Preparation method and application of plasma membrane targeted heavy atom-free photosensitizer
By introducing lipophilic n-octane and anthracene groups into silicon rhodamine dyes, a photosensitizer Mem-ANSiR that can specifically target cell membranes was developed, which solved the problems of short ROS half-life and limited diffusion distance, achieved efficient cancer cell killing under normoxic and hypoxic conditions, and improved the therapeutic effect of photodynamic therapy.
Patent Information
- Application Number
- CN202510720579.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-09-05
AI Technical Summary
Existing photodynamic therapy cannot effectively oxidize cell membrane structures in tumor treatment due to the short half-life and limited diffusion distance of ROS, resulting in poor treatment effect.
A plasma membrane-targeted heavy atom-free photosensitizer Mem-ANSiR was designed. By introducing a lipophilic n-octane group at the silicon bridge position of silorhodamine and an anthracene group at the central position, the SOCT-ISC mechanism was utilized to efficiently generate 1O2 and O2•− under light, achieving specific targeting and synergistic reaction to the cell membrane.
Mem-ANSiR can efficiently induce ferroptosis of cancer cells under normoxic and hypoxic conditions, reduce damage to normal tissues, and improve the therapeutic effect of photodynamic therapy.
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Figure CN120590430A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a preparation method and application of a cytoplasmic membrane-targeted heavy atom-free photosensitizer. Background Art
[0002] Ferroptosis is an iron-dependent form of programmed cell death. Its molecular mechanism involves the abnormal accumulation of lipid peroxidation (LPO) in cell membrane systems such as the plasma membrane, mitochondrial membrane, endoplasmic reticulum membrane and lipid droplets. Cancer cells have a greater demand for iron than normal cells and are more susceptible to ferroptosis. Therefore, inducing ferroptosis has important therapeutic potential in overcoming tumor drug resistance and metastasis. Given its advantages such as non-invasiveness, high spatiotemporal resolution and precise controllability, photodynamic therapy (PDT) has become an extremely attractive therapeutic strategy in the field of tumor treatment and an ideal method for inducing LPO to trigger ferroptosis. In recent years, researchers have developed a variety of photosensitizer systems that specifically activate ferroptosis pathways. However, due to the short half-life and limited diffusion distance of reactive oxygen species (ROS) generated during the treatment process [for example, the half-life of singlet oxygen (¹O2) in cells is less than 250 nanoseconds and the diffusion distance is less than 45 nanometers; superoxide anions (O2 • ⁻) Due to their small molecular size and negative charge (pKa = 4.8), they are unable to penetrate the cell membrane and effectively oxidize the membrane structure within the cell, thus preventing the PDT effect from being maximized. Therefore, the location of ROS generation within the cell is crucial for enhancing the PDT effect. The plasma membrane is a thin membrane structure surrounding the cell, responsible for separating the internal and external environments of the cell, regulating the entry and exit of substances, and participating in intercellular signaling and interaction. Therefore, the ROS generated by photosensitizers that can specifically target the plasma membrane under light can directly react with macromolecules such as phospholipids, enzymes, and polyunsaturated fatty acid side chains of membrane receptors and nucleic acids in the biological membrane, triggering lipid peroxidation reactions, leading to significant changes in the fluidity and permeability of the cell membrane, and ultimately inducing rapid cell death. Summary of the Invention
[0003] In view of this, the present invention introduces two lipophilic n-octane groups at the silicon bridge position of silorhodamine to design a dye Mem-SiR that can selectively target the cell membrane. On this basis, by introducing an anthracene group at its central position, a photosensitizer Mem-ANSiR that can selectively target the cell membrane was developed based on the SOCT-ISC mechanism. This photosensitizer can be efficiently generated simultaneously through type I and type II reactions. 1 O2 and O2 •− The synergistic effect of type I and type II pathways enables it to effectively induce ferroptosis in cancer cells under both normoxic (21% O2) and hypoxic (1% O2) conditions.
[0004] To achieve the above object, the technical solution of the present invention is as follows: A cell membrane-targeted heavy atom-free photosensitizer, the structural formula of which is: .
[0005] A method for preparing the above-mentioned plasma membrane-targeted heavy atom-free photosensitizer comprises the following steps: Step 1: Under nitrogen protection, compound 1 is dissolved in ultra-dry tetrahydrofuran, the temperature is controlled at -78°C, n-butyl lithium is added, and the temperature is maintained at this temperature and stirred to react to obtain a mixed solution; a tetrahydrofuran solution of dichlorodioctylsilane is added to the above mixed solution, and the stirring reaction is continued; after the reaction is completed, aqueous hydrochloric acid is added for neutralization, and the reaction product is extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and dried to obtain compound 2; Step 2: Compound 2 was dissolved in acetone, the temperature was controlled at 0°C, potassium permanganate powder was added, and the reaction was stirred while maintaining the temperature; the reaction product was filtered through diatomaceous earth, the yellow filtrate was dried, and the crude product was purified by column chromatography to obtain compound 3; Step 3: Under nitrogen protection, 2-methylbromobenzene or 9-bromoanthracene is dissolved in ultra-dry tetrahydrofuran, the temperature is controlled at -78°C, n-butyl lithium is added, and the temperature is maintained at this temperature and stirred to react to obtain a mixed solution; the tetrahydrofuran solution of compound 3 is added to the above mixed solution, and the stirring reaction is continued; after the reaction is completed, aqueous hydrochloric acid is added for neutralization, and the reaction product is extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, dried, and purified by column chromatography to obtain the target compound Mem-ANSiR; The structural formula of compound 1 is as follows: .
[0006] Furthermore, in step 1, the molar ratio of compound 1, n-butyl lithium and dichlorodioctylsilane is 1:4:1.8; and the concentration of the hydrochloric acid aqueous solution is 1 M.
[0007] Furthermore, the stirring reaction time in step 1 is 2 h; the stirring reaction temperature is continued to be room temperature and the time is 2 h.
[0008] Furthermore, in step 2, the molar ratio of compound 2 to potassium permanganate is 1:1.8.
[0009] Furthermore, the stirring reaction temperature in step 2 is room temperature and the time is 5 h; the column chromatography purification condition is EA / PE = 1 / 100, v / v.
[0010] Furthermore, in step 3, the molar ratio of 2-methylbromobenzene (or 9-bromoanthracene), n-butyllithium and compound 3 is 5:5:1; and the concentration of the hydrochloric acid aqueous solution is 1 M.
[0011] Furthermore, the stirring reaction time in step 3 is 30 minutes; the stirring reaction temperature is continued to be room temperature, and the time is overnight; the column chromatography purification condition is MeOH / CH2Cl2=1 / 30, v / v.
[0012] Application of the plasma membrane-targeted heavy atom-free photosensitizer as described above in the preparation of anti-tumor drugs.
[0013] An anti-tumor drug, the active ingredient of which contains the above-mentioned cell plasma membrane-targeted heavy atom-free photosensitizer.
[0014] Compared with the prior art, the present invention has the following beneficial effects: Photosensitizers with subcellular targeting can precisely accumulate in specific organelles, triggering specific cell death mechanisms, ensuring maximum therapeutic effect while minimizing damage to normal tissues. Traditional photosensitizers, due to their lack of targeting, can lead to uneven drug distribution and reduced therapeutic efficacy. The heavy atom-free photosensitizers provided by the present invention have excellent cell membrane targeting. The ROS generated during PDT can directly trigger lipid peroxidation reactions, damage the structure and function of cell membranes, and induce ferroptosis, showing great potential. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 Compound 3 of the present invention 1 HNMR spectrum.
[0016] Figure 2 Compound 3 of the present invention 13 CNMR image.
[0017] Figure 3 HRMS chart of compound 3 of the present invention.
[0018] Figure 4 The compound Mem-SiR of the present invention 1 HNMR spectrum.
[0019] Figure 5 The compound Mem-SiR of the present invention 13 CNMR image.
[0020] Figure 6 HRMS chart of Mem-SiR compound of the present invention.
[0021] Figure 7 The compound Mem-ANSiR of the present invention 1 HNMR spectrum.
[0022] Figure 8 The compound Mem-ANSiR of the present invention 13CNMR image.
[0023] Figure 9 HRMS chart of the compound Mem-ANSiR of the present invention.
[0024] Figure 10 Confocal images of A549 cells loaded with Mem-SiR (2 μM, 10 min).
[0025] Figure 11 (A) is the ionization of 1,3-diphenylisobenzofuran (DPBF) under laser (650 nm, 20 mW / cm 2 ) captures the singlet oxygen produced by Mem-SiR under the irradiation of UV-visible absorption spectrum; (B) DHR 123 is captured by laser (650 nm, 10 mW / cm 2 ) captures the fluorescence spectrum change of superoxide produced by Mem-SiR under irradiation; (C) is the fluorescence spectrum change of 1,3-diphenylisobenzofuran (DPBF) under laser (650 nm, 20 mW / cm 2 ) is the UV-visible absorption spectrum change of singlet oxygen produced by Mem-ANSiR under laser (650 nm, 10 mW / cm 2 ) captures the fluorescence spectrum changes of Mem-ANSiR producing superoxide under irradiation.
[0026] Figure 12 The phototoxicity and dark toxicity experiments were conducted under normoxic and hypoxic conditions at different concentrations of Mem-ANSiR.
[0027] Figure 13 Cell viability graph of A549 cells loaded with Mem-ANSiR and different death inhibitors.
[0028] Figure 14 Confocal images of A549 cells pre-incubated with Mem-ANSiR and the commercial lipid peroxidation probe C11-BODIPY before and after illumination. DETAILED DESCRIPTION
[0029] 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
[0030] A cell membrane-targeted heavy atom-free photosensitizer, the structural formula of which is: .
[0031] A method for preparing a plasma membrane-targeted heavy atom-free photosensitizer comprises the following steps:
[0032] (1) Under nitrogen protection, compound 1 (1.00 g, 2.4 mmol) was dissolved in ultra-dry tetrahydrofuran (15 mL) and the temperature was controlled at -78°C. n-Butyl lithium (1.6 M in n-hexane, 6.1 mL, 9.7 mmol) was added dropwise to the reaction solution over 30 min, and the mixture was stirred and reacted at this temperature for 2 hours. Dioctyldichlorosilane (1.5 mL, 4.4 mmol) was dissolved in 10 mL of tetrahydrofuran and the solution was slowly added dropwise to the mixture. The reaction solution was slowly warmed to room temperature and stirred for 2 hours. After the reaction was completed, 1 M aqueous hydrochloric acid was slowly added to the reaction flask to neutralize the reaction solution. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and dried to obtain compound 2.
[0033] (2) Compound 2 was dissolved in acetone (25 mL) and the temperature was maintained at 0°C. Potassium permanganate powder (1.5 g) was slowly added to the reaction solution in batches. The reaction solution was slowly warmed to room temperature and stirred for 5 hours. The reaction solution was filtered through diatomaceous earth, and the yellow filtrate was dried by rotary evaporation. The crude product was separated by column chromatography (EA:PE = 1:100) to obtain a light yellow solid compound 3 (176 mg, 14% yield). 1 H NMR (600 MHz, CDCl3) δ 8.40 (d, J = 9.0 Hz, 2H), 6.84 (dd, J = 9.0,2.7 Hz, 2H), 6.78 (d, J = 2.9 Hz, 2H), 3.09 (s, 12H), 1.28 – 1.12 (m, 24H), 0.98 – 0.93 (m, 4H), 0.83 (t, J = 7.2 Hz, 6H). 13 C NMR (151 MHz, CDCl3) δ185.54, 151.20, 139.02, 131.64, 130.56, 114.57, 113.09, 40.08, 33.37, 31.86,29.19, 29.05, 23.73, 22.64, 14.12, 14.10. ESI-MS [M+H] + : calcd for 521.3922,Found 521.39221. (3) Under nitrogen protection, 2-methylbromobenzene or 9-bromoanthracene (5 mmol) was dissolved in ultra-dry tetrahydrofuran (5 mL). The temperature was controlled at -78°C. n-Butyl lithium (1.6 M in n-hexane, 5 mmol) was added dropwise to the reaction solution over 15 minutes. The temperature was maintained and stirred for 30 minutes. Subsequently, a tetrahydrofuran solution of compound 3 (50 mg, 1 mmol) was slowly added dropwise to the reaction solution. The reaction solution was slowly warmed to room temperature and stirred overnight. After the reaction was completed, 1 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 a crude product. The crude product was purified by column chromatography (MeOH / CH2Cl2 = 1 / 30, v / v) to obtain the target compound.
[0034] Mem-SiR (43 mg, 75% yield). 1 H NMR (600 MHz, CD3OD) δ 7.47 (t, J = 11.4 Hz,1H), 7.38 (m, 2H), 7.33 (d, J = 4.8 Hz, 2H), 7.10 (t, J = 14.4 Hz, 3H), 6.81 (m, 2H), 3.35 (s, 12H), 2.03 (s, 3H), 1.21 (m, 28H), 0.84 (q, J = 10.8 Hz, 6H); 13 CNMR (150 MHz, CD3OD) δ 169.73, 154.20, 147.14, 141.06, 138.78, 135.51,129.97, 128.68, 127.89, 125.40, 120.68, 113.91, 39.52, 32.34, 31.54, 28.69,23.22, 22.32, 18.19, 13.66, 13.36, 13.02; ESI-MS [M]+: calcd for 595.4443, Found 595.4442. Mem-ANSiR (39 mg, 57% yield). 1H NMR (600 MHz, CDCl3) δ 8.64 (s, 1H), 8.10(d, J = 8.4 Hz, 2H), 7.48 (t, J = 8.8 Hz, 5H), 7.35 (t, J = 7.7 Hz, 2H), 7.20(s, 2H), 6.68 (d, J 0.84 (t, J = 6.8 Hz, 6H). 13 C NMR (151 MHz, CDCl3) δ 168.63, 153.99, 147.07,141.95, 132.38, 130.86, 130.12, 129.52, 128.78, 128.47, 127.15, 125.74,125.67, 120.71, 114.33, 40.97, 33.17, 31.87, 29.28, 29.19, 23.84, 22.65,14.56, 14.46, 14.19, 14.09. ESI-MS: calcd for 681.4599, Found 681.4589. Example 2
[0035] 1. Test solution preparation Mem-SiR and Mem-ANSiR were prepared as 2 mM stock solutions in chromatographically grade DMSO and subsequently diluted to the test concentrations in the appropriate solvents according to different test requirements. DPBF and DHR123 were each prepared as 2 mM stock solutions in chromatographically grade DMF.
[0036] 2. Cellular Targeting Studies of Dyes First, laser confocal microscopy was used to investigate the targeting of the dye Mem-SiR in A549 cells. Figure 10 As shown in the figure, A549 cells pre-loaded with Mem-SiR exhibited a strong fluorescence signal only on the cell membrane. These results indicate that the lipophilic n-octane structure of the Mem-SiR dye can be embedded in the cell membrane, while the positively charged silicon rhodamine skeleton can interact with the negatively charged phospholipids. This dual interaction gives the Mem-SiR dye excellent cell membrane targeting ability.
[0037] 3. In vitro ROS production capacity assessment Furthermore, in order to endow the dye Mem-SiR with photodynamic properties, an anthracene group was introduced at the center of silorhodamine, and a photosensitizer Mem-ANSiR that can selectively target the cell membrane was developed based on the SOCT-ISC mechanism. Then, the commercial DPBF and DHR123 probes were used to evaluate the photodynamic properties of Mem-ANSiR under light. 1 O2 and O2 •− situations such as Figure 11 As shown in A, the Mem-ANSiR / DPBF solution was exposed to 650 nm (10 mW / cm 2 ) laser irradiation can cause a significant decrease in the absorbance value at 410 nm, indicating that Mem-ANSiR produces a large amount of 1 O2. Figure 11 As shown in B, the Mem-ANSiR / DHR123 solution was exposed to 650 nm (10 mW / cm 2 ) laser irradiation can cause a significant increase in the fluorescence intensity at 530 nm, indicating that Mem-ANSiR produces a large amount of O2 •− In contrast, the dye Mem-SiR at 650 nm (10 mW / cm 2 ) laser irradiation will not cause any change in the spectrum ( Figure 11 C and Figure 11 D). The above results show that Mem-ANSiR can not only transfer energy with O2 under light, but also generate 1 O2 (Type I), and can undergo electron transfer with O2 to produce O2 •− (Type II), because Type I photosensitizers are hypoxia-dependent, Mem-ANSiR is expected to overcome the limitation of tumor cell hypoxia during photodynamic therapy.
[0038] 4. Photodynamic therapy research at the cellular level Given that Mem-ANSiR can synergistically produce a large amount of 1 O2 and O2 •− We further evaluated its photodynamic therapy effect using the cck8 experiment. Under normoxic (21% O2) and hypoxic (≤2% O2) conditions, cells were first treated with different concentration gradients of Mem-ANSiR for 1 hour, and then treated with (or without) a 650 nm laser light source (30 mW / cm 2 , 5 min) and then placed in a cell culture incubator for 24 hours. Figure 12As shown in Figure 3, Mem-ANSiR exhibited low dark toxicity and high phototoxicity under both normoxic and hypoxic conditions. To further elucidate the mechanism of cell death induced by Mem-ANSiR, we determined the cell survival rate when it was co-incubated with different death inhibitors. Figure 13 As shown in the figure, compared with the unincubated inhibitor group, when the cells were co-incubated with apoptosis inhibitors (Z-VAD-FMK), necrosis inhibitors (Nec-1), pyroptosis inhibitors (VX-765) and autophagy inhibitors (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. Figure 14 As shown in the figure, Mem-ANSiR can cause significant lipid peroxidation in cells after light exposure, further confirming that the mode of cell death induced by Mem-ANSiR is ferroptosis.
[0039] In summary, the present invention has developed a dye Mem-SiR that can specifically target the cell membrane by introducing two lipophilic n-octane groups at the silicon bridge position of the silicon rhodamine dye. The n-octane structure can be embedded in the lipid layer of the cell membrane through hydrophobic interaction, while the positively charged silicon rhodamine skeleton can electrostatically interact with the negatively charged phospholipid molecules. In order to give the dye Mem-SiR photodynamic properties, an anthracene group was introduced at the center position of silicon rhodamine, and a photodynamic photosensitizer Mem-ANSiR was developed based on the SOCT-ISC mechanism. This photosensitizer not only retains excellent cell membrane targeting properties, but also can produce through type I and type II pathways under light irradiation. 1 O2 and O2 •− , effectively triggering the ferroptosis pathway through lipid peroxide accumulation. Notably, Mem-ANSiR can still effectively kill cancer cells in a hypoxic microenvironment, providing a new photodynamic tool for solid tumor treatment.
[0040] 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 plasma membrane-targeted heavy atom-free photosensitizer, characterized in that: Its structural formula is: 。 2. A method for preparing the plasma membrane-targeted heavy atom-free photosensitizer according to claim 1, characterized in that: The following steps are involved: Step 1: Under nitrogen protection, compound 1 is dissolved in ultra-dry tetrahydrofuran, the temperature is controlled at -78°C, n-butyl lithium is added, and the temperature is maintained at this temperature and stirred to react to obtain a mixed solution; a tetrahydrofuran solution of dichlorodioctylsilane is added to the above mixed solution, and the stirring reaction is continued; after the reaction is completed, aqueous hydrochloric acid is added for neutralization, and the reaction product is extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and dried to obtain compound 2; Step 2: Compound 2 was dissolved in acetone, the temperature was controlled at 0°C, potassium permanganate powder was added, and the reaction was stirred while maintaining the temperature; the reaction product was filtered through diatomaceous earth, the yellow filtrate was dried, and the crude product was purified by column chromatography to obtain compound 3; Step 3: Under nitrogen protection, 2-methylbromobenzene or 9-bromoanthracene is dissolved in ultra-dry tetrahydrofuran, the temperature is controlled at -78°C, n-butyl lithium is added, and the temperature is maintained at this temperature and stirred to react to obtain a mixed solution; the tetrahydrofuran solution of compound 3 is added to the above mixed solution, and the stirring reaction is continued; after the reaction is completed, aqueous hydrochloric acid is added for neutralization, and the reaction product is extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, dried, and purified by column chromatography to obtain the target compound Mem-ANSiR; The structural formula of compound 1 is as follows: 。 3. The method for preparing a plasma membrane-targeted heavy atom-free photosensitizer according to claim 2, wherein: In step 1, the molar ratio of compound 1, n-butyl lithium and dichlorodioctylsilane is 1:4:1.8; and the concentration of the hydrochloric acid aqueous solution is 1 M.
4. The method for preparing a plasma membrane-targeted heavy atom-free photosensitizer according to claim 2, wherein: The stirring reaction time in step 1 is 2 h; the stirring reaction temperature is continued to be room temperature for 2 h.
5. The method for preparing a plasma membrane-targeted heavy atom-free photosensitizer according to claim 2, characterized in that: In step 2, the molar ratio of compound 2 to potassium permanganate is 1:1.
8.
6. The method for preparing a plasma membrane-targeted heavy atom-free photosensitizer according to claim 2, wherein: The stirring reaction temperature in step 2 was room temperature for 5 h; the column chromatography purification condition was EA / PE = 1 / 100, v / v.
7. The method for preparing a plasma membrane-targeted heavy atom-free photosensitizer according to claim 2, wherein: In step 3, the molar ratio of 2-methylbromobenzene (or 9-bromoanthracene), n-butyllithium and compound 3 is 5:5:1; and the concentration of the hydrochloric acid aqueous solution is 1 M.
8. The method for preparing a plasma membrane-targeted heavy atom-free photosensitizer according to claim 2, wherein: The stirring reaction time in step 3 was 30 minutes; the stirring reaction temperature was continued at room temperature and the time was overnight; the column chromatography purification conditions were MeOH / CH2Cl2 = 1 / 30, v / v.
9. Use of the plasma membrane-targeted heavy atom-free photosensitizer according to claim 1 in the preparation of anti-tumor drugs.
10. An anti-tumor drug, characterized in that: The active ingredient contains the cell plasma membrane-targeted heavy atom-free photosensitizer according to claim 1.