Photosensitizer and its preparation method and application
By designing a new photosensitizer that can induce non-programmed cell death after light and has the ability to self-report fluorescence signal, the complex problems of drug resistance, off-target toxicity and therapeutic response evaluation in photodynamic therapy are solved, and efficient and safe tumor treatment effects are achieved.
Patent Information
- Application Number
- CN202211610847.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-14
- Publication Date
- 2025-08-26
- Estimated Expiration
- 2042-12-14
AI Technical Summary
Existing photosensitizers have complex problems in photodynamic therapy, such as drug resistance, off-target toxicity, tumor hypoxia and treatment response evaluation, and lack the ability to self-report fluorescence signal, which affects the therapeutic effect and safety.
A new type of photosensitizer is designed, whose chemical structure can induce non-programmed death of cells after light and self-report through fluorescence signals. The specific preparation method includes a multi-step synthesis process, using specific intermediates and raw materials to react to generate photosensitizers with near-infrared fluorescence characteristics and convert to red fluorescence.
This photosensitizer has strong tumor cell phototoxicity under normal and hypoxia conditions, which can efficiently kill tumor cells, avoid drug resistance, and monitor the treatment process through fluorescence signals to improve treatment effect and safety.
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Figure CN116478189B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to a photosensitizer, a preparation method thereof, and an application thereof. Background Art
[0002] Currently, photodynamic therapy (PDT) has become a very promising method for treating cancer because photosensitizers can efficiently and rapidly produce reactive oxygen species (ROS) under light irradiation, leading to the death of cancer cells. Although more and more efficient photosensitizers (such as clinically approved porphyrin and phthalocyanine derivatives) have achieved considerable success (Li, XS, Lovell, JF, Yoon, J., Chen, XY, Clinical development and potential of photothermal and photodynamic therapies for cancer, Nat. Rev. Clin. Oncol., 2020, 17, 657-674), the main problems of photosensitizers still limit their choice as first-line treatment. Traditional PDT mainly causes programmed cell death, such as apoptosis, ferroptosis, pyroptosis, etc., and long-term use can easily cause drug resistance, thus limiting the clinical application of PDT.
[0003] In addition, photosensitizers are still caught in the problem of "off-target" toxicity, tumor hypoxia and overtreatment due to the lack of timely monitoring of treatment response. Although light has attractive advantages such as non-invasiveness, remote action and easy modulation of the energy involved, the assessment of cell damage is a prerequisite for PDT. More and more studies have shown that highly activated photosensitizers can damage normal cells and affect the therapeutic effect. Tumor ablation in PDT mainly involves ROS damage, destruction of tumor blood vessels and induction of inflammatory response (Zhao, XZ, Liu, JP, Fan, JL, Chao, H., Peng, XJ, Recent progress in photosensitizers for overcoming the challenges of photodynamic therapy: from molecular design to application, Chem. Soc. Rev., 2020, 50, 4185-4219). Therefore, off-target photosensitizers will inevitably damage a large number of normal cells. In addition, long-term exposure to high-power and short-wavelength light can damage the skin and organs. Clearly, real-time monitoring of photosensitizers in vivo and assessment of accurate therapeutic endpoints, guided by dosimetry, are crucial. Unfortunately, most current PDT approaches require the introduction of additional fluorescent probes, and the lack of photosensitizers capable of self-reporting fluorescent signals complicates and delays the monitoring process. Therefore, accurately assessing the location, duration, and on-demand dose of photosensitizers is crucial for precise treatment and minimizing toxic side effects.
[0004] Therefore, it is very meaningful for PDT to improve traditional photosensitizers and design photosensitizers that induce non-programmed cell death and can self-report through fluorescent signals.
[0005] In view of this, the present invention is proposed. Summary of the Invention
[0006] The purpose of the present invention is to provide a photosensitizer and its preparation method and application, so as to improve the above technical problems.
[0007] The present invention is achieved in that:
[0008] In a first aspect, the present invention provides a photosensitizer, the chemical structure of which is shown in Formula I:
[0009]
[0010] In formula I, R1 is selected from any one of substituted or unsubstituted alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted pyridine, substituted or unsubstituted benzoic acid, substituted or unsubstituted benzaldehyde, and substituted or unsubstituted phenol; R2 is selected from any one of substituted or unsubstituted phenol, substituted or unsubstituted N,N-dimethylaniline, substituted or unsubstituted methoxybenzene, substituted or unsubstituted fluorobenzene, and substituted or unsubstituted fused aromatic groups.
[0011] In a second aspect, the present invention further provides a method for preparing a photosensitizer, comprising:
[0012] (1) Prepare a first intermediate, the chemical structural formula of the first intermediate is:
[0013]
[0014] (2) Using the first intermediate as a raw material, a second intermediate is prepared. The chemical structure of the second intermediate is:
[0015]
[0016] (3) Using the second intermediate as a raw material, a third intermediate is prepared. The chemical structure of the third intermediate is:
[0017]
[0018] (4) Using the third intermediate as a raw material, a fourth intermediate is prepared. The chemical structure of the fourth intermediate is:
[0019]
[0020] A photosensitizer that induces rapid cell oncosis and self-reports fluorescence signals is prepared using the fourth intermediate and p-hydroxybenzaldehyde as raw materials. The chemical structure of the photosensitizer is:
[0021] Specifically, the method for preparing the photosensitizer includes:
[0022] Phosphorus oxybromide (20 mmol, 5.73 g) was dissolved in 5 mL of anhydrous dichloromethane. A mixture of N,N-dimethylformamide (20 mmol, 1.46 g) dissolved in 15 mL of anhydrous dichloromethane was added dropwise at 0°C. After the addition was complete, the reaction mixture was stirred at room temperature for 30 minutes. Subsequently, a solution of isoindolin-1-one (10 mmol, 1.33 g) dissolved in 50 mL of anhydrous dichloromethane was added dropwise at 0°C. Finally, the reaction mixture was refluxed for 6 hours. After the reaction was completed, the mixture was cooled and concentrated under reduced pressure to remove the solvent. Ice water was added with stirring, and the pH was adjusted to approximately 8 with 5 M aqueous sodium hydroxide solution to produce a black precipitate. Stirring was continued overnight, and the black precipitate was collected to obtain the first intermediate.
[0023] The first intermediate and 2-hydroxyphenylboronic acid were dissolved in 50 mL of toluene, and a 1 M aqueous sodium carbonate solution was added. Tetrakis(triphenylphosphine)palladium was used as a catalyst. Under inert gas protection, the reaction was carried out at 75°C for 24 hours. After the reaction, the crude product was extracted. The crude product was treated with a 4 M sodium hydroxide solution, and the second intermediate was obtained by extraction and column chromatography.
[0024] The molar ratio of the second intermediate, 2-hydroxyphenylboronic acid and tetrakis(triphenylphosphine)palladium is 8:17.5:0.25.
[0025] The crude product was dissolved in 100 mL of ethanol, and 4 M sodium hydroxide was added, followed by reflux reaction for 3 hours.
[0026] 3-Ethyl-2,4-dimethylpyrrole (10.4 mmol, 1.281 g) was dissolved in 15 mL of dichloromethane and phosphorus oxychloride (5.2 mmol, 0.797 g) was added dropwise at 0°C. Subsequently, a solution of the second intermediate (5.2 mmol, 1.233 g) dissolved in 25 mL of dichloromethane was added dropwise at 0°C. After the mixture was reacted at room temperature for 4 hours, the solution was concentrated by column chromatography to obtain the crude third intermediate. Without purification, the crude third intermediate was dissolved in 50 mL of ethyl acetate and methylboric acid (52 mmol, 3.112 g) was added, followed by reflux reaction for 3 hours. After the reaction, the solvent was evaporated and the solution was concentrated under reduced pressure to obtain the fourth intermediate by column chromatography.
[0027] It should be noted that the third intermediate does not need to be purified and can proceed to the next step, and the fourth intermediate can be synthesized by a one-step method.
[0028] The fourth intermediate (1 mmol, 366.19 mg) and 4-hydroxybenzaldehyde (1.2 mmol, 146.544 mg) were dissolved in 100 mL of toluene. 4 mL of piperidine and 100 mg of p-toluenesulfonic acid were added, and the reaction mixture was incubated at 140°C for 48 hours. After completion of the reaction, the mixture was cooled, concentrated under reduced pressure to evaporate the solvent, and column chromatography was performed to obtain a photosensitizer that induces rapid cell apoptosis and produces a self-reporting fluorescent signal.
[0029] In a third aspect, the present invention further provides a use of the above-mentioned photosensitizer in the preparation of anti-tumor drugs.
[0030] The present invention has the following beneficial effects: Upon illumination, the photosensitizer having the chemical structure represented by Formula I generates multiple ROS, which can induce unprogrammed cell oncosis, preventing the development of drug resistance with long-term use. It can effectively kill tumor cells and has great potential in anti-tumor applications. Furthermore, under conditions of both normal and low oxygen levels, it exhibits strong phototoxicity to tumor cells, overcoming the hypoxic environment of tumors and enhancing the effectiveness of photodynamic therapy. Furthermore, upon illumination, the photosensitizer cleaves specific carbon-carbon double bonds, converting its near-infrared fluorescence to red fluorescence, allowing the progress of photodynamic therapy to be monitored by changes in the fluorescence signal. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.
[0032] Figures 1 to 12 The H-NMR and C-NMR spectra of the photosensitizers synthesized in Examples 1 to 6 are shown;
[0033] Figures 13 to 18 is the mass spectrum of the photosensitizer synthesized in Examples 1 to 6;
[0034] Figures 19 to 22 The absorption spectrum (A) and fluorescence spectrum (B) of the photosensitizer in Experimental Example 1 of the present invention as a function of illumination time are shown;
[0035] Figure 23 The H NMR spectrum and mass spectrum of the photosensitizer after illumination in Experimental Example 2 of the present invention are shown;
[0036] Figure 24 This is the electron paramagnetic resonance signal diagram before and after illumination of the photosensitizer in Experimental Example 2 of the present invention;
[0037] Figure 25In Experimental Example 3 of the present invention, 2',7'-dichlorodihydrofluorescein diacetate, a singlet oxygen fluorescence probe, dihydrorhodamine 123, a hydroxyl radical and a peroxynitrite sensor were used to measure intracellular ROS; the purple channel represents the fluorescence of the photosensitizer, the red channel represents the fluorescence of the product generated after the photosensitizer is irradiated with light, and the green channel represents the fluorescence of the product after the ROS probe reacts with ROS;
[0038] Figure 26 The morphological changes of cells, mitochondria and endoplasmic reticulum after illumination of cells treated with photosensitizer in Experimental Example 4 of the present invention;
[0039] Figure 27 The changes in cell morphology after illumination of cells treated with photosensitizer in Experimental Example 4 of the present invention;
[0040] Figure 28 The changes in intracellular ATP and calcium ions after illumination in Experimental Example 5 of the present invention;
[0041] Figure 29 Western Blotting was used to detect the expression of cell apoptosis-related proteins in Experimental Example 6 of the present invention;
[0042] Figure 30 1 is the concentration-dependent cell viability curve in Experimental Example 7 of the present invention, A is HeLa cells, B is A549 cells, C is MCF-7 cells, and D is U87 cells. DETAILED DESCRIPTION
[0043] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.
[0044] The following is a detailed description of a photosensitizer provided by the present invention, its preparation method and application.
[0045] Some embodiments of the present invention provide a photosensitizer, the chemical structure of which is shown in Formula I:
[0046]
[0047] In formula I, R1 is selected from any one of substituted or unsubstituted alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted pyridine, substituted or unsubstituted benzoic acid, substituted or unsubstituted benzaldehyde, and substituted or unsubstituted phenol; R2 is selected from any one of substituted or unsubstituted phenol, substituted or unsubstituted N,N-dimethylaniline, substituted or unsubstituted methoxybenzene, substituted or unsubstituted fluorobenzene, and substituted or unsubstituted fused aromatic groups.
[0048] This photosensitizer can induce tumor cells to undergo unprogrammed cell death, also known as oncosis, triggering rapid cell death and self-reporting this process through fluorescence signals. Specifically, the photosensitizer generates multiple ROS upon illumination, which induce oncosis, a form of unprogrammed cell death. The generated ROS mediate the cleavage of specific carbon-carbon double bonds in the photosensitizer, converting near-infrared fluorescence to red fluorescence, enabling self-reporting of the fluorescence signal.
[0049] Specifically, the chemical structure of the photosensitizer is further optimized, R1 is selected from C1 to C10 alkyl groups, for example, R1 is selected from C1 to C5 alkyl groups; R2 is selected from substituted phenyl groups or substituted fused aryl groups.
[0050] Furthermore, when R1 is a methyl group and R2 is a substituted phenyl group, the substituent group is selected from any one of a tertiary amino group, a methoxy group, a hydroxyl group and a cyano group, and the specific substitution position may be the para position; when R2 is a substituted fused aryl group, the fused aryl group is a benzyl group or a heteroaryl group, and the substituent group on the benzyl group or the heteroaryl group is an alkyl group or a substituted alkoxy group.
[0051] Furthermore, the structural formula of the photosensitizer is selected from any one of the following structural formulas:
[0052]
[0053] In some embodiments, the chemical formula of the photosensitizer is:
[0054] Some embodiments of the present invention further provide a method for preparing the photosensitizer in the above embodiment, which comprises:
[0055] Prepare a first intermediate, the chemical structural formula of the first intermediate is:
[0056]
[0057] The first intermediate is used as a raw material to prepare a second intermediate, the chemical structure of which is:
[0058]
[0059] The third intermediate is prepared using the second intermediate as a raw material. The chemical structure of the third intermediate is:
[0060]
[0061] The fourth intermediate is prepared using the third intermediate as a raw material. The chemical structure of the fourth intermediate is:
[0062]
[0063] A photosensitizer that induces rapid cell oncosis and self-reports fluorescence signals is prepared using the fourth intermediate and p-hydroxybenzaldehyde as raw materials.
[0064] Furthermore, the step of preparing the first intermediate includes: dissolving phosphorus oxybromide in anhydrous dichloromethane, adding dropwise a mixed solution of N,N-dimethylformamide dissolved in anhydrous dichloromethane at a temperature of -5°C to 5°C, and stirring the mixed reaction solution at room temperature for 25 to 35 minutes after the addition is completed. Subsequently, adding dropwise a solution of isoindolin-1-one dissolved in anhydrous dichloromethane at a temperature of -5°C to 5°C, and finally, refluxing the reaction mixture for 5 to 7 hours. After the reaction is completed, cooling, concentrating under reduced pressure to remove the solvent, adding ice water with stirring, and adjusting the pH to about 8 to produce a black precipitate, which is the first intermediate;
[0065] The step of preparing the second intermediate includes: dissolving the first intermediate and 2-hydroxyphenylboronic acid in toluene, adding a sodium carbonate aqueous solution, using tetrakis(triphenylphosphine)palladium as a catalyst, reacting at a temperature of 70°C to 80°C under inert gas protection for 20 to 28 hours, extracting to obtain a crude product after the reaction is completed, treating the crude product with an alkaline solution, extracting and performing column chromatography to obtain the second intermediate;
[0066] The steps of preparing the third intermediate and the fourth intermediate include: dissolving 3-ethyl-2,4-dimethylpyrrole in dichloromethane, adding phosphorus oxychloride dropwise at a temperature of -5°C to 5°C, and then adding a solution of the second intermediate dissolved in dichloromethane dropwise at a temperature of -5°C to 5°C; after the mixed solution is reacted at room temperature for 3 to 5 hours, the solution is concentrated by column chromatography to obtain a crude product of the third intermediate without purification; the crude product of the third intermediate is dissolved in ethyl acetate, methylboric acid is added, and the reaction is refluxed for 2.5 to 3.5 hours. After the reaction is completed, the solvent is evaporated and concentrated under reduced pressure, and the fourth intermediate is obtained by column chromatography;
[0067] The step of preparing a photosensitizer through the fourth intermediate includes: dissolving the fourth intermediate and 4-hydroxybenzaldehyde in toluene, adding piperidine and p-toluenesulfonic acid, reacting the reaction mixture at a temperature of 130° C. to 150° C. for 45 to 52 hours, cooling after the reaction, concentrating under reduced pressure to evaporate the solvent, and obtaining the photosensitizer through column chromatography.
[0068] More specifically, some embodiments provide a method for preparing a photosensitizer comprising:
[0069] Phosphorus oxybromide (20 mmol, 5.73 g) was dissolved in 5 mL of anhydrous dichloromethane. A mixture of N,N-dimethylformamide (20 mmol, 1.46 g) dissolved in 15 mL of anhydrous dichloromethane was added dropwise at 0°C. After the addition was complete, the reaction mixture was stirred at room temperature for 30 minutes. Subsequently, a solution of isoindolin-1-one (10 mmol, 1.33 g) dissolved in 50 mL of anhydrous dichloromethane was added dropwise at 0°C. Finally, the reaction mixture was refluxed for 6 hours. After the reaction was completed, the mixture was cooled and concentrated under reduced pressure to remove the solvent. Ice water was added with stirring, and the pH was adjusted to approximately 8 with 5 M aqueous sodium hydroxide solution to produce a black precipitate. Stirring was continued overnight, and the black precipitate was collected to obtain the first intermediate.
[0070] The first intermediate and 2-hydroxyphenylboronic acid were dissolved in 50 mL of toluene, and a 1 M aqueous sodium carbonate solution was added. Tetrakis(triphenylphosphine)palladium was used as a catalyst. Under inert gas protection, the reaction was carried out at 75°C for 24 hours. After the reaction, the crude product was extracted. The crude product was treated with a 4 M sodium hydroxide solution, and the second intermediate was obtained by extraction and column chromatography.
[0071] The molar ratio of the second intermediate, 2-hydroxyphenylboronic acid and tetrakis(triphenylphosphine)palladium is 8:17.5:0.25.
[0072] The crude product was dissolved in 100 mL of ethanol, and 4 M sodium hydroxide was added, and the mixture was refluxed for 3 hours.
[0073] 3-Ethyl-2,4-dimethylpyrrole (10.4 mmol, 1.281 g) was dissolved in 15 mL of dichloromethane and phosphorus oxychloride (5.2 mmol, 0.797 g) was added dropwise at 0°C. Subsequently, a solution of the second intermediate (5.2 mmol, 1.233 g) dissolved in 25 mL of dichloromethane was added dropwise at 0°C. After the mixture was reacted at room temperature for 4 hours, the solution was concentrated by column chromatography to obtain the crude third intermediate. Without purification, the crude third intermediate was dissolved in 50 mL of ethyl acetate and methylboric acid (52 mmol, 3.112 g) was added, followed by reflux reaction for 3 hours. After the reaction, the solvent was evaporated and the solution was concentrated under reduced pressure to obtain the fourth intermediate by column chromatography.
[0074] It should be noted that the third intermediate does not need to be purified and can proceed to the next step, and the fourth intermediate can be synthesized by a one-step method.
[0075] The fourth intermediate (1 mmol, 366.19 mg) and 4-hydroxybenzaldehyde (1.2 mmol, 146.544 mg) were dissolved in 100 mL of toluene. 4 mL of piperidine and 100 mg of p-toluenesulfonic acid were added, and the reaction mixture was incubated at 140°C for 48 hours. After completion of the reaction, the mixture was cooled, concentrated under reduced pressure to evaporate the solvent, and column chromatography was performed to obtain a photosensitizer that induces rapid cell apoptosis and produces a self-reporting fluorescent signal.
[0076] The first intermediate is prepared in step (1), specifically as follows: phosphorus oxybromide (20 mmol, 5.73 g) is dissolved in 5 mL of anhydrous dichloromethane, and a mixed solution of N,N-dimethylformamide (20 mmol, 1.46 g) dissolved in 15 mL of anhydrous dichloromethane is added dropwise at 0°C. After the addition is completed, the mixed reaction solution is stirred at room temperature for 30 minutes. Subsequently, a solution of isoindolin-1-one (10 mmol, 1.33 g) dissolved in 50 mL of anhydrous dichloromethane is added dropwise at 0°C. Finally, the reaction mixture is refluxed for 6 hours. After the reaction is completed, the mixture is cooled and concentrated under reduced pressure to remove the solvent. Ice water is added with stirring, and the pH is adjusted to about 8 with a 5M aqueous sodium hydroxide solution to produce a black precipitate. Stirring is continued overnight, and the black precipitate is collected to obtain the first intermediate.
[0077] The second intermediate is prepared in step (2), specifically as follows: the first intermediate and 2-hydroxyphenylboronic acid are dissolved in 50 mL of toluene, and a 1 M aqueous sodium carbonate solution is added. The mixture is reacted at 75° C. for 24 hours under an inert gas atmosphere using tetrakis(triphenylphosphine)palladium as a catalyst. After the reaction is completed, a crude product is obtained by extraction. The crude product is treated with a 4 M sodium hydroxide solution, and the second intermediate is obtained by extraction and column chromatography.
[0078] The molar ratio of the second intermediate, 2-hydroxyphenylboronic acid and tetrakis(triphenylphosphine)palladium is 8:17.5:0.25.
[0079] The crude product was dissolved in 100 mL of ethanol, and 4 M sodium hydroxide was added, followed by reflux reaction for 3 hours.
[0080] The third and fourth intermediates can be synthesized in one step. The intermediates are prepared in steps (3) and (4), specifically as follows: 3-ethyl-2,4-dimethylpyrrole (10.4 mmol, 1.281 g) is dissolved in 15 mL of dichloromethane, and phosphorus oxychloride (5.2 mmol, 0.797 g) is added dropwise at 0°C. Subsequently, a solution of the second intermediate (5.2 mmol, 1.233 g) dissolved in 25 mL of dichloromethane is added dropwise at 0°C. After the mixed solution is reacted at room temperature for 4 hours, the solution is concentrated by column chromatography to obtain a crude product of the third intermediate. Without purification, the crude product of the third intermediate is dissolved in 50 mL of ethyl acetate, methylboric acid (52 mmol, 3.112 g) is added, and the mixture is refluxed for 3 hours. After the reaction is completed, the solvent is evaporated and concentrated under reduced pressure, and the fourth intermediate is obtained by column chromatography.
[0081] The third intermediate does not need to be purified and can be continued to the next step, and the fourth intermediate can be synthesized by a one-step method.
[0082] The photosensitizer prepared in step (4) is specifically prepared by dissolving the fourth intermediate (1 mmol, 366.19 mg) and 4-hydroxybenzaldehyde (1.2 mmol, 146.544 mg) in 100 mL of toluene, adding 4 mL of piperidine and 100 mg of p-toluenesulfonic acid, and reacting the reaction mixture at 140°C for 48 hours. After the reaction is completed, the mixture is cooled, concentrated under reduced pressure to evaporate the solvent, and column chromatography is performed to obtain a photosensitizer that induces rapid cell apoptosis and self-reports a fluorescent signal.
[0083] Some embodiments of the present invention also provide use of the photosensitizer according to any of the above embodiments in the preparation of anti-tumor drugs.
[0084] Specifically, the above-mentioned anti-tumor drug is a drug that induces tumor cell apoptosis.
[0085] The features and performance of the present invention are further described in detail below with reference to the embodiments.
[0086] Example 1
[0087] This embodiment provides a photosensitizer, the chemical structure of which is: Its preparation method is:
[0088] (1) Phosphorus oxybromide (20 mmol, 5.73 g) was dissolved in 5 mL of anhydrous dichloromethane. A mixed solution of N,N-dimethylformamide (20 mmol, 1.46 g) dissolved in 15 mL of anhydrous dichloromethane was added dropwise at 0°C. After the addition was completed, the mixed reaction solution was stirred at room temperature for 30 minutes. Subsequently, a solution of isoindolin-1-one (10 mmol, 1.33 g) dissolved in 50 mL of anhydrous dichloromethane was added dropwise at 0°C. Finally, the reaction mixture was refluxed for 6 hours. After the reaction was completed, the mixture was cooled and concentrated under reduced pressure to remove the solvent. Ice water was added and stirred, and the pH was adjusted to about 8 with a 5 M aqueous sodium hydroxide solution to produce a black precipitate. The stirring was continued overnight and the black precipitate was collected, which was the first intermediate.
[0089] (2) The first intermediate and 2-hydroxyphenylboronic acid were dissolved in 50 mL of toluene, and a 1 M aqueous sodium carbonate solution was added. Tetrakis(triphenylphosphine)palladium was used as a catalyst, and the mixture was reacted at 75°C for 24 hours under inert gas protection. After the reaction, a crude product was obtained by extraction. The crude product was treated with a 4 M sodium hydroxide solution, and the second intermediate was obtained by extraction and column chromatography. The molar ratio of the second intermediate, 2-hydroxyphenylboronic acid and tetrakis(triphenylphosphine)palladium was 8:17.5:0.25. The crude product was dissolved in 100 mL of ethanol, and 4 M sodium hydroxide was added, and the mixture was refluxed for 3 hours.
[0090] (3) 3-Ethyl-2,4-dimethylpyrrole (10.4 mmol, 1.281 g) was dissolved in 15 mL of dichloromethane, and phosphorus oxychloride (5.2 mmol, 0.797 g) was added dropwise at 0°C. Subsequently, a solution of the second intermediate (5.2 mmol, 1.233 g) dissolved in 25 mL of dichloromethane was added dropwise at 0°C. After the mixed solution was reacted at room temperature for 4 hours, the solution was concentrated by column chromatography to obtain a crude product of the third intermediate. Without purification, the crude product of the third intermediate was dissolved in 50 mL of ethyl acetate, methylboric acid (52 mmol, 3.112 g) was added, and the mixture was refluxed for 3 hours. After the reaction was completed, the solvent was evaporated and concentrated under reduced pressure, and the fourth intermediate was obtained by column chromatography. The third intermediate did not need to be purified and could be continued to the next step. The fourth intermediate could be synthesized by a one-step method.
[0091] (4) The fourth intermediate (1 mmol, 366.19 mg) and 4-hydroxybenzaldehyde (1.2 mmol, 146.544 mg) were dissolved in 100 mL of toluene, and 4 mL of piperidine and 100 mg of p-toluenesulfonic acid were added. The reaction mixture was reacted at 140°C for 48 hours. After the reaction, the mixture was cooled, concentrated under reduced pressure to evaporate the solvent, and column chromatography was performed to obtain a photosensitizer that induced rapid cell apoptosis and self-reported fluorescence signals in a yield of 35%. 1H NMR (400MHz, DMSO) δ = 9.80 (s, 1H), 8.38 (d, J = 8.3, 1H), 8.26 (dd, J = 7.9, 1.4, 1H), 8 .20(d,J=8.0,1H),7.94(s,1H),7.78(d,J=16.8,1H),7.61(t,J=7.6,1H),7.54–7. 43(m,4H),7.28(d,J=16.8,1H),7.14(dd,J=8.2,0.9,1H),7.11–7.05(m,1H),6.89 (d,J=8.6,2H),2.82–2.64(m,2H),2.29(s,3H),1.19(t,J=7.5,3H),-0.15(s,3H). 13 C NMR (101MHz, DMSO) δ = 158.61, 156.90, 147.18, 142.90, 134.99, 134.35, 133.91, 133.38, 133.26, 130.82, 129.42, 129.10, 128.61, 128.4 6,127.19,126.70,126.61,124.03,121.20,120.62,120.43,118.79,117.51,116.80,116.55,18.75,14.58,9.35.HRMS(ESI)m / z:calcd for C 31 H 27 BN2O2[M],470.2166; found,470.2222.[M-CH3] + 455.1931;found,455.1976.
[0092] Example 2 to Example 6
[0093] By replacing the substituent of benzaldehyde in step (4), other photosensitizers can be synthesized.
[0094] The H NMR spectra, C NMR spectra and MS spectra of the photosensitizers prepared in Examples 1 to 6 (N1 to N6 in order) are as follows: Figures 1 to 18 shown.
[0095] Test Example 1
[0096] Photosensitizer spectral testing
[0097] The photosensitizers (N1-N4) prepared in Examples 1-4 were dissolved in DMSO to prepare 10 mM stock solutions. Unless otherwise specified, the samples used in the following experiments were obtained by diluting the stock solutions. The photosensitizer stock solutions were diluted with 2 mL of a mixed solution of ethanol and water (v / v=2 / 1, containing 2% PEG 400) to a final concentration of 10 μM. A 680 nm laser was used as the light source (0.6 W / cm 2 ), after 30 seconds of illumination, the absorption spectrum and fluorescence spectrum were collected once. When collecting the fluorescence spectrum, the excitation wavelength was 550nm. The results are shown in Figure 2. Figures 19 to 22 As shown in the figure, after illumination, the near-infrared fluorescence of the photosensitizer turns into red fluorescence, and the red fluorescence increases with the increase of illumination time. This shows that during the process of photodynamic therapy, the change of fluorescence can achieve real-time monitoring of the treatment process.
[0098] Test Example 2
[0099] Mechanism of photo-cleavage of double bonds
[0100] (1) Determination of the reaction solution's nuclear magnetic hydrogen spectrum and mass spectrum
[0101] The specific operation is as follows: the photosensitizer N1 prepared in Example 1 was dissolved in a mixed solution of deuterated methanol and deuterated chloroform (v / v=5 / 1), stirred at room temperature, and irradiated with light for 5 minutes (680nm, 0.6W / cm 2 ), after the irradiation, the nuclear magnetic hydrogen spectrum and mass spectrum of the reaction solution were measured. Figure 23 As shown, left: mass spectrometry, right: H-NMR spectrum. The results show that after light irradiation, the carbon-carbon double bond is cleaved and converted into an aldehyde group, generating the corresponding product.
[0102] (2) EPR determination of free radical species
[0103] The specific operation is as follows: 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) is used as a free radical scavenger. DMPO (10 mM) is added to the ethanol solution of the photosensitizer N1 (2 mM) prepared in Example 1 under dark conditions, and the electron paramagnetic resonance signal is measured using an electron paramagnetic resonance spectrometer. Stirring, irradiation for 1 minute (680 nm, 0.6 W / cm 2 ) and then the electron paramagnetic resonance signal was measured again. Figure 24 As shown in the figure, the solution after illumination showed obvious electron paramagnetic resonance signal, indicating that illumination can cause the photosensitizer to produce free radical substances.
[0104] Test Example 3
[0105] Determination of intracellular reactive oxygen species
[0106] The specific steps are as follows: 2',7'-dichlorodihydrofluorescein diacetate, singlet oxygen fluorescence probe, dihydrorhodamine 123, hydroxyl radical and peroxynitrite sensors were used as indicators to measure the generation of ROS. HeLa cells were seeded in 24-well plates (2 mL, 2.5 × 10 5 cells / well) and cultured overnight in a 37°C incubator containing 5% CO2. The cells were then divided into different groups:
[0107] 1) Blank control group.
[0108] 2) Photosensitizer control group (photosensitizer incubation for 24 hours).
[0109] 3) ROS probe group (10 μM 2',7'-dichlorodihydrofluorescein diacetate, or 10 μM singlet oxygen fluorescence probe, or 10 μM dihydrorhodamine 123, or 10 μM hydroxyl radical and peroxynitrite sensor) was prepared in serum-free culture medium and incubated with cells in the dark for 30 minutes.
[0110] 4) Photosensitizer + ROS probe group: After incubation with photosensitizer for 24 hours, different ROS probes were incubated in the dark for 30 minutes.
[0111] 5) ROS probe illumination group: cells were incubated with different ROS probes in the dark for 30 minutes, washed three times with PBS, and then illuminated for 1 minute (680 nm, 0.6 W / cm 2 Then, complete medium was added and incubated for 4 hours.
[0112] 6) Photosensitizer + ROS probe illumination group: After incubation with photosensitizer for 24 hours, different ROS probes were incubated in the dark for 30 minutes, washed three times with PBS, and then illuminated for 1 minute (680nm, 0.6W / cm 2 Then, complete medium was added and incubated for 4 hours.
[0113] It should be noted that the above photosensitizer is the photosensitizer prepared in Example 1.
[0114] The results are as follows Figure 25 As shown, compared with other groups, the green channel fluorescence of the photosensitizer + ROS probe group was significantly enhanced after illumination, indicating that multiple ROS were generated.
[0115] Test Example 4
[0116] Cell oncosis morphology assay
[0117] The specific steps are as follows: HeLa cells were seeded in a confocal dish (2 mL, 1.5 × 10 5Cells were cultured overnight at 37°C in a 5% CO2 incubator. The cells were then divided into different groups: 1) blank control group. 2) illumination control group, blank cells were illuminated for 1 minute (680 nm, 0.6 W / cm 2 ) and then continued to incubate for 24 hours. 3) Photosensitizer group, photosensitizer incubation for 24 hours. 4) Photosensitizer illumination group, after incubation for 24 hours, light was irradiated for 1 minute (680nm, 0.6W / cm 2 ) and continue incubation for 24 hours.
[0118] For colocalization studies, cells were incubated with photosensitizer for 24 h and then illuminated for 1 min (680 nm, 0.6 W / cm 2 ) and then continue to incubate for 24 hours. Then use the co-localization dye Mito-Tracker TM Green FM dye (10 μM) or ER-Tracker TM The cells were incubated with Green dye (10 μM) for 30 minutes and stained with DAPI for 30 minutes. Figure 26 and Figure 27 As shown, compared with the other groups, the photosensitizer-exposed group showed cellular membrane blebbing, nuclear swelling, and chromatin aggregation. Colocalization results also showed swelling of the mitochondria and endoplasmic reticulum. These results are consistent with the main characteristics of cell apoptosis.
[0119] Test Example 5
[0120] Intracellular ATP and calcium ion determination
[0121] The specific steps are as follows: HeLa cells were seeded in 6-well plates (2 mL, 1.2 × 10 6 Cells were cultured overnight at 37°C in a 5% CO2 incubator. The cells were then divided into different groups: blank group and photosensitizer-exposed group. After incubating the cells with photosensitizer for 24 hours, they were illuminated for 1 minute (680 nm, 0.6 W / cm 2 ), and then continue to incubate for different time periods. After the end, the cells were collected. The changes in intracellular ATP and calcium ions were measured according to the ATP and calcium ion determination kits. Figure 28 As shown, after illumination, the intracellular ATP content decreased and the calcium ion concentration increased. The photosensitizer was the photosensitizer prepared in Example 1.
[0122] Test Example 6
[0123] Determination of changes in cell oncogenic proteins
[0124] The specific steps are as follows: HeLa cells were seeded in 6-well plates (2 mL, 1.2 × 10 6Cells were cultured overnight at 37°C in a 5% CO2 incubator. The cells were then divided into different groups: blank group and photosensitizer-exposed group. After incubating the cells with photosensitizer for 24 hours, they were illuminated for 1 minute (680 nm, 0.6 W / cm 2 ), and then incubated for 0, 6, 12, and 24 hours. After the incubation, the cells were collected, and protein was extracted and measured using a BCA protein assay kit.
[0125] Protein samples were denatured by boiling and separated by electrophoresis. The stacking gel voltage was 80 V for 30 minutes, then the resolving gel voltage was increased to 100 V for approximately 90 minutes. Electrophoresis was stopped at approximately 0.5 cm from the bottom of the resolving gel. The gel was removed and equilibrated in transfer buffer for 15 minutes. Transfer was then performed at a constant current of 200 mA for 100 minutes. The NC membrane was blocked in 5% nonfat dry milk blocking buffer (4°C, overnight). The blocking buffer was discarded without washing. Primary antibodies against porimin, calpain 1, bcl-2, and bax were diluted 1:200 and incubated overnight at 4°C on a shaker. The membrane was washed four times (10 minutes each) with PBST. The corresponding secondary antibodies were diluted 1:2000 and incubated at room temperature for 4 hours. The membrane was washed four times (10 minutes each) with PBST. The developer solution was applied to the NC membrane and allowed to stand at room temperature for 1 minute. The exposure time was adjusted until the optimal band appeared. GAPDH and β-actin were used as internal controls.
[0126] The results are as follows Figure 29 As shown, compared with the blank control group, the levels of the cell membrane protein Porimin and the calcium-activated protease family member Calpain 1 were significantly increased. This indicates changes in cell membrane permeability and expansion, as well as mitochondrial dysfunction and even morphological collapse. Concomitantly, the ratios of Bcl-2 and Bax proteins increased, a protein ratio change that is contrary to apoptosis and consistent with cell oncosis.
[0127] Test Example 7
[0128] Cytotoxicity test
[0129] The specific operation steps are as follows: HeLa cells, A549 cells, MCF-7 cells and U87 cells in the logarithmic growth phase were taken, digested and the cell density was adjusted to 1×10 5 / mL, and were evenly inoculated on 96-well plates, and cultured in a 37°C, 5% CO2 incubator for 24 hours. The culture medium was removed and photosensitizer intervention experiments were performed: the final concentration of 0.5, 10, 100 μL of complete DMEM medium containing 50 μM photosensitizer was added, and 6 replicate wells were set up for each concentration. After the plate was cultured in an incubator at 37°C and 5% CO2 for 24 hours, the medium was aspirated and washed three times with PBS. Each well was illuminated for 1 minute, and complete medium was added to continue incubation for 24 hours. After adding 10 μL of CCK-8 solution to each well and culturing in an incubator at 37°C and 5% CO2 for 4 hours, it was shaken at a constant temperature of 37°C for 10 minutes, and the absorbance A of each well was measured by an automatic microplate reader. Based on the experimental results, the cell survival rate was calculated. The results are shown in Table 1, and the concentration-dependent cell survival rate curve is shown in Table 1. Figure 30 As shown (A is HeLa cells, B is A549 cells, C is MCF-7 cells, and D is U87 cells).
[0130] Table 1 The half-lethal concentration of the photosensitizer in Example 1 in various cells
[0131]
[0132]
[0133] a The illumination conditions are: 680nm, 0.6W / cm 2 , 60s, and then the cells were incubated under normal conditions for 24h.
[0134] In summary, compared with the prior art, the advantages and beneficial effects of the present invention are as follows:
[0135] 1. The photosensitizer prepared in the embodiment of the present invention generates multiple ROS after illumination, which can induce cell apoptosis, a non-programmed cell death, avoiding the occurrence of drug resistance after long-term use. At the same time, it can effectively kill tumor cells and has great potential in anti-tumor applications.
[0136] 2. The photosensitizer prepared in the embodiment of the present invention has strong phototoxicity to tumor cells under conditions of normal oxygen content and low oxygen content, can overcome the hypoxic environment of the tumor, and improve the effect of photodynamic therapy.
[0137] 3. The photosensitizer prepared in the embodiment of the present invention can cut specific carbon-carbon double bonds after exposure to light, and the near-infrared fluorescence turns into red fluorescence. The progress of photodynamic therapy can be monitored by the change of the fluorescence signal.
[0138] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.
Claims
1. A photosensitizer, characterized in that The chemical structural formula of the photosensitizer is selected from any one of the following structural formulas: <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (N1)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (N2)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (N3)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (N4) 2. The photosensitizer according to claim 1, characterized in that The structural formula of the photosensitizer is: (N1).
3. Use of the photosensitizer according to claim 1 or 2 in the preparation of anti-tumor drugs.
4. A method for preparing a photosensitizer, characterized in that: The following steps are involved: Prepare a first intermediate, the chemical structural formula of the first intermediate is: The first intermediate is used as a raw material to prepare a second intermediate, and the chemical structure of the second intermediate is: The third intermediate is prepared using the second intermediate as a raw material. The chemical structure of the third intermediate is: The fourth intermediate is prepared using the third intermediate as a raw material. The chemical structure of the fourth intermediate is: A photosensitizer that induces rapid oncosis of tumor cells and self-reports fluorescence signals is prepared using the fourth intermediate and p-hydroxybenzaldehyde as raw materials. The chemical structure of the photosensitizer is: .
5. The preparation method according to claim 4, characterized in that The steps of preparing the first intermediate include: dissolving phosphorus oxybromide in anhydrous dichloromethane; ° C~5 ° C temperature, add dropwise a mixed solution of N,N-dimethylformamide dissolved in anhydrous dichloromethane, after the addition is completed, the mixed reaction solution is stirred at room temperature for 25 to 35 minutes, and then at -5 ° C~5 ° C, dropwise adding a solution of isoindolin-1-one dissolved in anhydrous dichloromethane, and finally, reflux the reaction mixture for 5 to 7 hours. After the reaction is completed, the mixture is cooled, concentrated under reduced pressure to remove the solvent, and ice water is added with stirring to adjust the pH to about 8 to produce a black precipitate, which is the first intermediate; The steps of preparing the second intermediate include: dissolving the first intermediate and 2-hydroxyphenylboronic acid in toluene, adding a sodium carbonate aqueous solution, using tetrakis(triphenylphosphine)palladium as a catalyst, under inert gas protection, 70 ° C~80 ° C temperature for 20 to 28 hours, after which the reaction is completed, extracting to obtain a crude product, treating the crude product with an alkaline solution, extracting and column chromatography to obtain the second intermediate; The steps of preparing the third intermediate and the fourth intermediate include: dissolving 3-ethyl-2,4-dimethylpyrrole in dichloromethane; ° C~5 ° C temperature, add phosphorus oxychloride dropwise, then at -5 ° C~5 ° C temperature, dropwise adding a solution of the second intermediate dissolved in dichloromethane; the mixed solution is reacted at room temperature for 3 to 5 hours, the solution is subjected to column chromatography, and concentrated to obtain a crude product of the third intermediate, which does not need to be purified. The crude product of the third intermediate is dissolved in ethyl acetate, methylboric acid is added, and the reaction is refluxed for 2.5 to 3.5 hours. After the reaction is completed, the solvent is evaporated and concentrated under reduced pressure, and the fourth intermediate is obtained by column chromatography; The steps of preparing the photosensitizer by using the fourth intermediate include: dissolving the fourth intermediate and 4-hydroxybenzaldehyde in toluene, adding piperidine and p-toluenesulfonic acid, and reacting the mixture at 130 ° C~150 ° The reaction was carried out at a temperature of 45 to 52 hours. After the reaction was completed, the mixture was cooled, concentrated under reduced pressure to evaporate the solvent, and the photosensitizer was obtained by column chromatography.