Heavy-atom-free photosensitizer based on silicon-pyridine rale dye as well as preparation method and application of heavy-atom-free photosensitizer

Abstract

The invention relates to the technical field of fluorescent probes, in particular to a heavy-atom-free photosensitizer based on silicon-piraline red dye as well as a preparation method and application of the heavy-atom-free photosensitizer. Aiming at the problems that most photosensitizers are strong in dependence on oxygen, high in needed dosage, have toxic and side effects and the like, a near-infrared photosensitizer AN-SiPy is constructed based on a J aggregation-induced intersystem crossing mechanism, the photosensitizer has no PDT effect in the presence of a monomer, and after a J-aggregate is formed, ROS can be generated through a type I mechanism and a type II mechanism, and the photosensitizer can be used for preparing a near-infrared photosensitizer A-SiPy. NADH oxidation and Cyt c reduction can also be catalyzed in an oxygen-independent mode, cell death is induced in a ferroptosis mode under illumination, and the median lethal concentrations (IC50) in normal oxygen (21% O2) and hypoxia (1% O2) are 6.2 nM and 9.4 nM respectively. Therefore, the photosensitizer AN-SiPy disclosed by the invention has a very important application value in the field of tumor photodynamic therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a heavy atom-free photosensitizer based on silapyronine dye, and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) is an emerging minimally invasive tumor therapy that has been widely used in the treatment of epidermal cancer or cavity cancer. This treatment method produces toxic reactive oxygen species (ROS) with the participation of photosensitizers (PSs), light and oxygen (or other adjacent substrates), thereby killing cancer cells. After absorbing light of a specific wavelength, the photosensitizer transitions from the ground state (S0) to the excited singlet state (S1), and then reaches the excited triplet state (T1) through intersystem crossing (ISC). The triplet state photosensitizer can react with oxygen ( 3 O2) generates singlet oxygen ( 1 O2) (Type I) or generate superoxide anion (O2 •− ) (Type II), as the main precursor of other ROS, O2 •− It can be catalyzed by superoxide dismutase (SOD) to form H2O2, which can react with Fe 2+ The Fenton reaction generates highly oxidizing hydroxyl radicals (·OH). Most currently developed PSs exert their PDT effects through a type II mechanism, which is highly dependent on oxygen. This characteristic limits their application in hypoxic malignancies. Compared with the high-oxygen-consuming type II PDT, type I PDT, while less oxygen-dependent, also requires oxygen. Nicotinamide adenine dinucleotide (NADH) is a key intracellular reduced metabolite. It serves as an important coenzyme in the respiratory electron transport chain, carrying out electron transfer and participating in maintaining intracellular redox balance. Over 400 biocatalytic reactions in cells rely on NADH as a coenzyme. Recent studies have shown that photosensitizer-mediated photocatalytic NADH / NAD+ conversion can lead to cellular metabolic dysfunction and kill cancer cells without the presence of oxygen, providing an innovative strategy for addressing therapeutic resistance in hypoxic regions of solid tumors. Summary of the Invention

[0003] In order to address the problems of most photosensitizers being highly dependent on oxygen, requiring high dosages, and having toxic side effects, this paper constructs a photodynamic photosensitizer AN-SiPy based on silanol dye. In PBS, AN-SiPy exists in two forms: monomers and J-aggregates. The appearance of J-aggregates effectively reduces its singlet to triplet energy gap (ΔE ST), which promotes the generation of ROS. Under 660 nm laser irradiation, AN-SiPy not only generates a large amount of ROS through type I and type II mechanisms, but also catalyzes NADH oxidation and Cyt c reduction in an oxygen-independent manner. Cytotoxicity experiments confirmed that AN-SiPy induces cell death via ferroptosis, with half-lethal concentrations (IC50) of 6.2 nM and 9.4 nM under normoxia (21% O2) and hypoxia (1% O2), respectively.

[0004] To achieve the above object, the technical solution of the present invention is as follows: The present invention provides a heavy atom-free photosensitizer based on silapyronine dye, the structural formula of which is: .

[0005] The present invention also provides a method for preparing the above-mentioned heavy atom-free photosensitizer based on silapyronine dye, comprising the following steps: Under nitrogen protection, 9-bromoanthracene is dissolved in ultra-dry tetrahydrofuran, and n-butyl lithium is then added dropwise, and the reaction is stirred. Subsequently, a tetrahydrofuran solution of silapyrrolidone is added dropwise, and the reaction is continued with stirring. After the reaction is completed, aqueous hydrochloric acid is added for neutralization. The resulting reaction mixture is extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, dried, and purified by column chromatography to obtain the target compound AN-SiPy.

[0006] Furthermore, the molar ratio of 9-bromoanthracene, n-butyl lithium and silapyrrolidone is 3:3:1.

[0007] Furthermore, the stirring reaction temperature is -78°C and the time is 1 h.

[0008] Furthermore, the temperature for continuing to stir the reaction is room temperature and the time is 4 h.

[0009] Furthermore, the concentration of the hydrochloric acid aqueous solution is 2 M.

[0010] Furthermore, the column chromatography purification condition is MeOH / CH2Cl2=1 / 10, v / v.

[0011] The present invention also provides the use of the above-mentioned heavy atom-free photosensitizer based on silapyronine dye in the preparation of tumor photodynamic therapy drugs.

[0012] Furthermore, the photodynamic therapy is a synergistic therapy of type I photodynamic therapy, type II photodynamic therapy and photoredox catalysis.

[0013] Compared with the prior art, the present invention has the following beneficial effects: Heavy atom effects are a classic approach to improving the ISC efficiency of organic photosensitizers. However, the introduction of heavy atoms is often associated with increased toxicity and decreased photostability, severely limiting their application in biomedical applications. Therefore, the development of novel, heavy-atom-free, highly efficient, and biocompatible photosensitizer systems has become a key research focus. J-aggregation can effectively reduce the singlet-to-triplet energy level difference (ΔEST), promoting intersystem crossing (ISC), and thus generating ROS. The photosensitizer AN-SiPy developed in this paper is constructed based on this J-aggregation-induced ISC mechanism. While the monomer has no PDT effect, the J-aggregates formed not only generate significant ROS via type I and type II mechanisms but also catalyze NADH oxidation and Cyt c reduction in an oxygen-independent manner, thereby killing cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The compound AN-SiPy of the present invention 1 H NMR spectrum.

[0015] Figure 2 The compound AN-SiPy of the present invention 13 C NMR spectrum.

[0016] Figure 3 HRMS chart of the compound AN-SiPy of the present invention.

[0017] Figure 4 These are the absorption spectra of compound AN-SiPy in different solvents, where (A) is PBS, (B) is H2O, and (C) is CH3CN.

[0018] Figure 5 (A) shows DCFH under laser (650 nm, 10 mW / cm 2 ) is the fluorescence spectrum change diagram of the total ROS generated by the captured photosensitizer under irradiation; (B) is the fluorescence spectrum change diagram of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) under laser (650 nm, 20 mW / cm 2 ) is the UV-visible absorption spectrum change of singlet oxygen produced by the captured photosensitizer under laser (650nm, 10 mW / cm 2 ) is the fluorescence spectrum change diagram of superoxide produced by the captured photosensitizer under irradiation; (D) is the fluorescence spectrum change diagram of HPF under laser (650 nm, 10 mW / cm 2 ) shows the fluorescence spectrum changes of hydroxyl radicals produced by the captured photosensitizer under irradiation.

[0019] Figure 6 (A) DCFH in the presence of Triton-100 under laser (650 nm, 10 mW / cm 2) is the fluorescence spectrum change diagram of the total ROS generated by the captured photosensitizer under irradiation; (B) is the fluorescence spectrum change diagram of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) under laser (650 nm, 20 mW / cm 2 ) is the UV-visible absorption spectrum change of singlet oxygen produced by the captured photosensitizer under laser (650 nm, 10 mW / cm 2 ) is the fluorescence spectrum change diagram of superoxide produced by the captured photosensitizer under irradiation; (D) is the fluorescence spectrum change diagram of HPF under laser (650 nm, 10 mW / cm 2 ) shows the fluorescence spectrum changes of hydroxyl radicals produced by the captured photosensitizer under irradiation.

[0020] Figure 7 (A) shows an oxygen-saturated PBS solution of AN-SiPy (15 μM) / NADPH (240 μM) under illumination (650 nm LED, 40 mW / cm 2 ) UV-visible absorption spectrum of AN-SiPy (15 μM) / NADPH (240 μM) / Cyt c (60 μM) in nitrogen-saturated PBS solution under illumination (650 nm LED, 40 mW / cm 2 )’s UV-visible absorption spectrum changes.

[0021] Figure 8 The phototoxicity and dark toxicity experiments of AN-SiPy at different concentrations under normoxic and hypoxic conditions were conducted respectively.

[0022] Figure 9 Figure 2 is the cell viability graph of A549 cells loaded with AN-SiPy and different cell death inhibitors. DETAILED DESCRIPTION

[0023] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. Example 1

[0024] A heavy atom-free photosensitizer based on silpyronine dye, the structural formula of which is: .

[0025] A method for preparing a heavy atom-free photosensitizer based on silpyronine dye comprises the following steps: Under nitrogen, 9-bromoanthracene (501 mg, 1.95 mmol) was dissolved in ultra-dry tetrahydrofuran (10 mL) and the temperature was maintained at -78°C. n-Butyllithium (1.2 mL, 1.95 mmol, 1.6 M in n-hexane) was added dropwise over 15 minutes, and the reaction was stirred at this temperature for 1 hour. Subsequently, a solution of silpyronine (211 mg, 0.65 mmol) in tetrahydrofuran was slowly added dropwise to the reaction mixture. The reaction mixture was slowly warmed to room temperature and stirred for another 4 hours. After the reaction, 2 M aqueous hydrochloric acid was slowly added to the reaction flask to neutralize the reaction. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and dried to obtain the crude product. The crude product was purified by column chromatography (MeOH / CH2Cl2 = 1 / 10, v / v) to obtain the target compound AN-SiPy (288 mg, 85% yield). 1H NMR (600 MHz, Chloroform-d) δ 8.62 (s, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.48 (dd, J = 12.9, 8.3 Hz, 4H), 7.39 – 7.35 (m, 2H),7.29 (d, J = 2.8 Hz, 2H), 6.66 (d, J = 9.7 Hz, 2H), 6.34 (dd, J = 9.7, 2.5Hz, 2H), 3.34 (s, 12H), 0.77 (s, 6H). 13C NMR (150 MHz, Chloroform-d) δ168.31, 154.28, 148.35, 141.92, 132.48, 130.96, 130.23, 129.03, 128.85,128.52, 127.34, 125.92, 125.89, 121.24, 114.35, 41.32. [M] + : calculated for485.2413, Found 485.2410. Example 2

[0026] 1. Test solution preparation AN-SiPy was prepared in chromatographic-grade acetonitrile to a 2 mM stock solution, which was then diluted to the test concentration using the appropriate solvent according to test requirements. HPF, DHR123, and DCFH were each prepared in chromatographic-grade DMF to a 2 mM stock solution. NADPH and Cyt c were prepared in ultrapure water to 50 mM and 5 mM stock solutions, respectively.

[0027] 2. UV-Vis absorption spectroscopy The absorption spectra of AN-SiPy in different solvents (PBS, H2O and CH3CN) with increasing concentration (1-15 μM) are shown in Figure 2. Figure 4 As shown. In CH3CN and H2O, the maximum absorption peak of AN-SiPy is 660 nm, and the absorbance at 660 nm shows a good linear relationship with the concentration. In PBS, AN-SiPy has only a monomer absorption peak at 660 nm at low concentrations. As the titration concentration increases, a J aggregation peak appears at 706 nm. The appearance of J aggregation can effectively reduce the energy gap from singlet to triplet state (ΔE ST ), thereby improving ISC efficiency, which is beneficial to enhance ROS production.

[0028] 3. In vitro ROS production capacity assessment To ensure that AN-SiPy can produce J aggregation in PBS, we selected 6 μM as the test concentration. First, the DCFH probe was used to evaluate the total ROS generation of AN-SiPy under light irradiation, as shown in Figure 2. Figure 5 As shown in A, AN-SiPy / DCFH solution was exposed to 650 nm (10 mW / cm 2 ) laser irradiation can cause a significant increase in DCF fluorescence signal, indicating that AN-SiPy produces a large amount of ROS under light. In order to determine the contribution of specific reactive oxygen species, ABDA, DHR123 and HPF probes were used to evaluate 1 O2, O2 •− and •OH production efficiency, e.g. Figure 5 As shown in B-5D, AN-SiPy can not only generate energy transfer with O2 under light, 1 O2, and can produce O2 by electron transfer with O2 •− and •OH, indicating that AN-SiPy produces a large amount of ROS through both Type I and Type II pathways. Considering that Type I photosensitizers are hypoxia-dependent, AN-SiPy is expected to overcome the limitation of tumor cell hypoxia during photodynamic therapy. Furthermore, to verify the effect of J aggregation on ROS production, the generation of ROS by AN-SiPy in the presence of TritonX-100 was evaluated. Figure 6 As shown, the AN-SiPy / Triton X-100 / DCFH solution was detected at 650 nm (10 mW / cm 2 ) laser irradiation did not cause any spectral changes, indicating that Triton X-100 disaggregates J aggregates into monomers, thereby eliminating their photodynamic effect. These results suggest that the presence of aggregates can cause excited-state energy level splitting, reducing the ΔEST energy level difference, promoting ISC, and thus leading to ROS generation.

[0029] 4. In vitro photoredox catalysis Furthermore, the effect of AN-SiPy on NADPH / NAPD was tested. + Under normal oxygen conditions, the photocatalytic effect of 650 nm laser (40 mW / cm 2 After irradiation of AN-SiPy (15 μM) / NADH (240 μM) PBS solution, the characteristic absorption peak of NADH at 339 nm decreased rapidly, while NAPD + The characteristic absorption peak at 260 nm increased significantly, which means that AN-SiPy can photocatalyze NADPH oxidation, thereby changing the intracellular NADH / NAD + balance( Figure 7 A). It is worth noting that under normoxic conditions, when AN-SiPy catalyzes the oxidation of NADH, oxygen serves as the terminal electron acceptor. When the oxygen concentration decreases, the rate and efficiency of NADH oxidation will also decrease. Therefore, under hypoxic conditions, cytochrome c (Cyt c) is selected as the electron acceptor, such as Figure 7 As shown in B, a 650 nm laser (40 mW / cm 2 After irradiation of a PBS solution of AN-SiPy (15 μM) / NADH (240 μM) / Cyt c (60 μM) with light, the absorption peaks of Cyt c at 520 nm and 550 nm increased, demonstrating that AN-SiPy catalyzes the oxidation of NADH and the reduction of Cyt c under light, and that this process is oxygen-independent. These results demonstrate that AN-SiPy can catalyze the oxidation of NADH and the reduction of Cyt c under light in an oxygen-independent manner, overcoming the limitation of traditional photodynamic therapy on the oxygen concentration of the tumor microenvironment.

[0030] 5. Photodynamic therapy research at the cellular level A549 cells were cultured in DMEM medium containing 10% FBS (fetal bovine serum), 100 U / mL penicillin G sodium, and 100 μg / mL streptomycin at 37°C in a humidified atmosphere of 5% carbon dioxide. Before the cck8 experiment, cells were plated in 96-well cell culture plates and allowed to rest for 12 hours until the cells attached to the wall. After treatment with different concentrations of AN-SiPy for 1 hour under normoxic (21% O2) and hypoxic (≤2% O2) conditions, the cells were illuminated with a 650 nm laser light source (30 mW / cm 2 , 20 min) and then placed in a cell culture incubator for 24 hours. Figure 8As shown, AN-SiPy exhibits low dark toxicity and high phototoxicity, with half-inhibitory concentrations (IC50) of 6.2 nM and 9.4 nM under normoxic and hypoxic conditions, respectively. The low half-lethal dose can reduce the systemic distribution of the drug and significantly reduce the risk of toxicity, making it a promising therapeutic agent for cancer phototherapy.

[0031] 6. Exploring the Mode of Cell Death To further elucidate the mechanism of AN-SiPy-induced cell death in A549 cells, we determined the cell survival rate when AN-SiPy was co-incubated with different cell death inhibitors. Figure 9 As shown, compared with the group without inhibitor incubation, when A549 cells were co-incubated with apoptosis inhibitor (Z-VAD-FMK), necrosis inhibitor (Nec-1) and autophagy inhibitor (3-MA), the cell survival rate was not significantly improved; in contrast, the experimental group treated with ferroptosis inhibitor (Fer-1) showed a significant increase in cell viability, indicating that ferroptosis may be the main cell death pathway.

[0032] In summary, the present invention successfully constructed a near-infrared photosensitizer, AN-SiPy, based on the fluorescence of silpyronine. This photosensitizer not only combines traditional Type I and Type II photodynamic effects, but also exhibits unique oxygen-independent photoredox catalytic capabilities. The synergistic effect of Type I photodynamic and photoredox catalytic mechanisms enables this photosensitizer to maintain potent antitumor activity even in the hypoxic microenvironment unique to tumor tissue. Notably, the combined action of these three photodynamic therapy mechanisms enables this series of drugs to achieve superior tumor ablation effects at ultra-low concentrations (nM level).

[0033] The above description is only for better explanation of the embodiments of the present invention and is not intended to limit the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention are intended to be within the scope of the present invention.

Claims

1. A heavy atom-free photosensitizer based on silpyronine dye, characterized in that: Its structural formula is: 。 2. The method for preparing the heavy atom-free photosensitizer based on silapyronine dye according to claim 1, characterized in that: The following steps are involved: Under nitrogen protection, 9-bromoanthracene is dissolved in ultra-dry tetrahydrofuran, and n-butyl lithium is then added dropwise, and the reaction is stirred. Subsequently, a tetrahydrofuran solution of silapyrrolidone is added dropwise, and the reaction is continued with stirring. After the reaction is completed, aqueous hydrochloric acid is added for neutralization. The resulting reaction mixture is extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, dried, and purified by column chromatography to obtain the target compound AN-SiPy.

3. The method for preparing a heavy atom-free photosensitizer based on silapyronine dye according to claim 2, wherein: The molar ratio of the 9-bromoanthracene, n-butyl lithium and silapyrrolidone is 3:3:

1.

4. The method for preparing a heavy atom-free photosensitizer based on silapyronine dye according to claim 2, wherein: The stirring reaction was carried out at a temperature of -78°C and for 1 h.

5. The method for preparing a heavy atom-free photosensitizer based on silpyronine dye according to claim 2, wherein: The stirring reaction was continued at room temperature for 4 h.

6. The method for preparing a heavy atom-free photosensitizer based on silpyronine dye according to claim 2, wherein: The concentration of the hydrochloric acid aqueous solution is 2 M.

7. The method for preparing a heavy atom-free photosensitizer based on silpyronine dye according to claim 2, wherein: The column chromatography purification conditions were MeOH / CH2Cl2 = 1 / 10, v / v.

8. Use of the heavy atom-free photosensitizer based on silapyronine dye according to claim 1 in the preparation of drugs for photodynamic therapy of tumors.

9. The use of the heavy atom-free photosensitizer based on silapyronine dye according to claim 8, characterized in that: The photodynamic therapy is a synergistic therapy of type I photodynamic therapy, type II photodynamic therapy and photoredox catalysis.

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