Xanthene nitroreductase-responsive fluorescent dye compound as well as preparation method and application thereof

Abstract

The invention discloses a xanthene nitroreductase-responsive fluorescent dye compound as well as a preparation method and application thereof, and belongs to the technical field of xanthene derivatives. The fluorescent dye compound can be used for quickly and specifically recognizing overexpressed nitroreductase in tumor cells; after the fluorescent probe acts with nitroreductase, the fluorescence intensity of the fluorescent probe at 718 nm is obviously enhanced, and the fluorescent probe has excellent targeting capability on mitochondria; meanwhile, under the excitation of illumination with the specific wavelength of 660 nm, an acting product of the fluorescent dye compound and nitroreductase can efficiently generate reactive oxygen species (ROS), and then the effect of specifically killing tumor cells is achieved. The xanthene nitroreductase response type fluorescent dye compound provided by the invention can achieve the goal of integration of diagnosis and treatment of solid tumors, and has important clinical application value.

Description

Fluorescent dye compounds responsive to xanthones and their preparation methods and applications Technical Field

[0001] This invention relates to the field of xanthan derivatives. More specifically, this invention relates to a xanthan nitroreductase-responsive fluorescent dye compound, its preparation method, and its applications. Background Technology

[0002] The hypoxic tumor microenvironment is a hallmark feature of solid tumors, and its formation is closely related to the oxygen supply-demand imbalance caused by the rapid proliferation of tumor cells. This microenvironment not only accelerates tumor invasion and metastasis but also significantly reduces the sensitivity of tumor cells to radiotherapy and chemotherapy, thus severely limiting the clinical treatment efficacy of tumors. Nitroreductase (NTR), as a specific marker of hypoxia, is highly expressed in hypoxic tumor cells but extremely poorly expressed in normal aerobic cells, making it a core target for targeted detection and treatment of hypoxic tumor cells.

[0003] Currently, NTR-responsive fluorescent probes have been applied in the field of tumor hypoxia detection, but existing technologies still have many significant shortcomings: First, the response rate is slow and the detection limit is relatively high, making it difficult to meet the actual needs of rapid detection and real-time imaging of trace NTRs; second, the targeting performance is poor, with most probes lacking precise subcellular localization capabilities and failing to efficiently accumulate in key organelles of tumor cells such as mitochondria, cell membranes, and endoplasmic reticulum; third, the water solubility is poor, and the fluorescence absorption and emission wavelengths are short, making it difficult to adapt to the application requirements of in vivo detection; fourth, the functions are relatively limited, with most existing probes only having hypoxia detection functions, unable to simultaneously detect and treat hypoxic sites in tumors, making it difficult to meet the trend of integrated diagnosis and treatment in clinical applications.

[0004] Meanwhile, photodynamic therapy, as a minimally invasive tumor treatment technique, works by using photosensitizers to generate reactive oxygen species (ROS) under specific light conditions to kill tumor cells. However, traditional photosensitizers have significant drawbacks: their treatment efficiency drops drastically in hypoxic environments, and they are prone to systemic toxicity due to insufficient targeting.

[0005] In summary, developing novel probes that combine mitochondrial targeting, highly sensitive NTR response, and multifunctional integration is of paramount importance for overcoming existing technological bottlenecks and improving the level of precision diagnosis and treatment of tumors. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0007] Another objective of this invention is to provide a xanthanid nitroreductase-responsive fluorescent dye compound. This fluorescent dye compound uses xanthanid derivatives as its core framework and incorporates mitochondrial targeting units and nitroreductase-responsive units to construct a fluorescent probe that combines targeted recognition and enzyme response functions. This fluorescent probe can specifically locate in cellular mitochondria and, under the action of nitroreductase overexpressed in tumor cells, can rapidly induce specific changes in fluorescence signals. After being taken up by tumor cells, it achieves efficient fluorescence imaging mediated by nitroreductase in hypoxic tumor cells, while also exerting a photodynamic killing effect on tumor cells, thereby enabling real-time monitoring of the diagnosis and treatment process of tumor cell hypoxia.

[0008] To achieve these objectives and other advantages according to the present invention, a thallium-based nitro reductase-responsive fluorescent dye compound is provided, having the structure of formula (I): (I); where R1 is an N-alkylated nitrogen heterocyclic structural unit and R2 is a benzene ring structural unit containing a substituent.

[0009] Preferably, R1 has the structure of the following formula (II): (II); where R3 is .

[0010] Preferably, R2 is .

[0011] The objective of this invention can also be further achieved by a method for preparing xanthones-type nitroreductase-responsive fluorescent dye compounds, comprising the following steps: S1, reacting 5-iodo-2,3,3-trimethyl-3H-indole with 1,2-oxothiacyclopentane 2,2-dioxide to prepare compound (III); S2, preparing compound (IV) by formylation reaction with cyclohexanone as a raw material, wherein compound (IV) is further reacted with 2-hydroxy-4-methoxybenzaldehyde, boron tribromide, and 1-(bromomethyl)-4-nitro-2-(trifluoromethyl)benzene to prepare compound (V); S3, condensing compound (III) and compound (V) to obtain xanthones-type nitroreductase-responsive fluorescent dye compounds; (III) (IV) (V).

[0012] Preferably, in step S1, the molar ratio of 5-iodo-2,3,3-trimethyl-3H-indole to 1,2-oxothiacyclopentane 2,2-dioxide is 1:1 to 2, and the reaction conditions are: under argon protection, toluene as solvent, and reflux for 10 to 15 h with stirring.

[0013] Preferably, in step S2, the preparation of compound (IV) by formylation of cyclohexanone as a raw material specifically includes: dissolving anhydrous N,N-dimethylformamide in dichloromethane to obtain a mixed solution, adding PBr3 dropwise to the mixed solution at 0 °C, stirring for 0.5–1 h, adding cyclohexanone to the reaction solution, continuing stirring for 15–20 h, after the reaction is completed, pouring the reaction mixture into ice water, adjusting the pH to neutral with sodium carbonate, extracting with dichloromethane, combining the organic phases, evaporating the solvent, and obtaining compound (IV).

[0014] Preferably, in step S2, the molar ratio of compound (IV) to 2-hydroxy-4-methoxybenzaldehyde is 1 to 2:1, and the reaction conditions are stirring at room temperature for 15 to 20 h.

[0015] Preferably, in step S3, the molar ratio of compound (III) to compound (V) is 1:1, the solvent for the condensation reaction is ethanol, and the reaction conditions are heating at 80-90°C for 10-15 h.

[0016] The objective of this invention can also be further achieved by the application of xanthanase-responsive fluorescent dye compounds in the preparation of nitroreductase-responsive detection probes.

[0017] The objective of this invention can also be further achieved through the application of xanthannae nitroreductase-responsive fluorescent dye compounds in the preparation of photosensitizers for photodynamic diagnosis and treatment of tumors.

[0018] The present invention has at least the following beneficial effects: 1. The xanthanid nitroreductase-responsive fluorescent dye compound of the present invention can rapidly respond to the overexpressed nitroreductase in tumor cells by targeting the core biological characteristics of the hypoxic tumor microenvironment. It can rapidly trigger changes in fluorescence signal through specific enzymatic reactions, thereby achieving rapid and accurate identification of the hypoxic tumor microenvironment. Moreover, the response time is short (currently reported response times are generally over 30 min, while the response time of this compound is 7 min) and the signal discrimination is high, which can timely capture the dynamic changes of tumor hypoxia.

[0019] 2. The fluorescent dye compound of the present invention, which is responsive to nitroreductase, has excellent bioselectivity and exhibits a high degree of specificity in recognizing nitroreductase overexpressed in tumor cells. It can effectively avoid interference from low-level enzyme activity in normal tissue cells and can still accurately target hypoxic tumor cells in complex biological systems. It has strong anti-interference ability and ensures the specificity and accuracy of the diagnosis and treatment process.

[0020] 3. The mitochondrial targeting unit (R1 unit is an N-alkylated nitrogen heterocyclic structural unit with a positive charge, which can efficiently pass through the negatively charged mitochondrial membrane to achieve the purpose of targeting mitochondria) introduced into the xanthanid nitroreductase-responsive fluorescent dye compound of the present invention can specifically locate and effectively enrich the mitochondria of tumor cells, so that the action site of photodynamic therapy is precisely focused on the key organelles of tumor cells, significantly improving the targeting accuracy of photodynamic therapy, while greatly reducing damage to normal tissue cells, and further optimizing the balance between therapeutic effect and safety.

[0021] 4. The method of preparing xanthanna-type nitro reductase-responsive fluorescent dye compounds of the present invention has the advantages of low synthesis cost, simple synthesis steps, and low toxicity of the synthesis process.

[0022] 5. The xanthanna nitroreductase-responsive fluorescent dye compound of this invention innovatively integrates hypoxia diagnosis, fluorescence imaging and photodynamic therapy into a single molecular system. It does not require the use of other reagents and can achieve accurate identification, real-time imaging monitoring and targeted killing of hypoxic tumors through the same probe. It truly realizes an advanced diagnosis-treatment integrated model, which not only simplifies the clinical application process, but also improves the efficiency of diagnosis and treatment and optimizes the treatment plan, demonstrating unique technological innovation value.

[0023] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0024] Figure 1 shows the 1H NMR spectrum of the xanthium-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 prepared in Example 1 of this invention; Figure 2 shows the 1C NMR spectrum of the xanthium-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 prepared in Example 1 of this invention; Figure 3 shows the high-resolution mass spectrum of the xanthium-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 prepared in Example 1 of this invention; Figure 4 shows the interaction mechanism of the xanthium-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 prepared in Example 1 of this invention with nitroreductase; Figure 5 shows the UV-Vis absorption spectra of the xanthium-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 (5 μM) prepared in Example 1 of this invention with different concentrations of nitroreductase (0~5 μg / mL); Figure 6 shows the UV-Vis absorption spectra of the xanthium-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 (5 μM) prepared in Example 1 of this invention. Figure 7 shows the fluorescence emission spectrum at 718 nm of the xanthium-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 (5 μM) prepared in Example 1 of this invention reacting with different concentrations of nitroreductase; Figure 8 shows the reaction of the xanthium-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 (5 μM) prepared in Example 1 of this invention with Na... + K + Mg 2+ ,Hcy,GSH,Cys,Lys,Ser,Arg,AcO - ,ClO - ClO4 - Cl - SO3 2- HSO3 - Fluorescence response diagrams of different interfering substances, including vitamin C, esterase, NADH, and NTR+NADH. The concentrations of NTR (5 μg / mL), NADH (500 μg / mL), Hcy (10 mM), GSH (10 mM), and Cys (10 mM) were all 100 μM, while the concentrations of other analytes were all 100 μM. Figure 9 shows the fluorescence response of the xanthanid nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 (5 μM) prepared in Example 1 of this invention in the detection of singlet oxygen (…) using DPBF. 1 Experimental UV absorption spectrum of O2. The values ​​of DPBF (40 μM), NTR (5 μg / mL), NADH (500 μg / mL), and photoexcitation parameters (660 nm, 50 mW / cm²) are shown. 2Figure 10 shows the fluorescence emission of the xanthanid nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 (5 μM) prepared in Example 1 of this invention at 525 nm using a DHR123 probe to detect superoxide anion radicals. The parameters include DHR123 (5 μM), NTR (5 μg / mL), NADH (500 μg / mL), and photoexcitation parameters (660 nm, 50 mW / cm²). 2 Figure 11 shows the imaging effect of the xanthium-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 (5 μM) prepared in Example 1 of this invention in HeLa cells under different oxygen concentrations; Figure 12 shows the imaging effect of the xanthium-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 (5 μM) prepared in Example 1 of this invention in HeLa cells under 21% and 1% oxygen concentrations over time; Figure 13 shows the mitochondrial targeting experiment of the xanthium-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 (5 μM) prepared in Example 1 of this invention in HeLa cells; Figure 14 shows the fluorescence imaging effect of the xanthium-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 (5 μM) prepared in Example 1 of this invention in HeLa cells after different treatments, using the DA-DCFH probe to detect reactive oxygen species (ROS). The DA-DCFH (10 μM) and photoexcitation parameters (660 nm, 50 mW / cm²) are also shown. 2 Figure 15 shows the cytotoxicity of the xanthones-based nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 (5 μM) prepared in Example 1 of this invention at different concentrations under dark and light conditions. The photoexcitation parameters are shown in Figure 15 (660 nm, 50 mW / cm²). 2 Figure 16 shows the fluorescence imaging of HeLa cells double-stained with Calcein-AM / PI after different treatments of the xanthones-like nitroreductase-responsive fluorescent dye compound IX-NO2-CF3 (5 μM) prepared in Example 1 of this invention. The photoexcitation parameters of Calcein-AM (2 μL) and PI (2 μL) are 660 nm, 50 mW / cm². 2 ); Detailed Implementation

[0025] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0026] It should be understood that terms such as "having," "comprising," and "including" used in the embodiments of this application do not exclude the presence or addition of one or more other elements or combinations thereof. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are only used to explain the relative positional relationship and movement of components in a specific posture. If the specific posture changes, the directional indication will also change accordingly. When an element is referred to as "fixed to" or "set on" another element, it can be directly on the other element or may have an intervening element present. When an element is referred to as "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element through an intervening element. Descriptions involving "first," "second," etc., in the embodiments of this application are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features.

[0027] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0028] It should be noted that the technical solutions of the various embodiments of this application can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this application.

[0029] Main equipment: UV-Vis spectrophotometer (Edinburgh FLS1000, UK), fluorescence spectrophotometer (Shimadzu U-3900, Japan), confocal fluorescence microscope (Nikon A1R, Japan); Main chemical reagents: 5-iodo-2,3,3-trimethyl-3H-indole (AR, Aladdin, Shanghai, China), 1,2-oxothiacyclopentane 2,2-dioxide (AR, Aladdin, Shanghai, China), toluene (AR, Aladdin, Shanghai, China), N,N-dimethylformamide (AR, Aladdin) (AR, Aladdin, Shanghai, China), dichloromethane (AR, Tongguang, Beijing, China), boron tribromide (AR, Aladdin, Shanghai, China), cyclohexanone (AR, Aladdin, Shanghai, China), 2-hydroxy-4-methoxybenzaldehyde (AR, Aladdin, Shanghai, China), CsCO3 (AR, Aladdin, Shanghai, China), 1-(bromomethyl)-4-nitro-2-(trifluoromethyl)benzene (AR, Aladdin, Shanghai, China), piperidine (AR, Aladdin, Shanghai, China), NTR (Sigma-Aldrich, USA).

[0030] Example 1: A xanthanid nitroreductase-responsive fluorescent dye compound (IX-NO2-CF3) has the following structure: The specific synthesis route is as follows: The specific synthetic steps are as follows: S1, Under argon protection, 5-iodo-2,3,3-trimethyl-3H-indole (0.57 g, 2 mmol) and 1,2-oxothiacyclopentane 2,2-dioxide (0.31 g, 2.6 mmol) were dissolved in 10 mL of toluene and refluxed with stirring for 12 hours. After the reaction was completed, the reaction solution was filtered and dried to obtain red solid compound 1 (0.74 g, yield: 90.5%). 1 H NMR (600MHz, DMSO-d6) δ 8.18 (s, 1H), 7.92 (d,J= 8.4 Hz, 1H), 7.74 (d,J= 8.4 Hz, 1H), 4.51 (t,J= 7.9 Hz, 2H), 2.72 (s, 3H), 2.63 (t,J= 6.7 Hz, 2H), 2.13–2.02 (m,2H), 1.46 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 197.22, 144.70, 141.64, 138.14,132.93, 117.93, 96.50, 54.79, 47.89, 47.23, 24.17, 22.37, 14.40.ESI-MS: Calcdfor C 14 H 19 INO3S [M + H] +408.01, found 408.03.S2. Anhydrous N,N-dimethylformamide (DMF, 1.46 g, 20 mmol) was dissolved in 20 mL of dichloromethane. PBr3 (4.88 g, 18 mmol) was added dropwise to the mixture at 0 °C, and the mixture was stirred for 0.5 hours. Cyclohexanone (0.78 g, 8 mmol) was added to the reaction mixture, and the mixture was stirred for another 18 hours. After the reaction was complete, the reaction mixture was poured into ice water, the pH was adjusted to neutral with sodium carbonate, and the mixture was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, and the solvent was evaporated to give a pale yellow oily compound 2 (1.04 g, yield: 68.7%). Compound 2 (0.9 g, 4.8 mmol), 2-hydroxy-4-methoxybenzaldehyde (0.61 g, 4 mmol), and CsCO3 (2.31 g, 12 mol) were dissolved in 50 mL of anhydrous DMF and stirred at room temperature for 18 hours. After the reaction was complete, the mixture was filtered and extracted with dichloromethane (3 × 50 mL). The organic phases were combined, the solvent was evaporated, and the mixture was purified by column chromatography (petroleum ether / ethyl acetate, V / V = 3 / 1) to give a yellow solid compound 3 (0.52 g, yield: 53.6%). 1 H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 7.29 (d,J= 8.5 Hz, 1H), 6.96 (s, 1H), 6.88 (d,J= 2.4 Hz, 1H), 6.76 (dd,J= 8.5, 2.5Hz, 1H), 3.81 (s, 3H), 2.62–2.52 (t,J= 5.8 Hz, 2H), 2.29 (t,J= 6.0 Hz, 2H), 1.68–1.57 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 186.78, 161.60, 160.64,153.23,128.46,127.69, 126.27, 114.75, 112.18, 111.51, 101.01, 56.19, 29.49, 21.71,20.45. ESI-MS: Calcd for C 15 H 15 O3[M + H] +243.10, found 243.10. Under argon protection, compound 3 (0.48 g, 2 mmol) was dissolved in 10 mL of dichloromethane. BBr3 (0.77 mL, 8 mmol) was added dropwise to the reaction solution at 0 °C, and the mixture was stirred for 1 hour. After the reaction was completed, the mixture was quenched with water and extracted with dichloromethane (3 × 50 mL). The organic phases were combined, the solvent was evaporated, and the mixture was purified by silica gel column chromatography (dichloromethane / methanol, V / V = 20 / 1) to give a yellow solid compound 4 (0.21 g, yield: 45.8%). 1 H NMR (600 MHz, DMSO-d6) δ 10.15 (d,J= 4.4Hz, 1H), 7.15 (dd,J= 8.4, 1.9 Hz, 1H), 6.88 (s, 1H), 6.58 (d,J= 2.2 Hz, 1H),6.57 – 6.54(dd,J= 2Hz, 1H), 2.54–2.43 (t,J= 6.0 Hz, 2H), 2.24 (t,J= 6.0 Hz,1H), 1.57 (m,J= 6.1 Hz, 1H). 13 C NMR (151 MHz, DMSO-d6) δ 186.59, 160.89,160.40, 153.26, 128.62, 128.22, 125.14, 113.51, 112.61, 111.77, 102.42,29.48, 21.76, 20.53. ESI-MS: Calcd for C 14 H 13 O3[M + H] + 229.09, found 229.09. Under argon protection, 1-(bromomethyl)-4-nitro-2-(trifluoromethyl)benzene (0.39 g, 1.8 mmol), compound 4 (0.2 g, 0.9 mol), and K2CO3 (0.19 g, 1.35 mmol) were added to a round-bottom flask containing 10 mL of acetonitrile and heated at 55 °C for 6 h. After the reaction was complete, the mixture was extracted with dichloromethane (3 × 50 mL), the organic phases were combined, the solvent was evaporated, and the mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate, V / V = 10 / 1) to give a yellow solid compound 5 (0.14 g, yield: 43.9%). 1HNMR (600 MHz, DMSO-d6) δ 10.19 (s, 1H), 8.16 (t,J= 8.1 Hz, 1H), 7.62 (d,J=12.0 Hz, 1H), 7.47 (d,J= 8.4 Hz, 1H), 7.30 (d,J= 8.4 Hz, 1H), 7.02 – 6.90 (m,2H), 6.84 (dd,J= 8.5, 2.3 Hz, 1H), 2.51 (d,J= 6.1 Hz, 2H), 2.25 (t,J= 6.1 Hz,2H), 1.58 (t,J= 6.2 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 186.88, 160.55,159.94, 153.15, 147.20, 143.53, 133.36, 128.68, 127.54, 127.19,127.16,126.86, 126.44, 121.73, 115.44, 112.45, 112.08, 102.19, 68.37, 29.51, 21.71,20.43.ESI-MS: Calcd for C 22 H 18 F3NO5[M + H] + 432.11, found 432.11.S3. Compound 5 (0.11 g, 0.3 mmol) and compound 1 (0.12 g, 0.3 mol) were dissolved in 10 mL of ethanol. After adding 10 μL of piperidine, the mixture was heated at 85 °C for 12 hours. After the reaction was completed, the solvent was evaporated, and the mixture was purified by silica gel column chromatography (dichloromethane / methanol, V / V = 10 / 1) to give the blue-green compound IX-NO2-CF3 (0.13 g, yield: 54.6%). 1H NMR (600 MHz, DMSO-d6) δ 8.52 (d,J= 14.8 Hz, 1H), 8.22 (d,J= 8.4 Hz, 1H), 8.15 (s, 1H), 8.09 (d,J= 1.7 Hz, 1H), 8.07 (d,J= 8.3 Hz, 1H), 7.84 (dd,J=8.4, 1.7 Hz, 1H), 7.55 (t,J= 9.0 Hz, 2H), 7.49 (s, 1H), 7.24 (d,J= 2.3 Hz,1H), 7.13 – 7.05 (m, 1H), 6.71 (d,J= 14.9 Hz, 1H), 5.48 (s, 2H), 4.50 (t,J=8.0 Hz, 2H), 2.68 (s, 4H), 2.56 (t,J= 6.7 Hz, 2H), 2.10 – 1.96 (m, 2H), 1.78(t,J= 6.2 Hz, 2H), 1.70 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 177.33, 161.36,161.25, 154.38, 147.30, 145.74, 144.85, 143.26, 141.95, 138.04, 133.81,133.65, 132.57, 131.96, 129.59, 128.11, 127.42,126.46, 126.02, 124.00,116.61, 115.78, 115.54, 114.04, 105.41, 102.70, 92.76, 68.80, 50.95, 48.04,44.56, 29.07, 27.73, 24.35, 20.52.HRMS: Calcd for C 36 H 33 F3IN2O7S [M + H] + 821.1000, found 821.1000. Among them, the proton spectrum of compound IX-NO2-CF3 is shown in Figure 1, the carbon spectrum is shown in Figure 2, and the high-resolution mass spectrum is shown in Figure 3.

[0031] In Example 2, a 10 mM PBS buffer solution with pH 7.4 was prepared. As the NTR concentration increased from 0 to 5 μg / mL (NADH 500 μg / mL), the UV absorption of compound IX-NO2-CF3 (5 μM) at 690 nm gradually increased. The UV-Vis absorption spectrum is shown in Figure 5.

[0032] Example 3: A 10 mM PBS buffer solution at pH 7.4 was prepared. Compound IX-NO2-CF3 (5 μM) showed almost no fluorescence at 718 nm. After adding 0–5 μg / mL NTR (containing 500 μg / mL NADH), its fluorescence at 718 nm significantly increased by a factor of 77.69. Kinetic experiments showed that compound IX-NO2-CF3 reached equilibrium in 7 min after the addition of 5 μg / mL NTR (containing 500 μg / mL NADH). The fluorescence emission spectrum is shown in Figure 6, and the kinetic response is shown in Figure 7.

[0033] Example 4: Preparation of 10 mM PBS buffer solution, pH 7.4, and 5 μM MIX-NO2-CF3 for Na+. + K + Mg 2+ ,Hcy,GSH,Cys,Lys,Ser,Arg,AcO - ,ClO - ClO4 - Cl - SO3 2- HSO3 - The fluorescence intensity of the detection system was increased by various interfering agents, including vitamin C, esterase, NADH, and NTR+NADH. The concentrations of NTR (5 μg / mL), NADH (500 μg / mL), Hcy (10 mM), GSH (10 mM), and Cys (10 mM) were used, while the concentrations of other analytes were all 100 μM. The results are shown in Figure 8. Figure 8 shows that only the fluorescence intensity at 718 nm was significantly enhanced when interacting with NTR and NADH. This indicates good selectivity, and the other analytes did not significantly affect the fluorescence intensity of the detection system.

[0034] Example 5: A 10 mM PBS buffer solution with pH 7.4 was prepared. After reacting DPBF (40 μM), IX-NO2-CF3 (5 μM), and 5 μg / mL NTR (containing 500 μg / mL NADH) for 10 min, the solution was irradiated with light. UV spectroscopy measurements were performed every 30 s. The photoexcitation parameters were: 660 nm, 50 mW / cm². 2 The test results are shown in Figure 9. As can be seen from Figure 9, the absorption of IX-NO2-CF3 at 410 nm is significantly reduced, proving that after reacting with nitroreductase, it generates [a specific substance] under light. 1 O2. The response mechanism of IX-NO2-CF3 with nitroreductase is shown in Figure 4.

[0035] Example 6: A 10 mM PBS buffer solution with pH 7.4 was prepared. DHR123 (5 μM), IX-NO2-CF3 (5 μM), and 5 μg / mL NTR (containing 500 μg / mL NADH) were reacted for 10 min, followed by illumination. Fluorescence spectroscopy was measured every 60 s. The photoexcitation parameters were: 660 nm, 50 mW / cm². 2 The test results are shown in Figure 10. Figure 10 shows that the fluorescence of IX-NO2-CF3 at 525 nm is significantly enhanced, proving that it generates O2 under illumination after interacting with NTR. •⁻ .

[0036] Example 7: To verify the response performance of compound IX-NO2-CF3 to the hypoxic environment of tumor cells, HeLa cells were divided into 5 groups and incubated for 12 h at oxygen concentrations of 21%, 10%, 5%, and 1%, respectively. Then, 5 μL of IX-NO2-CF3 was added to each group and incubated for 1 h. Confocal microscopy (λ) was then performed. ex =647 nm, λ em Imaging detection at 680~740 nm was performed, and the results are shown in Figure 11. As can be seen from Figure 11, as the oxygen concentration decreased from 21% to 1%, the fluorescence intensity of the red channel gradually increased, indicating that IX-NO2-CF3 can respond rapidly and specifically to the hypoxic environment of tumor cells.

[0037] Example 8 investigated the response rate of compound IX-NO2-CF3 to a hypoxic environment in tumor cells. HeLa cells were incubated for 12 h at 21% and 1% oxygen concentrations, respectively, followed by incubation with 5 μMIX-NO2-CF3 for 1 h. The results were analyzed using a confocal microscope (λ). ex =647nm, λ em Cell imaging was performed every 10 minutes at a wavelength of 680~740 nm. The results are shown in Figure 12. As can be seen from Figure 12, the red fluorescence enhancement rate of this compound is much faster in a 1% hypoxic environment than in a 21% normoxic environment, indicating that this compound can respond rapidly to the hypoxic state of tumor cells.

[0038] Example 9 investigated the specific targeting of the compound IX-NO2-CF3 to mitochondria in hypoxic tumor cells. HeLa cells were incubated at 1% oxygen for 12 h, then incubated for 1 h with 5 μM IX-NO2-CF3, followed by incubation with 2.5 μM Mito-tracker Green (a commercially available mitochondrial dye) for another 30 min. Detection was performed using confocal fluorescence microscopy (red channel: λ). ex =647 nm, λ em =680~740 nm; Green channel: λ ex=488 nm, λ em =500-550 nm), the detection results are shown in Figure 13. As can be seen from Figure 13, the red fluorescence of compound IX-NO2-CF3 highly overlaps with the green fluorescence of Mito-tracker Green, proving that the compound can specifically target and locate mitochondria.

[0039] Example 10 verifies the ability of compound IX-NO2-CF3 to generate reactive oxygen species (ROS) under hypoxic conditions and light exposure. HeLa cells were cultured under hypoxic conditions for 12 h and then divided into four groups: Group 1 was the control group; Group 2 was incubated with 5 μM IX-NO2-CF3 and 10 μM fluorescein dichlorodihydroacetate (DA-DCFH) for 1 h (without light); Group 3 was incubated with 10 μM DA-DCFH for 1 h followed by light treatment (50 mW / cm², 2 min); and Group 4 was incubated with 5 μM IX-NO2-CF3 and 10 μM DA-DCFH for 1 h followed by light treatment (50 mW / cm², 2 min). The results were obtained using confocal fluorescence microscopy (λ). ex =488 nm, λ em The detection results (500-550nm) are shown in Figure 14. As can be seen from Figure 14, the first, second and third green channels showed no obvious fluorescence, while only the fourth group showed strong green fluorescence, indicating that IX-NO2-CF3 can generate a large amount of reactive oxygen species under light conditions after being treated with oxygen deficiency.

[0040] Example 11 evaluated the cytotoxicity and photodynamic therapeutic activity of compound IX-NO2-CF3 using the MTT assay. In the dark group, HeLa cells were treated with different concentrations of compound IX-NO2-CF3 under normal oxygen and hypoxia environments for 8 h, and cell viability was tested. The results are shown in Figure 15. As can be seen from Figure 15, cell viability remained at a high level, even at a concentration of 20 μM, the viability was still over 80%, indicating that compound IX-NO2-CF3 has low dark toxicity. In the light group, cells in the normal oxygen group were incubated with different concentrations of compound IX-NO2-CF3 for 1 h, then treated with light (660 nm, 50 mW / cm², 10 min), and cultured for another 8 h before cell viability was measured. Cells in the hypoxia group were first cultured under hypoxia for 12 h, then incubated with different concentrations of compound IX-NO2-CF3 for 1 h, then treated with light (660 nm, 50 mW / cm², 10 min), and cultured for another 8 h before cell viability was measured. The results showed that in a normal oxygen environment, cell survival rate decreased to a certain extent with increasing compound concentration; after treatment in a hypoxic environment, cytotoxicity was significantly enhanced compared to the normal oxygen group, indicating that compound IX-NO2-CF3 can significantly improve the efficiency of photodynamic killing of tumor cells after treatment in a hypoxic environment.

[0041] Example 12 investigated the photodynamic killing ability of compound IX-NO2-CF3 on tumor cells after hypoxia treatment using Calcein-AM and PI double staining. HeLa cells were first treated with hypoxia for 12 h and then divided into four groups: Group 1 was the control group; Group 2 received only 5 μMIX-NO2-CF3 (no light); Group 3 received only light treatment (50 mW / cm², 10 min); and Group 4 received 5 μMIX-NO2-CF3 followed by light treatment (50 mW / cm², 10 min). Cells were then incubated with Calcein-AM (2 μL) and PI (2 μL) for 30 min before imaging (green channel: λ). ex =488 nm, λ em =500-550 nm; Red channel: λ ex =561 nm, λ em =590–640 nm). The test results are shown in Figure 16. As can be seen from Figure 16, a large number of cells died in the fourth group, indicating that the compound can kill tumors only after hypoxia treatment and light irradiation, demonstrating its potential for diagnosis and treatment in hypoxic environments.

[0042] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Other modifications can be readily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. A fluorescent dye compound responsive to xanthanyl nitroreductase, characterized in that, It has the structure of the following formula (I): (I); where R1 is an N-alkylated nitrogen heterocyclic structural unit and R2 is a benzene ring structural unit containing a substituent.

2. The oxanthanna-based nitroreductase-responsive fluorescent dye compound as described in claim 1, characterized in that, R1 has the structure of the following equation (II): (II); Among them, R3 is 。 3. The xanthanid nitroreductase-responsive fluorescent dye compound as described in claim 1, characterized in that, R2 is 。 4. A method for preparing a xanthannaphthyl nitroreductase-responsive fluorescent dye compound as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Compound (III) is prepared by reacting 5-iodo-2,3,3-trimethyl-3H-indole with 1,2-oxothiacyclopentane 2,2-dioxide; S2. Compound (IV) is prepared by formylation of cyclohexanone; Compound (IV) is further reacted with 2-hydroxy-4-methoxybenzaldehyde, boron tribromide, and 1-(bromomethyl)-4-nitro-2-(trifluoromethyl)benzene to prepare compound (V); S3. Compound (III) and compound (V) are condensed to obtain a thallium-based nitro reductase-responsive fluorescent dye compound; (III), (IV), （V）。 5. The method for preparing oxanthannavid nitroreductase-responsive fluorescent dye compounds as described in claim 4, characterized in that, In step S1, the molar ratio of 5-iodo-2,3,3-trimethyl-3H-indole to 1,2-oxothiacyclopentane 2,2-dioxide is 1:1 to 2. The reaction conditions are: under argon protection, toluene as solvent, and reflux for 10 to 15 h with stirring.

6. The method for preparing oxanthannavid nitroreductase-responsive fluorescent dye compounds as described in claim 4, characterized in that, In step S2, the preparation of compound (IV) from cyclohexanone as a raw material via formylation reaction specifically includes: dissolving anhydrous N,N-dimethylformamide in dichloromethane to obtain a mixed solution; adding PBr3 dropwise to the mixed solution at 0 °C and stirring for 0.5–1 h; adding cyclohexanone to the reaction solution and continuing stirring for 15–20 h; after the reaction is completed, pouring the reaction mixture into ice water; adjusting the pH to neutral with sodium carbonate; extracting with dichloromethane; combining the organic phases; evaporating the solvent to obtain compound (IV).

7. The method for preparing oxanthannavid nitroreductase-responsive fluorescent dye compounds as described in claim 4, characterized in that, In step S2, the molar ratio of compound (IV) to 2-hydroxy-4-methoxybenzaldehyde is 1 to 2:1, and the reaction conditions are stirring at room temperature for 15 to 20 h.

8. The method for preparing oxanthannavid nitroreductase-responsive fluorescent dye compounds as described in claim 4, characterized in that, In step S3, the molar ratio of compound (III) to compound (V) is 1:1, the solvent for the condensation reaction is ethanol, and the reaction conditions are heating at 80-90°C for 10-15 h.

9. The use of the xanthanid nitroreductase-responsive fluorescent dye compound as described in any one of claims 1 to 3 in the preparation of nitroreductase-responsive detection probes.

10. The use of the xanthanid nitroreductase-responsive fluorescent dye compound as described in any one of claims 1 to 3 in the preparation of photosensitizers for photodynamic diagnosis and treatment of tumors.

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