Mitochondrial targeting photosensitizer capable of being activated by glutathione as well as preparation method and application of mitochondrial targeting photosensitizer
By designing a Mito-ProPS, a photosensitizer that is targeted and activated by glutathione, the inaccurate positioning of photosensitizers and damage to normal cells in photodynamic therapy is solved, and efficient and accurate tumor treatment is achieved.
Patent Information
- Application Number
- CN202510106073.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-23
- Publication Date
- 2025-05-13
AI Technical Summary
In existing photodynamic therapies, the positioning of photosensitizers in cells is inaccurate, resulting in low treatment efficiency and easy damage to normal cells, and lacks suborganism targeting of tumor cells.
Mito-ProPS, a photosensitive agent that is targeted and activated by glutathione, is designed to ensure that photosensitizer accumulates on the mitochondria of tumor cells and activates under light, producing ROS, resulting in tumor cell death by combining it with specific conditions in the tumor microenvironment.
It improves the accuracy and efficiency of photodynamic therapy in the tumor treatment direction, reduces damage to normal tissues, and significantly improves the effectiveness of cancer treatment.
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Figure CN119977868A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of photosensitizers, and in particular to a photosensitizer that is targeted to mitochondria and can be activated by glutathione, and a preparation method and application thereof. Background Art
[0002] Photodynamic therapy (PDT) is a new light-activated, non-destructive treatment method. It uses an effective light source to excite photosensitizers, converting oxygen in cells into cytotoxic singlet oxygen, thereby killing tumor cells and achieving the effect of treating tumors. However, in practical applications, photodynamic therapy still has great limitations. For example, because the reactive oxygen species (ROS) produced by photosensitizers have a short lifetime (30-180ns) and a small action distance (<50nm), the localization of photosensitizers in cells greatly affects the therapeutic efficiency of PDT. On the other hand, while photosensitizers damage tumor cells, they also indiscriminately damage normal cells. How to use the differences between normal cells and tumor cells to selectively design photosensitizers that are only activated in tumor cells is of great significance. Based on this, designing a photosensitizer that targets organelles and activates in the tumor cell environment is crucial to improving the accuracy and efficiency of tumor treatment.
[0003] Mitochondria are the center of cell metabolism and are closely related to cell apoptosis. By designing mitochondria-targeted photosensitizers, it is possible to ensure that the photosensitizers accumulate on the mitochondria in tumor cells, thereby generating a large amount of ROS under laser irradiation, effectively leading to mitochondrial dysfunction and cell apoptosis, thereby significantly improving the effect of cancer treatment. However, most mitochondria-targeted photosensitizers require the connection of a triphenylphosphine group (PPh 3 ), which makes the structure and synthesis of this type of photosensitizer molecules particularly complex. It is of practical significance to design a simple photosensitizer that targets mitochondria and can be activated by the tumor cell microenvironment.
[0004] In traditional photodynamic therapy, photosensitizer drugs are usually delivered to the tumor site to achieve the purpose of treatment. However, this delivery method has a significant disadvantage: the drug not only accumulates in the tumor area, but also inevitably spreads to the normal tissue site, causing damage to the normal tissue when activated by light. Activatable photosensitizers utilize the unique physiological conditions of tumor cells, such as acidic microenvironment, overexpressed enzymes, and GSH, as specific triggering factors. These unique tumor environments enable photosensitizers to be activated only at the tumor site, thereby showing their photosensitivity. Under light conditions, the activated photosensitizers can produce singlet oxygen, effectively killing tumor cells, while greatly reducing damage to surrounding normal tissues.
[0005] However, the existing technology still has the problems of poor tumor targeting of photosensitizers and non-specific activation in non-tumor environments. When producing therapeutic effects, they are prone to side effects on normal tissues, and lack of tumor cell subcellular organelle targeting, resulting in low photosensitizer treatment efficiency. Summary of the invention
[0006] In order to comprehensively solve the above problems, the present invention aims to design a new photosensitizer Mito-ProPS, the fluorescence produced by which overlaps greatly with the fluorescence of commercial mitochondrial probes, and its Pearson correlation coefficient is as high as 0.876. Mito-ProPS can selectively and efficiently kill tumor cells (HepG2) under light, without obvious toxicity to normal cells (CHO-K1). The present invention can give full play to the advantages of both by combining mitochondrial targeting and photodynamic therapy activated by the tumor microenvironment. ROS are generated under the irradiation of external light sources, causing mitochondrial dysfunction and causing the death of tumor cells, thereby achieving the purpose of treatment. This strategy not only improves the efficacy of the drug at the tumor site, but also reduces damage to normal tissue sites, and is a more accurate treatment method.
[0007] In order to achieve the above-mentioned purpose, the first aspect of the present invention provides a mitochondrial-targeted photosensitizer that can be activated by glutathione, the chemical name of which is Mito-ProPS, and the structural formula is:
[0008]
[0009] The second aspect of the present invention provides a method for preparing a mitochondrial-targeted photosensitizer that can be activated by glutathione, comprising:
[0010] Step 1: Synthesize Mito-PS using 1,1,2,3-tetramethyl-1H-benzo[e]indole and 4-hydroxy-3,5-diiodobenzaldehyde as raw materials;
[0011] Step 2: Dissolve Mito-PS and 2-(bromomethyl)cyclohex-2-en-1-one in acetonitrile, then add N,N-diisopropylethylamine, react under argon protection at room temperature, remove the solvent by rotary evaporation, and purify by column chromatography using dichloromethane and methanol as eluents to obtain compound Mito-ProPS, with the structural formula
[0012] Preferably, step 1 comprises:
[0013] Step 1.1: Dissolve 1,1,2,3-tetramethyl-1H-benzo[e]indole and 4-hydroxy-3,5-diiodobenzaldehyde in ethanol;
[0014] Step 1.2: piperidine was then added, and the reaction solution was heated to reflux under argon protection. After cooling to room temperature, the solvent was removed by rotary evaporation, and column chromatography was performed using dichloromethane and methanol as eluents to obtain the pure purple compound Mito-PS.
[0015] Preferably, the molar ratio of 1,1,2,3-tetramethyl-1H-benzo[e]indole to 4-hydroxy-3,5-diiodobenzaldehyde is 1:1.5.
[0016] Preferably, step 2 comprises:
[0017] Step 2.1: Dissolve Mito-PS and 2-(bromomethyl)cyclohex-2-en-1-one in acetonitrile, then add N,N-diisopropylethylamine, react under argon protection at room temperature, and remove the solvent by rotary evaporation;
[0018] Step 2.2: Column chromatography was performed using dichloromethane and methanol as eluents to purify the compound Mito-ProPS.
[0019] Preferably, the molar ratio of Mito-PS to 2-(bromomethyl)cyclohex-2-en-1-one is 1:2.
[0020] The third aspect of the present invention provides the use of the above-mentioned mitochondrial-targeted photosensitizer that can be activated by glutathione as a preparation of an anti-tumor photosensitizer drug.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] 1. The fluorescence produced by the Mito-ProPS prepared by the present invention after being activated by tumor cells has a great overlap with the fluorescence of commercial mitochondrial probes, and the Pearson correlation coefficient is as high as 0.876, indicating that the activated photosensitizer can be well positioned in the mitochondria of tumor cells, thereby improving the accuracy and efficiency of photodynamic therapy in tumor treatment.
[0023] 2. The Mito-ProPS prepared by the present invention can be activated by GSH in the tumor cell environment, and then kill the tumor cells under the action of light, but cannot be activated in normal cells, thus avoiding side effects on normal tissues, improving the selectivity of photodynamic therapy, and reducing the occurrence of side effects.
[0024] 3. The Mito-ProPS prepared by the present invention can selectively and efficiently kill tumor cells (HepG2) under light irradiation, but has no obvious toxicity to normal cells (CHO-K1). BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.
[0026] In the attached picture:
[0027] Figure 1 .The structure of Mito-ProPS, a photosensitizer targeted to mitochondria and activated by GSH;
[0028] Figure 2 .Structure of Mito-PS, a mitochondrial-targeted, non-activating photosensitizer;
[0029] Figure 3 .Synthesis of Mito-PS and Mito-ProPS;
[0030] Figure 4 .The fluorescence intensity of Mito-ProPS before and after activation by GSH;
[0031] Figure 5 .The ROS production efficiency of Mito-ProPS before and after activation by GSH using DPBF as an indicator;
[0032] Figure 6 .Specific localization of Mito-ProPS in tumor cells (compared with commercial mitochondrial probes);
[0033] Figure 7 .Mito-ProPS and Mito-PS kill tumor cells and normal cells. Figure 7 In the figure, (a) dark toxicity and phototoxicity (MTT) of Mito-ProPS and Mito-PS on tumor cells HepG2; (b) dark toxicity and phototoxicity (MTT) of Mito-ProPS and Mito-PS on normal cells CHO-K1. DETAILED DESCRIPTION
[0034] The following combination Figure 1-Figure 7 The preferred embodiments of the present invention are described. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.
[0035] Embodiment 1:
[0036] A photosensitizer that targets mitochondria and can be activated by glutathione, with the chemical name: Mito-ProPS, and the structural formula is: The chemical name is (E)-2-(3,5-diiodo-4-((6-oxocyclohex-1-en-1-yl)methoxy)phenylvinyl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium.
[0037] Embodiment 2:
[0038] A method for preparing a mitochondrial-targeted photosensitizer that can be activated by glutathione, comprising:
[0039] Step 1: 1,1,2,3-tetramethyl-1H-benzo[e]indole and 4-hydroxy-3,5-diiodobenzaldehyde Mito-PS was synthesized as raw materials;
[0040] Step 1.1: (0.22 g, 1 mmol) 1,1,2,3-tetramethyl-1H-benzo[e]indole and (0.56 g, 1.5 mmol) 4-hydroxy-3,5-diiodobenzaldehyde Dissolve in 20 mL of ethanol;
[0041] Step 1.2: Then add 2 mL of piperidine, heat the reaction solution under argon protection and reflux for 12 hours, cool to room temperature, remove the solvent by rotary evaporation, and purify by column chromatography using dichloromethane and methanol as eluents to obtain a pure purple compound Mito-PS, the structural formula of which is:
[0042] Mito-PS (0.3 g, 51.2%). 1 H NMR (400 MHz, CDCl 3 )δ8.21(s,2H),8.00(dd,J=8.0,1.2Hz,1H),7.88(d,J=7.2Hz 1H),7.61-7.55(m,1H),7.52(td,J=7.2,1.2Hz,1H),7.49(s,2H),7.42-7.38(m,1H),6.57(d,J=14.4Hz,1H),3.07(s,3H),1.81(s,6H). 13 C NMR (100 MHz, DMSO-d 6 )δ154.22,144.36,142.88,141.24,137.15,132.03,131.36,129.72,129.52,128.75,127. 73,125.86,125.14,124.18,122.66,117.16,88.51,54.01,32.50,21.80.LCMS(ESI):mass calcd.for C 23 H 20 I 2 NO + 580.0,m / z found 580.0[M] + .
[0043] Step 2: (0.29 g, 0.5 mmol) Mito-PS and (0.19 g, 1 mmol) 2-(bromomethyl)cyclohex-2-en-1-one Dissolve in 10 mL of acetonitrile, then add 0.5 mL of N,N-diisopropylethylamine (DIPEA), react under argon protection at room temperature for 12 hours, remove the solvent by rotary evaporation, and purify by column chromatography using dichloromethane and methanol as eluents to obtain the compound Mito-ProPS (0.19 g, 55.9%), with the structural formula The chemical name is (E)-2-(3,5-diiodo-4-((6-oxocyclohex-1-en-1-yl)methoxy)phenylvinyl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium.
[0044] 1 H NMR (400 MHz, DMSO-d 6 )δ8.23-8.18(m,2H),8.00(dd,J=8.0,1.2Hz,1H),8.01(d,J=14.4Hz,1H),7.81(d,J=7.2Hz,1H),7.55-7.49(m,3H),7.43-7.35( m,1H),6.55(d,J=14.4Hz,1H),6.48-6.42(m,1H),4.61(s,2H),3.05(s,3H),2.52-2.44(m,2H),2.05-1.96(m,4H),1.83(s,6H). 13 C NMR(100MHz,DMSO-d6)δ201.96,161.22,158.49,146.88,145.58,143.62,134.03,133.15,132.91,132.78,131.84,131.68,130 .28,127.53,127.32127.17,123.49,120.00,119.37,87.87,68.53,54.09,39.19,36.73,28.03,26.94,22.13.LCMS(ESI):mass calcd.for C 30 H 28 I 2 NO 2+ 688.0,m / z found 688.1[M] + .
[0045] Embodiment 3:
[0046] The mitochondria-targeted photosensitizer that can be activated by glutathione prepared in the above example is used as an anti-tumor photosensitizer drug.
[0047] experiment:
[0048] 1. Fluorescence intensity of Mito-ProPS before and after activation by GSH
[0049] The specific operation is to add GSH (final concentration 5 mM) to the PBS solution of Mito-ProPS (1 μM), and record the fluorescence intensity of Mito-ProPS at the maximum excitation wavelength (620 nm) as the reaction time increases.
[0050] 2. ROS production efficiency of Mito-ProPS before and after activation by GSH using DPBF as an indicator
[0051] The specific operation is to add GSH (final concentration 5mM) to the PBS solution of Mito-ProPS (1μM), react for 4 hours, then add 1,3-diphenylisobenzofuran (DPBF, final concentration 30μM) thereto, and then illuminate the above solution. The light source consists of a 300-watt halogen lamp, a water tank for cooling, and a colored glass filter, which cuts off at 520nm. The luminous flux rate for wavelengths greater than 520nm is 18 milliwatts / square centimeter. The rate of DPBF degradation by Mito-ProPS before and after GSH treatment at different illumination times was investigated, that is, the rate at which the absorption of DPBF at 415nm decreases. This rate reflects the efficiency of ROS produced by the photosensitizer. The faster the degradation rate, the higher the efficiency of ROS production by the photosensitizer. By Figure 5 The experimental results show that after Mito-ProPS is activated by GSH, the degradation rate of DPBF is greatly increased and its ROS production efficiency is greatly improved.
[0052] 3. Specific localization of Mito-ProPS in tumor cells
[0053] The specific operation is to co-incubate Mito-ProPS (2μM) with human liver cancer cell HepG2 cells for 4 hours, then wash with PBS (2ml×3). Then co-incubate with commercial mitochondrial probe (0.5μM) with HT-29 cells for 15 minutes, wash with PBS (2ml×3) and observe under a fluorescence microscope. As can be seen from the results in the figure, the fluorescence generated by Mito-ProPS and the fluorescence of the commercial mitochondrial probe have a great overlap, and the Pearson correlation coefficient is as high as 0.876.
[0054] 4. Mito-ProPS and Mito-PS kill tumor cells and normal cells
[0055] The specific operation of the dark cell toxicity test is to incubate different concentrations of Mito-ProPS or Mito-PS (4μM, 3μM, 2μM, 1μM, 0.5μM) with human liver cancer cells HepG2 (high GSH) or Chinese hamster ovary cells CHO-K1 (low GSH) for 4 hours, then wash with PBS, and then continue to culture for 20 hours, and use thiazolyl blue (MTT) to determine the degree of cell killing at different concentrations. Figure 7 (a) and 7(b), it can be seen that neither Mito-ProPS nor Mito-PS has obvious dark toxicity to tumor cells (HepG2) or normal cells (CHO-K1), indicating that the material itself has good biocompatibility.
[0056] The specific operation of the cell phototoxicity test is to incubate different concentrations of Mito-ProPS or Mito-PS (4μM, 3μM, 2μM, 1μM, 0.5μM) with human liver cancer cells HepG2 or Chinese hamster ovary cells CHO-K1 for 4 hours, then wash with PBS, and then illuminate with the above light source for 1 minute, and then continue to culture for 20 hours, and use thiazolyl blue (MTT) to determine the degree of cell killing at different drug concentrations. Figure 7 (a) and 7(b) show that Mito-ProPS can selectively and efficiently kill tumor cells (HepG2) under light irradiation, but has no obvious toxicity to normal cells (CHO-K1); while Mito-PS can indiscriminately kill tumor cells (HepG2) and normal cells (CHO-K1). This result shows that the GSH activation strategy of Mito-ProPS is of great significance for improving the targeting and specificity of photodynamic therapy.
[0057] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.
Claims
1. A photosensitizer that targets mitochondria and can be activated by glutathione, characterized in that: The chemical name is: Mito-ProPS, and the structural formula is:
2. A method for preparing a mitochondrial-targeted photosensitizer that can be activated by glutathione, characterized in that: include Step 1: Synthesize Mito-PS using 1,1,2,3-tetramethyl-1H-benzo[e]indole and 4-hydroxy-3,5-diiodobenzaldehyde as raw materials; Step 2: Dissolve Mito-PS and 2-(bromomethyl)cyclohex-2-en-1-one in acetonitrile, then add N,N-diisopropylethylamine, react under argon protection at room temperature, remove the solvent by rotary evaporation, and purify by column chromatography using dichloromethane and methanol as eluents to obtain compound Mito-ProPS, with the structural formula 3. The method for preparing a mitochondrial-targeted photosensitizer that can be activated by glutathione according to claim 2, characterized in that: Step 1 includes: Step 1.1: Dissolve 1,1,2,3-tetramethyl-1H-benzo[e]indole and 4-hydroxy-3,5-diiodobenzaldehyde in ethanol; Step 1.2: piperidine was then added, and the reaction solution was heated to reflux under argon protection. After cooling to room temperature, the solvent was removed by rotary evaporation, and column chromatography was performed using dichloromethane and methanol as eluents to obtain the pure purple compound Mito-PS.
4. The method for preparing a mitochondrial-targeted photosensitizer that can be activated by glutathione according to claim 3, characterized in that: The molar ratio of 1,1,2,3-tetramethyl-1H-benzo[e]indole to 4-hydroxy-3,5-diiodobenzaldehyde is 1:1.
5.
5. The method for preparing a mitochondrial-targeted photosensitizer that can be activated by glutathione according to claim 4, characterized in that: Step 2 includes: Step 2.1: Dissolve Mito-PS and 2-(bromomethyl)cyclohex-2-en-1-one in acetonitrile, then add N,N-diisopropylethylamine, react under argon protection at room temperature, and remove the solvent by rotary evaporation; Step 2.2: Column chromatography was performed using dichloromethane and methanol as eluents to purify the compound Mito-ProPS.
6. The use of a mitochondrial-targeted photosensitizer that can be activated by glutathione as claimed in claim 1, characterized in that: The molar ratio of Mito-PS to 2-(bromomethyl)cyclohex-2-en-1-one was 1:
2.
7. The use of a mitochondrial-targeted photosensitizer that can be activated by glutathione as claimed in claim 1, characterized in that: Use as a preparation of anti-tumor photosensitizer drugs.