RNA-responsive fluorescent probe and its preparation method and application

Abstract

This invention discloses an RNA-reactive fluorescent probe, its preparation method, and its application in tumor imaging and treatment. Upon response to ALP enzyme, the probe exhibits enhanced fluorescence, with a corresponding increase in photoacoustic intensity at 685 nm. Simultaneously, after enzyme response, the probe reacts with cytoplasmic RNA under red light irradiation, prolonging its residence time in tumor cells and achieving long-window tumor imaging. More importantly, this cross-linking reaction causes mitochondrial damage, leading to severe tumor cell apoptosis and thus inhibiting tumor growth. This invention's probe not only enables long-window near-infrared and photoacoustic dual-modal imaging of tumors but, more importantly, inhibits tumor growth, achieving integrated tumor diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the fields of detection technology and imaging, specifically relating to an enzyme-light dual-controlled RNA reactive fluorescent probe and its preparation method, as well as the application of the probe in tumor imaging and treatment. Background Technology

[0002] Cancer, also known as malignant tumor, is one of the major diseases that seriously threaten human life and health. According to data from the National Cancer Center in September 2021, with the increasing aging population, continuous deterioration of the ecological environment, unhealthy lifestyles, and food safety issues, the incidence and mortality rates of cancer in my country are showing a significant upward trend, which has become a public health and even social problem that must be taken very seriously. Molecular probes, as one of the core elements of molecular imaging technology, play an important role in the precision diagnosis and treatment of cancer. Although a large number of molecular imaging probes have been developed for cancer imaging and treatment, their poor tumor specificity and the potential for damage to normal tissues often prevent them from achieving precise diagnosis and treatment. Based on the characteristics of the tumor microenvironment (TME), scientists have developed a variety of activatable molecular probes that can be triggered by endogenous substances (such as overexpressed enzymes, acidic pH, highly reduced glutathione, and hypoxia) and can specifically destroy cancer cells, thereby reducing the toxic side effects of traditional chemotherapy or radiotherapy on normal tissues. However, most activatable diagnostic and treatment strategies are triggered by only a single factor and are usually insufficient to effectively avoid normal cells and precisely ablate cancer cells, leading to potential false positive or false negative diagnostic results. Summary of the Invention

[0003] To overcome the shortcomings and deficiencies of existing technologies, this invention provides an enzyme-light dual-controlled RNA-reactive fluorescent probe, its preparation method, and its applications. This probe not only prolongs the retention time in tumor cells, but more importantly, the cross-linking of the probe with RNA causes mitochondrial damage, leading to severe tumor cell apoptosis and thus inhibiting tumor growth. Furthermore, among numerous triggering modes, light has proven to be an attractive tool for precise activation of therapy with spatiotemporal controllability, without requiring physical contact.

[0004] To achieve the above objectives, the technical solution of the present invention is as follows:

[0005] An RNA-reactive fluorescent probe has the following structural formula:

[0006]

[0007] The preparation method of the above-mentioned RNA reactive fluorescent probe includes the following steps:

[0008] (1) Compound 1 was reacted with 5-chloropentyne to give compound 2;

[0009] (2) Compound 2 is reacted with raw material A to obtain compound 3;

[0010] (3) Compound 3 was reacted with resorcinol to obtain compound 4;

[0011] (4) Compound 4 was reacted with phosphorus trichloride to obtain compound 5;

[0012] (5) React N-alpha-tert-butoxycarbonyl-L-lysine with raw material B and then react with trifluoroacetic acid to obtain compound 6;

[0013] (6) Compound 6 was reacted with azidoacetic acid to give compound 7;

[0014] (7) Compound 7 was reacted with RGD-NH2 to give compound 8;

[0015] (8) React compound 5 with compound 8 to obtain compound 9, which is the RNA reactive probe.

[0016] Furthermore, the preparation method of the above-mentioned RNA reactive probe includes the following steps:

[0017] (1) Compound 1 and 5-chloropentyne were reacted in an organic solvent in the presence of a metal halide compound at 90–120 °C. o The reaction proceeds for 20–30 hours at step C to yield compound 2; the metal halide compound is selected from potassium halide, and the organic solvent is selected from acetonitrile, etc.

[0018] (2) Mix compound 2 with raw material A in a mixed solvent at 90–130 °C. o The reaction at C lasts for 5–12 hours to yield compound 3; the mixed solvent is a mixture of alcohol, toluene, and other solvents.

[0019] (3) Compound 3 was reacted with resorcinol at room temperature for 8 to 15 hours in the presence of an inorganic base and an organic solvent to obtain compound 4; the inorganic base was potassium carbonate and the organic solvent was selected from acetonitrile, etc.

[0020] (4) Compound 4 and phosphorus oxychloride are reacted in an organic solvent at 0–25 °C. o The reaction at C was carried out for 10–16 hours to obtain compound 5; the organic solvent was selected from pyridine, etc.

[0021] (5) React raw material B with NHS at room temperature for 10-16 hours in the presence of an organic solvent and a condensing agent, then react with N-alpha-tert-butoxycarbonyl-L-lysine at room temperature for 10-16 hours in the presence of an organic base and an organic solvent, and then react with trifluoroacetic acid in an organic solvent at -4 to 4 o The reaction at C lasted 15–45 minutes to yield compound 6. The organic solvent used in the above reaction was dichloromethane, the condensing agent was EDC, and the organic base was DIPEA, etc.

[0022] (6) Azideacetic acid and NHS were reacted at room temperature for 10-16 hours in the presence of an organic solvent and a condensing agent, and then reacted with compound 6 in the presence of an organic base and an organic solvent for 10-16 hours to obtain compound 7. The organic solvent used in the above reaction was dichloromethane, the condensing agent was EDC, and the organic base was DIPEA, etc.

[0023] (7) Compound 7 was reacted with NHS at room temperature for 5 to 12 hours in the presence of an organic solvent and a condensing agent, and then reacted with compound 7 in the presence of an organic base and an organic solvent for 5 to 12 hours at room temperature to obtain compound 8; the organic solvent used in the above reaction was N,N-dimethylformamide, the condensing agent was EDC, and the organic base was DIPEA, etc.

[0024] (8) Compound 5 and compound 8 were reacted at room temperature for 3 to 8 hours in the presence of an organic solvent, an inorganic copper salt and a reducing agent to obtain compound 9, which is the RNA reactive fluorescent probe; the organic solvent was dimethyl sulfoxide, the inorganic copper salt was copper sulfate pentahydrate and the reducing agent was sodium ascorbate.

[0025] As an example, the preparation method of the above-mentioned RNA reactive fluorescent probe includes the following steps:

[0026] (1) Dissolve 5-chloropentyne in acetonitrile, add KI to it, 80 o The mixture was heated under reflux for 1 h, then compound 1 was added, and the mixture was heated under reflux for another 24 h to obtain compound 2 after purification.

[0027] (2) Dissolve compound 2 and raw material A in n-butanol and toluene, 110 o After heating under reflux for 8 h, compound 3 was obtained after purification.

[0028] (3) Dissolve resorcinol and potassium carbonate in acetonitrile, stir at room temperature for 30 minutes, and then add compound 3,50. o After heating at C for 12 h, compound 4 was purified to obtain compound 4.

[0029] (4) Compound 4 was dissolved in pyridine, and phosphorus oxychloride was slowly added dropwise under ice bath conditions. After the addition was completed, the mixture was stirred at room temperature for 12 h and purified to obtain compound 5.

[0030] (5) Dissolve 3-(2-furan)propionic acid in dichloromethane, add NHS and EDC, stir at room temperature for 12 h, then extract and evaporate to dryness; then dissolve the evaporated product in dichloromethane, add N-alpha-tert-butoxycarbonyl-L-lysine and DIPEA, stir at room temperature for 12 h; after the reaction is complete, add 2 mL of trifluoroacetic acid under ice bath conditions, 0 oAfter stirring at C for 30 min, compound 6 was obtained after purification.

[0031] (6) Dissolve azidoacetic acid in dichloromethane, add NHS and EDC to it and stir at room temperature for 12 h, then extract and evaporate to dryness; then dissolve the evaporated product in dichloromethane, add compound 6 and DIPEA to it, stir at room temperature for 12 h, and purify to obtain compound 7;

[0032] (7) Compound 7 was dissolved in N,N-dimethylformamide, NHS and EDC were added and stirred at room temperature for 8 h, then extracted and evaporated to dryness. The evaporated product was then dissolved in N,N-dimethylformamide, RGD-NH2 and DIPEA were added and stirred at room temperature for 8 h, and the mixture was purified to obtain compound 8;

[0033] (8) Compound 5 and compound 8 were dissolved in DMSO, and then a mixture of copper sulfate pentahydrate solution and sodium ascorbate solution was added. The mixture was stirred at room temperature for 5 h and purified to obtain compound 9, which is the RNA reactive fluorescent probe.

[0034] In the above technical solution, the equivalent ratio of compound 1, 5-chloropentyne, and KI in step (1) is 1:2:(3-5), preferably 1:2:4; the equivalent ratio of compound 2 and raw material A in step (2) is 2:1, and the volume ratio of solvent n-butanol and toluene is 7:1; the equivalent ratio of compound 3, resorcinol, and potassium carbonate in step (3) is 1:2:(2-3), preferably 1:2:2.5; the equivalent ratio of compound 4 and phosphorus oxychloride in step (4) is 1:5; the equivalent ratio of Boc-Lys-OH, raw material B, NHS, and EDC in step (5) is 1:(1-1.5):(1-1.5):(1-1.5), preferably 1:1.1:1.2:1.3.

[0035] In this invention, the chemical structural formula of RGD-NH2 is as follows:

[0036]

[0037] In this invention, the chemical structural formulas of compounds 1, 2, 3, 4, 5, Boc-Lys-OH, 6, 7, 8, raw material A, and raw material B are as follows:

[0038]

[0039] In this invention, the chemical structural formulas of raw material A and raw material B are as follows:

[0040]

[0041] Compounds 2, 3, 4, 5, and 9 of this invention ( f -RCP (RNA reactive fluorescent probe) and compound 11 (RCP, control group probe) are both in ionic form, which is a conventional representation in this field. They are often coordinated with halogen anions, such as I... - Cl - The specific coordination method is also a conventional technique and does not affect the realization of the technical effect of the present invention.

[0042] This invention discloses the application of the above-mentioned RNA reactive fluorescent probe in the preparation of long-window fluorescence imaging reagents or photoacoustic imaging reagents; or the application of the above-mentioned RNA reactive fluorescent probe in long-window fluorescence imaging or photoacoustic imaging; or the application of the above-mentioned RNA reactive fluorescent probe in the preparation of tumor-targeted therapy reagents; or the application of the above-mentioned RNA reactive fluorescent probe in tumor-targeted therapy.

[0043] The method for long-window fluorescence imaging using the above-mentioned RNA reactive fluorescent probe includes the following steps: ... f - An aqueous solution of RCP was injected into the tail vein of tumor-bearing nude mice, and the in vivo fluorescence imaging effect was observed at different time points under anesthesia; in vivo fluorescence imaging was completed.

[0044] The method for long-window photoacoustic imaging using the above-mentioned RNA reactive fluorescent probe includes the following steps: ... f - An aqueous solution of RCP was injected into the tail vein of tumor-bearing nude mice, and the in vivo photoacoustic imaging effect was observed at different time points under anesthesia; in vivo photoacoustic imaging was completed.

[0045] The method for tumor-targeted therapy using the above-mentioned RNA reactive fluorescent probe includes the following steps: ... f -RCP aqueous solution was injected into tumor-bearing nude mice via the tail vein, and the tumor site was irradiated with a 660 nm laser 4 h later (0.1 W / cm²). 2 (3 min) Record changes in tumor volume and mouse weight to observe the effect of tumor treatment.

[0046] Due to the application of the above technical solutions, the advantages of this invention are:

[0047] 1. This invention designs and synthesizes an enzyme-light dual-controlled RNA reaction fluorescent probe. After activation by the ALP enzyme, the probe reacts with RNA molecules in the cytoplasm under red light irradiation, prolonging the retention time of the probe in the tumor and realizing long-window tumor fluorescence imaging and photoacoustic imaging.

[0048] 2. The RNA-reactive fluorescent probe in this invention reacts with cytoplasmic RNA and causes mitochondrial damage, leading to severe tumor cell apoptosis and thus inhibiting tumor growth.

[0049] 3. The RNA-reactive fluorescent probe in this invention has good targeting and responsiveness to tumor tissue, resulting in significant tumor treatment effects and realizing integrated diagnosis and treatment of tumors. Attached Figure Description

[0050] Figure 1 This is a schematic diagram of the synthesis of RNA reactive fluorescent probes.

[0051] Figure 2 This is a schematic diagram of the synthesis of the control probe.

[0052] Figure 3 For example, in Example 2 (a), the RNA reactive fluorescent probe f -RCP (5 μM) or control probe RCP (5 μM) with ALP enzyme (200 UL) -1 (a) Changes in absorption spectra before and after incubation; (b) Probe f -RCP (5 μM) or control probe f-RCP (5 μM) in the presence or absence of Na3VO4 (1 mM) with ALP enzyme (200 μL) -1 (a) Changes in fluorescence emission spectra before and after incubation; (b) Probe f -RCP (5 μM) and different concentrations of ALP enzyme (0~200 μL) -1 In Tris-HCl buffer at 37°C o (a) UV-Vis absorption spectrum after 1 h of incubation at C; (b) Ratio-dependent photoacoustic PAS of the probe. 685 Relationship with different concentrations of ALP enzyme (Figure: Photoacoustic images containing probes and different concentrations of ALP enzyme).

[0053] Figure 4 In Example 3(a), RNA reactive fluorescent probe was injected via tail vein. f -RCP (1.5 μg g) -1 ) or control probe RCP (1.5 μg g -1 (a) Real-time fluorescence imaging in HepG2 tumor-bearing nude mice that were untreated or pretreated with the ALP enzyme inhibitor Na3VO4 (10 mM, 50 μL); (b) Quantitative analysis of tumor fluorescence intensity corresponding to (a).

[0054] Figure 5 In Example 4(a), RNA reactive fluorescent probe was injected via tail vein. f -RCP (1.5 μg g) -1) or control probe RCP (1.5 μg g -1 (a) Real-time photoacoustic imaging in HepG2 tumor-bearing nude mice; (b) Quantitative analysis of the photoacoustic intensity of the tumor corresponding to (a).

[0055] Figure 6 The RNA reactive fluorescent probe in Example 5 (a) f - Schematic diagram of RCP or control probe RCP treatment for HepG2 tumors; (b) Nude mouse photographs before treatment and on days 3, 5, 7, 9, and 11 after treatment; 660 nm laser irradiation (0.1 W cm⁻¹) -2 Tumor growth curve over 3 minutes (c); tumor weight after treatment (d). Dosage: f -RCP = RCP = 1.5 ng g -1 **P < 0.01 (n = 3).

[0056] Figure 7 The fluorescence intensity ratio of the probe (5 μM) after adding various biological species is (I-I0) / I0.

[0057] Figure 8 (g) Quantification of colocalization coefficients of different groups of HepG2 cells. (h) Normalized fluorescence intensity of cellular RNA extracted from different cell groups previously. (i) RNA agarose gel electrophoresis images of cellular RNA extracted from different groups under excitation at 546 nm (EB) and 632 nm (CyOH), respectively. Statistical significance level was defined as ***p<0.001 (n=3).

[0058] Figure 9 The results of co-incubation with different existing dyes.

[0059] Figure 10 For in vitro assessment of apoptosis and mitochondrial damage. (a) Live / dead staining images of HepG2 cells under different treatments (scale bar = 100 μm). (b) Received f - Cell viability of HepG2 cells with RCP or RCP under 660 nm laser irradiation and no irradiation. (c) Mitochondrial membrane potential analysis of HepG2 cells under different treatments using JC-1 dye (scale bar = 25 μm). (d) Quantitative analysis of intracellular ATP concentration in various treatments. (e) TEM ultrathin section images of mitochondrial integrity in different groups (scale bar = 500 nm). f -RCP]=[RCP]=5 μM; 660 nm laser: 0.1 W cm -2 , 3 min. The statistical significance level is **p<0.01 (n=3).

[0060] Figure 11 To assess the migration ability of cells under different treatments. Detailed Implementation

[0061] Existing probes crosslink with RNA by adding a photosensitizer and then irradiating it with light. This invention solves this problem by eliminating the need for an additional photosensitizer. Moreover, the probe of this invention is an activating type. It can only crosslink with RNA under light after being digested by an enzyme. If the probe is not digested by an enzyme, it cannot crosslink with RNA even under light. This means that crosslinking is controllable, which is something that existing probes cannot achieve.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Tumor-bearing female nude mice were modeled using conventional methods and met the animal experiment requirements of Soochow University.

[0063] Example 1: The preparation method of the RNA reactive fluorescent probe of the present invention, the synthesis steps are as follows: Figure 1 As shown, the coordinating anion in the compound is an iodide ion, as detailed below:

[0064] (1) 5-Chloropentyne (2.05 g, 20 mmol) was dissolved in 60 mL of acetonitrile, and KI (6.64 g, 40 mmol) was added. The mixture was heated under nitrogen protection and refluxed for 1 h. Then, 2,3,3-trimethyl-3H-indole (1.59 g, 10 mmol) was added, and the mixture was heated under reflux for another 24 h. After the reaction was completed, the mixture was cooled to room temperature, and the acetonitrile was removed by rotary evaporation. Water was added, and the mixture was extracted three times with dichloromethane. The mixture was dried over anhydrous magnesium sulfate, and most of the dichloromethane was removed by rotary evaporation. The mixture was then concentrated, and anhydrous diethyl ether was added for recrystallization. A large amount of gray solid precipitated out. The solid was filtered and washed three times with anhydrous diethyl ether to obtain a gray solid, which was compound 2 (1.24 g, yield 35%). 1 H NMR (600 MHz, DMSO- d6 ) δ 7.99-7.95 (m, 1H), 7.87-7.83(m, 1H), 7.66-7.60 (m, 2H), 4.54-4.46 (m, 2H), 2.96 (s, 1H), 2.85 (s, 3H), 2.45-2.38 (m, 2H), 2.11-2.02 (m, 2H), 1.55 (s, 6H). 13 C NMR (151 MHz, DMSO- d6)δ 197.97, 142.76, 142.04, 130.35, 129.86, 124.47, 116.16, 84.01, 73.39,55.19, 47.75, 26.97, 22.92, 16.18, 15.05 . Calculated for C 16 H 20 N + , ([M] + ): 226.16, found ESI-MS: m / z 226.27.

[0065] (2) Compound 2 (706 mg, 2 mmol) and 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde (172 mg, 1 mmol) were dissolved in n-butanol (10 mL) and toluene (2 mL) under nitrogen protection. o The mixture was heated under reflux for 12 h. After the reaction was complete, it was cooled to room temperature, the reaction solvent was removed by an oil pump, a small amount of methanol was added to dissolve the residue, and anhydrous diethyl ether was added dropwise under stirring. A large amount of dark brown solid precipitated out. The solid was filtered and washed twice with anhydrous diethyl ether. The dark brown solid obtained was compound 3 (550 mg, yield 77%). 1 H NMR (600 MHz, DMSO- d6 ) δ 8.27 (d, J = 14.1 Hz,2H), 7.66-7.62 (m, 2H), 7.49-7.41 (m, 4H), 7.30 (t, J = 7.2 Hz, 2H), 6.37 (d, J = 14.1 Hz, 2H), 4.26 (t, J = 7.3 Hz, 4H), 2.99 (s, 2H), 2.71 (t, J = 5.9Hz, 4H), 2.38-2.31 (m, 4H), 1.96-1.89 (m, 4H), 1.90-1.84 (m, 2H), 1.77-1.71(m, 2H), 1.68 (s, 12H), 1.57-1.52 (m, 2H). 13 C NMR (151 MHz, DMSO- d6) δ 173.35,149.09, 144.10, 142.93, 142.00, 129.55, 127.28, 126.15, 123.51, 112.25,102.47, 84.47, 73.22, 49.99, 43.76, 28.42, 26.87, 22.92, 21.26, 16.24. Calcd.for C 40 H 44 ClN2 + , ([M] + ): 587.32, found ESI-MS: m / z 587.44.

[0066] (3) Resorcinol (110 mg, 1.00 mmol) was dissolved in 8 mL of acetonitrile, potassium carbonate (173 mg, 1.25 mmol) was added, and the mixture was stirred at room temperature for 30 min. Then, compound 3 (357 mg, 0.50 mmol) was added. o The mixture was stirred at C for 12 h. After the reaction was complete, the mixture was cooled to room temperature, acetonitrile was removed by rotary evaporation, water was added, and the mixture was extracted three times with dichloromethane. The mixture was dried over anhydrous magnesium sulfate, evaporated to dryness, and purified by silica gel column chromatography (dichloromethane:methanol = 20:1, v / v) to give a dark blue solid, which was compound 4 (231 mg, 82% product). 1 H NMR (600 MHz, DMSO- d6 ) δ 8.18 (d, J = 13.9Hz, 1H), 7.61 (s, 1H), 7.55 (d, J = 7.3 Hz, 1H), 7.40 (d, J = 8.9 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 7.29 (d, J = 7.9 Hz, 1H), 7.18 (t, J = 7.4 Hz, 1H),6.63-6.57 (m, 1H), 6.46 (s, 1H), 6.10 (d, J = 13.9 Hz, 1H), 4.13 (t, J= 7.3Hz, 2H), 3.00-2.94 (m, 1H), 2.69-2.65 (m, 2H), 2.65-2.58 (m, 2H), 2.35-2.29(m, 2H), 1.92-1.84 (m, 2H), 1.85-1.76 (m, 2H), 1.68 (s, 6H). 13 C NMR (151 MHz, DMSO- d6 , overlapping peaks) δ 159.85, 157.24, 142.97, 140.68, 140.65, 138.47,138.45, 130.37, 128.79, 124.13, 122.76, 115.92, 115.69, 110.39,106.59,102.70,98.22,84.04,72.61,48.56,42.55,28.44,28.00,25.99,24.39,20.98,15.75. Calcd. for 30 H 30 NO2 + , ([M] + ): 436.23, found ESI-MS: m / z 436.40.

[0067] (4) Compound CyOH (113 mg, 0.2 mmol) was dissolved in 2 mL of pyridine, and phosphorus oxychloride (152 mg, 1.0 mmol) was added dropwise under ice bath conditions. After the addition was complete, the mixture was stirred at room temperature for 12 h. After the reaction was complete, the pyridine was removed by rotary evaporation, and the mixture was prepared by preparative high performance liquid chromatography (HPLC) to obtain a blue solid, which was compound 5 (84 mg, yield 65%). 1 H NMR (600 MHz, DMSO- d6 ) δ 8.61 (d, J = 15.0 Hz, 1H), 7.77 (d, J = 7.4Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.60-7.53 (m, 2H), 7.51-7.46 (m, 2H), 7.35(d, J = 0.8 Hz, 1H), 7.19-7.14 (m, 1H), 6.63 (d, J = 15.1 Hz, 1H), 4.46 (t,J = 7.4 Hz, 2H), 3.00 (s, 1H), 2.77-2.71 (m, 2H), 2.67 (t, J = 5.9 Hz, 2H), 2.41-2.35 (m, 2H), 2.04-1.95 (m, 2H), 1.89-1.81 (m, 2H), 1.77 (s, 6H). 13 C NMR (151 MHz, DMSO-) d6 ) δ 179.22, 160.79, 159.03 (d, J C-P = 34.5 Hz), 155.07 (d, J C-P = 5.9 Hz), 153.85, 146.42, 143.22, 142.26, 132.93, 129.85, 129.55,128.36, 123.78, 118.72, 118.66 (d, J C-P = 4.2 Hz), 115.17, 114.22, 108.37 (d, J C-P = 5.3 Hz), 106.13, 84.31, 73.40, 51.62, 45.07, 29.48, 28.30, 27.32,24.61, 20.76, 16.23. Calcd. for C 30 H 29 NO5P - , ([M-2H] - ): 514.1789, found ESI-MS: m / z 514.1792.

[0068] (5) Dissolve 3-(2-furan)propionic acid (154 mg, 1.1 mmol) in 5 mL of dichloromethane, add NHS (138 mg, 1.2 mmol) and EDC (249 mg, 1.3 mmol), and stir at room temperature for 12 h. After the reaction is complete, add water, extract three times with dichloromethane, dry to anhydrous magnesium sulfate, and evaporate to dryness. Then dissolve the evaporated product in 6 mL of dichloromethane, add N-alpha-tert-butoxycarbonyl-L-lysine (246 mg, 1.0 mmol) and N,N-diisopropylethylamine (330 μL, 2 mmol), and stir at room temperature for 12 h. After the reaction is complete, add 2 mL of trifluoroacetic acid under ice bath conditions. oThe mixture was stirred at C for 30 min. After the reaction was complete, dichloromethane and trifluoroacetic acid were removed by rotary evaporation. The product was then separated and purified by preparative high performance liquid chromatography to obtain a white solid product, which was compound 6 (185 mg, with an overall yield of 69% for the two steps). 1 H NMR (600 MHz, DMSO- d6 ) δ 8.24 (s, 3H), 7.88 (t, J = 5.5 Hz, 1H), 7.44 (d, J = 0.6 Hz, 1H), 6.30-6.25 (m, 1H), 6.03-5.98 (m, 1H), 3.81 (t, J = 6.0 Hz, 2H), 3.00-2.97 (m, 2H),2.77 (t, J = 7.6 Hz, 2H), 2.47-2.44 (m, 1H), 2.33 (t, J = 7.7 Hz, 2H), 1.77-1.65 (m, 4H). 13 C NMR (151 MHz, DMSO- d6 ) δ 172.06, 171.70, 155.74, 142.25,111.32, 105.98, 52.93, 41.36, 39.09, 30.70, 29.61, 24.53, 22.73. Calcd. forC 13 H 19 N2O4 - , ([MH] - ): 267.13, found ESI-MS: m / z 267.08.

[0069] (6) Azideacetic acid (56 mg, 0.55 mmol) was dissolved in 4 mL of dichloromethane, and NHS (69 mg, 0.6 mmol) and EDC (125 mg, 0.65 mmol) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, water was added, and the mixture was extracted three times with dichloromethane. The extract was dried over anhydrous magnesium sulfate and then evaporated to dryness. The evaporated product was then dissolved in 4 mL of dichloromethane, and compound 6 (134 mg, 0.5 mmol) and N,N-diisopropylethylamine (165 μL, 1.0 mmol) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the reaction solvent was removed by rotary evaporation, and the product was purified by preparative high-performance liquid chromatography to obtain a colorless liquid product, which was compound 7 (144 mg, yield 82%). 1H NMR (600 MHz, DMSO- d6 ) δ 12.64 (s, 1H), 8.34-8.32 (m, 2H), 7.85-7.83 (m, 1H), 7.44 (d, J = 0.7 Hz, 1H), 6.31-6.26 (m, 1H), 6.01 (d, J = 2.9 Hz, 1H), 4.15-4.12 (m, 2H), 2.97 (d, J = 5.7 Hz, 2H), 2.77 (t, J =7.6 Hz, 2H), 2.56-2.55 (m, 1H), 2.47-2.46 (m, 2H), 2.34-2.31 (m, 2H), 1.67-1.64 (m, 2H), 1.56-1.53 ​​(m, 2H). 13 C NMR (151 MHz, DMSO- d6 ) δ 174.22, 171.61,168.48, 155.72, 142.21, 111.28, 105.94, 52.88, 51.36, 41.36, 39.16, 31.61,29.64, 24.51, 23.73. Calcd. for C 15 H 20 N5O5 - , ([MH] - ): 350.15, found ESI-MS: m / z350.16.

[0070] (7) Compound 7 (35 mg, 0.1 mmol) was dissolved in 2 mL of N,N-dimethylformamide, and NHS (13 mg, 0.11 mmol) and EDC (23 mg, 0.12 mmol) were added. The mixture was stirred at room temperature for 8 h. After the reaction was complete, N,N-dimethylformamide was removed by an oil pump, and the mixture was extracted three times with water and dichloromethane. The extract was dried over anhydrous magnesium sulfate and then evaporated to dryness. The evaporated extract was then dissolved in 4 mL of N,N-dimethylformamide, and RGD-NH2 (60 mg, 0.1 mmol) and N,N-diisopropylethylamine (33 μL, 0.2 mmol) were added. The mixture was stirred at room temperature for 8 h. After the reaction was complete, the reaction solvent was removed by rotary evaporation, and the product was purified by preparative high-performance liquid chromatography to obtain a white solid product, which was compound 8 (54 mg, yield 58%). 42 H 59 N14 O 11 - , ([MH] - ): 935.4493, found ESI-MS: m / z 935.4504.

[0071] (8) Compound 5 (13 mg, 0.02 mmol) and compound 8 (19 mg, 0.02 mmol) were dissolved in 1 mL of DMSO. Then, copper sulfate pentahydrate solution (400 μL, 0.1 M) and sodium ascorbate solution (440 μL, 0.1 M) were mixed, sonicated for five minutes, and then added dropwise to the above solution. The mixture was stirred at room temperature for 5 h. After the reaction was complete, the product was separated and purified using preparative high-performance liquid chromatography to obtain a blue solid product, which was the RNA reactive fluorescent probe (20 mg, yield 63%). 72 H 90 N 15 O 16 P, ([MH]): 1451.6428, found ESI-MS: m / z 1451.6460.

[0072] Preparation of the control probe as follows Figure 2 Azideacetic acid (11 mg, 0.11 mmol) was dissolved in 4 mL of dichloromethane, and NHS (14 mg, 0.12 mmol) and EDC (25 mg, 0.13 mmol) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, water was added, and the mixture was extracted three times with dichloromethane. The extract was dried over anhydrous magnesium sulfate and then evaporated to dryness. The residue was then dissolved in 4 mL of N,N-dimethylformamide, and RGD-NH2 (60 mg, 0.1 mmol) and N,N-diisopropylethylamine (33 μL, 0.2 mmol) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, N,N-dimethylformamide was removed by rotary evaporation, and the product was purified by preparative high-performance liquid chromatography to give a white solid product, compound 10 (48 mg, 70% yield). Calcd. for C 29 H 41 N 12 O8 - ,([MH] -The chromatogram (m / z) was 935.45, and the ESI-MS result was 935.50. Then, compound 5 (13 mg, 0.02 mmol) and compound 10 (14 mg, 0.02 mmol) were dissolved in 1 mL of DMSO. Next, copper sulfate pentahydrate solution (400 μL, 0.1 M) was mixed with sodium ascorbate solution (440 μL, 0.1 M), sonicated for five minutes, and then added dropwise to the above solution. The mixture was stirred at room temperature for 5 h. After the reaction was complete, the product was purified by preparative high-performance liquid chromatography to obtain a blue solid product, which was the target probe RCP (15 mg, yield 58%). Calcd. for C 59 H 72 N 13 O 13 P, ([MH]): 1201.5110, found ESI-MS: m / z 1201.5079.

[0073] Example 2 Fluorescence Imaging of a Ratio Photoacoustic Probe

[0074] RNA reactive fluorescent probes f -RCP (5 μM) or control probe RCP (5 μM) were respectively mixed with 200 UL -1 ALP enzyme in Tris-HCl buffer (Ph = 8.0) at 37°C o C was co-incubated for 1 h; in the competitive experiment, ALP enzyme (200 μL) was first added. -1 ) with ALP enzyme inhibitor Na3VO4 (1 mM) in Tris-HCl buffer at 37°C o After co-incubating at C for 0.5 h, probes were added separately. f -RCP (5 μM) or control probe RCP (5 μM), in Tris-HCl buffer at 37°C o Incubation at C continued for 1 hour, followed by measurement of UV absorption and fluorescence spectra. Results are as follows: Figure 3 As shown in a and 3b, the probe f -RCP initially showed two absorption peaks at 605 and 653 nm with almost no fluorescence. After incubation with ALP enzyme, the absorption gradually red-shifted, and a new maximum absorption peak appeared at 685 nm; simultaneously, the fluorescence intensity at 710 nm was significantly enhanced, 30 times stronger than without ALP enzyme. Furthermore, if ALP enzyme was pretreated with the ALP enzyme inhibitor Na3VO4, the probe... f The fluorescence of -RCP was almost completely inhibited, indicating that the activity of ALP enzyme was effectively inhibited.

[0075] The probe f-RCP (5 μM) was mixed with different concentrations of ALP enzyme (0, 0.5, 1, 2, 3, 4, 5, 10, 20, 50, 75, 100 and 200 UL). -1 In Tris-HCl buffer solution 37 o After co-incubation at C for 1 h, the ultraviolet absorption spectrum was subsequently measured. From Figure 3 c shows that as the ALP enzyme concentration increases from 0 to 200 μL... -1 The absorbance of the probe gradually increased at 685 nm. After completing the above detection, the absorbance at 685 nm (PAS) was then measured. 685 The photoacoustic signal at 685 nm gradually increased with ALP enzyme concentration, and remained relatively constant within the range of 0–10 μL. -1 It exhibits a good linear relationship (R 2 = 0.96)( Figure 3 d).

[0076] Example 3 Fluorescence Imaging of RNA Reactive Fluorescent Probes

[0077] SPF-grade female athymic nude mice, 6-8 weeks old, weighing 18-20 g, were tested under standard conditions (temperature 25 ± 2°C). o They were raised at C (relative humidity 60 ± 10%) with a light / dark cycle of 12 h.

[0078] 2 × 10⁻⁶ HepG2 cells in good growth condition 6 The tumor was transplanted subcutaneously into the right back of a nude mouse. After one week of growth, the tumor reached a volume of 60 mm. 3 The probe was injected via tail vein. f -RCP (1.5 μg g) -1 ) or control probe RCP (1.5 μg g -1 The drug was injected into nude mice in each group, and the tumors were irradiated with a 660 nm laser for 3 minutes 4 hours later, with a power density of 0.1 W / cm². -2 (for f -RCP+660 nm group, RCP+660 nm group), the remaining two groups are not illuminated (for f -RCP group, RCP group), with 3 nude mice in each group. For the inhibitor experiment, the ALP enzyme inhibitor Na3VO4 (10 mM, 50 μL) was carefully injected into the tumor of nude mice via intratumoral injection, and the probe was injected again after 30 min. f -RCP (1.5 μg g) -1The probe was injected into nude mice via tail vein injection. Near-infrared fluorescence images were then recorded at 0, 2, 4, 8, 12, 24, and 36 h post-injection using a small animal in vivo optical imaging system (IVIS spectrum, Perkin Elmer), and the fluorescence intensity of the tumors was delineated using IVIS software. The results are as follows: Figure 4 As shown in Figure a, the fluorescence intensity of the tumor gradually increased over time, reaching its maximum at 4 hours after probe injection, after which the fluorescence signal in each group began to decrease. (Compared to the experimental group) f Compared to -RCP+660 nm, f The fluorescence signal at the tumor site rapidly decreased in the RCP group, the RCP+660 nm group, and the RCP group. Simultaneously, the experimental group... f -RCP+660nm showed strong fluorescence signals at tumor sites at 8, 12, 24, and 36 h time points, consistent with the results of long-stay cell assays. Figure 4 As shown by the quantitative fluorescence analysis of b, the experimental group f The fluorescence intensity of -RCP+660 nm remained at 15% of the maximum fluorescence intensity after 36 h, and it also had a stronger signal-to-noise ratio. Meanwhile, the fluorescence signal at the tumor site in the inhibitor group remained at a low level throughout.

[0079] Example 5: Photoacoustic Imaging of RNA-Reactive Fluorescent Probes

[0080] 2 × 10⁻⁶ HepG2 cells in good growth condition 6 The tumor was transplanted subcutaneously into the right back of a nude mouse. After one week, the tumor reached a size of 60 mm. 3 The probe was injected via tail vein. f -RCP (1.5 μg g) -1 ) or control probe RCP (1.5 μg g -1 The drug was injected into nude mice in each group, and the tumors were irradiated with a 660 nm laser for 3 minutes 4 hours later, with a power density of 0.1 W / cm². -2 (for f -RCP+660 nm group, RCP+660 nm group), the remaining two groups are not illuminated (for f -RCP group, RCP group), with 3 nude mice in each group. Photoacoustic images of the tumor were acquired at 0, 2, 4, 8, 12, 24 and 36 h time points at an excitation wavelength of 685 nm using a multispectral tomography system (MOST, iThera Medical GmbH, inSight / inVision 256). Image reconstruction and data analysis were performed using MOST imaging system software. Results are as follows: Figure 5As shown in Figure a, the photoacoustic intensity at 685 nm increased over time in all groups, reaching a maximum at 4 h after probe injection, and then began to decrease. However, compared with the experimental group... f Compared to -RCP+660 nm, f The photoacoustic signal at the tumor site decreased rapidly in the RCP group, the RCP+660 nm group, and the RCP group. The aforementioned fluorescence and photoacoustic imaging data demonstrate the effectiveness of the probe. f -RCP, after being activated by ALP enzymes in tumor cells, can also cross-link with cytoplasmic RNA under infrared irradiation, thus achieving the purpose of long-window tumor imaging.

[0081] Example 6: Tumor-targeted therapy using RNA-reactive fluorescent probes

[0082] 2 × 10⁻⁶ HepG2 cells in good growth condition 6 The tumor was transplanted subcutaneously on both sides of the back of nude mice (18-20 g). After one week, the tumor volume reached 40-50 mm. 3 The probe was injected via tail vein. f -RCP (1.5 μg g) -1 ) or control probe RCP (1.5 μg g -1 The drug was injected into nude mice in each group, and the right tumor was irradiated with a 660 nm laser for 3 minutes 4 hours later at a power density of 0.1 W / cm². -2 (for f -RCP+660 nm group, RCP+660 nm group), left side not illuminated (for f -RCP group, RCP group), with 3 nude mice in each group. The length and width of the tumor were measured daily using calipers, and the result was calculated using the formula V (volume) = 1 / 2 × L (length) × W (width). 2 Tumor volume changes were calculated, and tumor changes in nude mice were simultaneously recorded using a camera. Results are as follows: Figure 6 As shown in b, 6c, and 6d, only the experimental group... f -RCP+660 nm tumors showed significant tumor inhibition over time, indicating that the probe f -RCP cross-linked cytoplasmic RNA significantly inhibited tumor growth, and there was no statistically significant difference in body weight among the groups.

[0083] Example 7

[0084] For further research f -RCP's reaction selectivity detected various potential interfering species, such as MMP-2, MMP-9, GGT, RNase, trypsin, H2O2, and BSA, all at consistent dosages. Figure 7As shown, when these potential disturbance species are added f No significant fluorescence enhancement was observed when 200 UL was added to RCP (5 μM). Notably, when 200 UL was added... -1 During ALP, significant fluorescence enhancement was observed as before.

[0085] Example 8

[0086] HepG2 cells at 37ºC and f Incubate with RCP (5 μM) for 2 hours, then irradiate with a 660 nm laser (denoted as ). f The experimental group consisted of cells treated with RCP at +660 nm; cells treated with RCP were then irradiated with a 660 nm laser (labeled as RCP+660 nm), and other cells were treated under the same conditions except for the light irradiation (labeled as RCP+660 nm). f -RCP or RCP) was used as a control group. Figure 8 This indicates that acceptance f Cells irradiated with RCP+660nm laser produced bright intracellular fluorescence, while the fluorescence of the other three control groups was extremely weak, which means that the probe... f -RCP enhanced cellular uptake under 660 nm laser irradiation. Furthermore, HepG2 cells in all groups were pretreated with SYTORNASelect green, a commercial RNA staining agent, before CLSM imaging. Compared to the three control groups, f -RCP+660 nm group in f -Excellent overlap was observed between the red fluorescence of RCP and the green fluorescence of RNA. Figure 8 g, Pearson correlation coefficient high = 0.62), indicating that the probe f -RCP can effectively bind to intracellular RNA.

[0087] Next, the binding ability of the probe to active RNA was tested using the TRNzol-A total RNA extraction kit. + RNA was extracted from cells treated in the four groups described above. Figure 8 As shown in h, in the experimental analysis, strong fluorescence was measured only from the RNA portion of the cells, while almost no fluorescence was observed in the other control groups. To further investigate the specificity of the probe f-RCP for intracellular RNA, cytoplasmic and nuclear RNA were extracted from the four groups of cells mentioned above using cytoplasmic and nuclear purification kits, and they were separated by RNA gel electrophoresis. Figure 8 i). from f A distinct fluorescent band was collected from cytoplasmic RNA in the -RCP+660 nm group, while almost no red fluorescence of the probe was detected from nuclear RNA gels, indicating that the probe... f-RCP largely binds to cytoplasmic RNA rather than nuclear RNA. These results demonstrate that, under red light irradiation, the probe... f -RCP can specifically covalently label the cytoplasmic RNA of tumor cells.

[0088] To further study the probe f -RCP subcellular organelle localization, integrating various commercial trackers and f -RCP (5 μM) was applied to cells for 2 hours ( Figure 9 This indicates that ALP can be effectively triggered. f -RCP, and eventually accumulate in mitochondria and endoplasmic reticulum.

[0089] Example 9

[0090] HepG2 cells were seeded in 96-well cell culture plates (5000 cells / well) and incubated at 37ºC for 24 hours. Various concentrations were then added. f -RCP or RCP (0, 0.5, 1, 2, 5, 10, 15, and 20 μM), followed by incubation at 37ºC for 48 hours, and then MTT assay for probe cytotoxicity. f - Photocytotoxicity of RCP and RCP on 4T1 cells: After incubation in DMEM medium for 2 h, each well was removed and the cells were washed once with 1×PBS buffer, followed by irradiation at 660 nm (0.1 W cm⁻¹). -2 After incubation for 3 minutes and 48 hours, cell viability was assessed using MTT assay. Confocal imaging of live / dead cells using the AM / PI kit showed that... f -RCP+660 nm achieved highly effective treatment, and no significant cytotoxicity was observed in the control group cells, which was further confirmed by annexin V-FITC and PI staining. Figure 10 a). Subsequently, intracellular caspase-3 levels, a typical indicator of apoptosis, were assessed using an immunostaining strategy. f Cells in the -RCP+660 nm group showed higher caspase-3 levels, indicating that the ELMPRC effect led to severe apoptosis. The MTT assay further characterized the cytotoxic effect of the probe on HepG2 cells. Figure 10 As shown in b, when the concentration is below 20 μM, the probe f Both RCP and RCP showed negligible cytotoxicity to 4T1 cells, and the overall cell viability remained above 90% after 48 hours of culture. However, under 660 nm laser irradiation, f-RCP cells exhibited greater cytotoxicity than RCP cells at the same concentration. Furthermore, the transfer and migration abilities of cells treated differently were also observed. Figure 11 As shown, the other five groups of HepG2 cells all exhibited good proliferation and invasion capabilities within 48 hours, while f Cells in the RCP+660 nm group retained only about 10% of their cell migration ability. In summary, this evidence strongly supports... f -RCP has great potential in cancer treatment.

[0091] Changes in mitochondrial membrane potential (MMP) in HepG2 cells were assessed using JC-1 dye, indicating a potential for color-dependent emission changes in mitochondria. Figure 10 As shown in c, in accepting f Strong green fluorescence (JC-1 monomers) and weak red fluorescence (JC-2 aggregates) were detected in mitochondria in cells with RCP+660 nm, indicating mitochondrial membrane disruption. However, strong red fluorescence was recorded in control cells, suggesting that the mitochondria remained intact. ATP production was measured to examine mitochondrial function. Figure 10 d). Compared with the control group, using f Cells treated with RCP at 660 nm showed lower ATP productivity. TEM studies revealed changes in HepG2 cell morphology after different treatments. Figure 10 e). The results showed that 660 nm, RCP, RCP+660 nm and f Cells treated with RCP exhibited typical mitochondrial morphology similar to those in the PBS group. In stark contrast, f -RCP at 660 nm significantly induced marked morphological changes in mitochondrial microstructure in cells. These results undoubtedly confirm that... f Cross-linking of RCP with cytoplasmic RNA can lead to severe mitochondrial damage.

[0092] Based on the above research results, the probe of the present invention... f The cross-linking reaction between RCP and RNA not only enables long-window tumor imaging, but also inhibits tumor growth, thereby achieving the goal of tumor treatment.

Claims

1. An RNA-reactive fluorescent probe, the structural formula of which is as follows: 。 2. The method for preparing the RNA-reactive fluorescent probe according to claim 1, characterized in that, Includes the following steps: Compound 5 was reacted with compound 8 to obtain an RNA reactive probe; the chemical structures of compounds 5 and 8 are as follows: 。 3. The method for preparing the RNA reactive fluorescent probe according to claim 2, characterized in that, Compound 5 and compound 8 were reacted at room temperature for 5–10 hours in the presence of an organic solvent, an inorganic copper compound, and a reducing agent to obtain an RNA reactive probe.

4. The method for preparing the RNA-reactive fluorescent probe according to claim 2, characterized in that, Compound 4 was reacted with phosphorus oxychloride (POCl3) to give compound 5; compound 4 has the following chemical structure: 。 5. The method for preparing the RNA-reactive fluorescent probe according to claim 2, characterized in that, Compound 7 was activated in an organic solvent and then reacted with RGD-NH2 at room temperature for 4–12 hours to obtain compound 8; compound 7 has the following chemical structure: 。 6. The use of the RNA reactive fluorescent probe of claim 1 in the preparation of long-window-period fluorescent imaging reagents or photoacoustic imaging reagents.

7. The use of the RNA-reactive fluorescent probe of claim 1 in the preparation of reagents that react with cytoplasmic RNA and / or induce mitochondrial damage.

8. The application of the RNA-reactive fluorescent probe of claim 1 in the preparation of tumor imaging and therapeutic reagents.

9. The use of compounds 5 and 8 of claim 2 in the preparation of the RNA-reactive fluorescent probe of claim 1.

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