significant positive correlation with the APAP dosage, with the highest signal in the high-dose group (300 mg / kg). In mice that were pre-injected with the antidote NAC before APAP administration, the fluorescence enhancement in the liver region was effectively suppressed. This further confirms that the probe XDM-P can achieve real-time, non-invasive monitoring of ALP activity changes during drug-induced liver injury in vivo, and can be used to assess the degree of liver injury and drug efficacy.

[0050] In summary, the probe XDM-P prepared in this invention can achieve non-invasive, real-time, concentration-dependent fluorescence imaging of alkaline phosphatase in animal disease models, dynamically reflecting the distribution and changes of ALP in vivo, providing a powerful tool for early disease diagnosis and in vivo research on pathological processes such as tumors.

[0051] This invention, based on an innovative design strategy of "phosphodiester hydrolysis-intramolecular charge transfer (ICT) regulation," successfully constructed a novel alkaline phosphatase (ALP)-responsive near-infrared fluorescent probe, XDM-P. This probe cleverly utilizes an oxanthracene-cyanoisophorone conjugated system to construct a D–π–A type near-infrared fluorescent core, and introduces an ALP-specific recognition site through phosphate ester bonds. Under the catalysis of ALP, the phosphate ester bonds in the probe molecule undergo specific hydrolysis, releasing the strong electron-donating phenolic hydroxyl group, thereby restoring the ICT effect and inducing a sensitive switching of the fluorescence signal from an "OFF" to an "ON" state.

[0052] This probe possesses excellent photophysical properties, with an emission wavelength in the near-infrared region of 774 nm and a large Stokes shift of up to 194 nm. This significantly reduces autofluorescence interference from biological tissues, endowing it with excellent deep tissue penetration and high signal-to-noise ratio imaging performance. In vitro experimental data confirm that XDM-P exhibits a good linear response relationship (R0) in the ALP concentration range of 0–100 U / L. 2 =0.992); kinetic parameters showed a Michaelis constant (Km) of 8.05 μM and a maximum reaction rate (Vmax) of 1.76 μM / min, indicating that the probe has high affinity for ALP and high catalytic conversion efficiency. Furthermore, the probe showed no significant response to common biological interferences such as reactive oxygen species, metal ions, and bio-thiols, exhibiting excellent selectivity.

[0053] This invention validates the probe's practical performance through a multi-level model system from cells to living organisms. In HepG2 liver cancer cells and APAP-induced liver injury cell models, the probe successfully achieved real-time imaging of ALP activity, with fluorescence intensity positively correlated with the degree of cell damage, and this effect could be reversed by the hepatoprotective agent NAC. Furthermore, in liver cancer-bearing mouse models and drug-induced liver injury mouse models, after tail vein injection of the probe, both the tumor and liver lesion areas showed significant time-dependent fluorescence enhancement, achieving high-contrast in vivo visualization monitoring. Therefore, the probe XDM-P of this invention achieves significant improvements in overall performance in terms of response rate, specificity, detection sensitivity, and in vivo applicability. It can not only non-invasively, in real-time, and dynamically reflect the distribution and changes of ALP in vivo, but also provides a powerful molecular tool for the early diagnosis, pathological mechanism research, and drug screening of diseases such as liver cancer and drug-induced liver injury, possessing broad scientific research value and clinical translation prospects.

[0054] In the description of this application, the references to terms such as "an embodiment," "some embodiments," "in this embodiment," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0055] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A basic phosphatase-responsive near-infrared fluorescent probe, characterized by, The fluorescent probe is compound XDM-P, and the molecular structure of the compound comprises: a donor-π-conjugated-acceptor type near-infrared fluorophore constructed by using xanthene and cyanoisophorone as a mother nucleus; and a phosphate recognition group connected to a phenolic hydroxyl group of the fluorophore through a phosphate ester bond.

2. The alkaline phosphatase-responsive near-infrared fluorescent probe according to claim 1, wherein The fluorescent probe has a structure shown in formula (I): Formula (I).

3. A method for preparing a near-infrared fluorescent probe responsive to alkaline phosphatase according to claim 1 or 2, characterized by, The method comprises the following steps: S1: a Claisen-Schmidt condensation reaction is performed on 6-hydroxy-2,3-dihydro-1H-furan-4-carboxaldehyde and a dicyanoisophorone compound to obtain an intermediate XDM-OH; S2: dimethyl chlorophosphate is used for phosphate esterification on a phenolic hydroxyl group of the intermediate XDM-OH to obtain an intermediate XDM-PMe2; S3: bromotrimethylsilane is used for dealkylation on the intermediate XDM-PMe2 to obtain the probe XDM-P. The intermediate XDM-OH and the intermediate XDM-PMe2 have the following structural formulas, respectively. 。 4. The production method according to claim 3, characterized by, In the step S1, the molar ratio of the 6-hydroxy-2,3-dihydro-1H-furan-4-carboxaldehyde to the dicyanoisophorone compound is 1: (1±0.2), the reaction solvent is a polar solvent, and the polar solvent comprises ethanol, acetonitrile, methanol and tetrahydrofuran; the catalyst is piperidine; and the reaction temperature is 60-80 DEG C.

5. The preparation method according to claim 3, characterized in that, In the step S2, the molar ratio of the intermediate XDM-OH to dimethyl chlorophosphate is 1: (3.6±0.5), the phosphate esterification is performed in anhydrous solvent, and the anhydrous solvent comprises anhydrous dichloromethane, anhydrous acetonitrile and anhydrous chloroform; the catalyst is a nucleophilic base, and the nucleophilic base comprises triethylamine, 4-dimethylamino pyridine and 1,8-diazabicyclo[5.4.0]undec-7-ene; the reaction temperature is 0 DEG C, and then stirring is performed at room temperature.

6. The preparation method according to claim 3, characterized in that, In the step S3, the molar ratio of the bromotrimethylsilane to the intermediate XDM-PMe2 is (4.0-6.0):1, the reaction solvent is anhydrous dichloromethane, the bromotrimethylsilane is added at 0 DEG C, and then stirring is performed at room temperature.

7. Use of the fluorescent probe according to claim 1 or 2 for detecting alkaline phosphatase in different concentrations. When the fluorescent probe is reacted with different concentrations of ALP, a concentration-dependent fluorescence enhancement phenomenon occurs: under the action of ALP, the phosphate bond in the probe is specifically hydrolyzed to release a dephosphorylated product with strong fluorescence, the intramolecular charge transfer effect is restored, and an opening type response of fluorescence "OFF-ON" is realized; and in the range of 0-100 U / L of the ALP concentration, the fluorescence intensity and the ALP concentration present a linear relationship.

8. Use of the fluorescent probe according to claim 1 or 2 in near-infrared fluorescence imaging for detecting alkaline phosphatase in cells. The application The method comprises the following steps: S1: the fluorescent probe is co-incubated with cells to make the fluorescent probe enter the inside of the cells; S2: under the stimulation of different concentrations of ALP donors or the induction of damage by drugs, a near-infrared channel is used to detect a fluorescence signal, and the fluorescence signal intensity is enhanced with the increase of the ALP concentration or the degree of cell damage; S3: the fluorescent probe is used to realize semi-quantitative visual analysis of the concentration of alkaline phosphatase in cells.

9. The application of the fluorescent probe of claim 1 or 2 in the fluorescent imaging of alkaline phosphatase in the animal model of liver cancer disease, characterized in that, The application The method comprises the following steps: S1: a tumor-bearing mouse model is established, and the fluorescent probe is introduced into the mouse body by injection; S2: At different time points after the probe enters the tumor-bearing mice, the fluorescence signal in the near-infrared region of the tumor site is detected by a live imaging system; S3: According to the enhanced fluorescence signal of the tumor site over time, non-invasive and real-time visualization monitoring of liver cancer lesions and intratumoral high expression of ALP is achieved.

10. The use of the fluorescent probe of claim 1 or 2 in the fluorescence imaging of alkaline phosphatase in an animal model of drug-induced liver injury. The application Comprising the following steps: S1: Establish a drug-induced liver injury mouse model, and introduce the fluorescent probe into the mouse body by injection; S2: Detect the fluorescence signal in the near-infrared region of the liver area by a live imaging system; S3: According to the positive correlation between the fluorescence signal intensity and the dose of the inducing drug, the degree of drug-induced liver injury is evaluated and the efficacy of liver-protecting drugs is monitored.

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