CN108164570A - A kind of photosensitizer containing selenium and its preparation method and application - Google Patents

Abstract

The invention discloses a selenium-containing photosensitizer and its preparation method and application. The invention synthesizes a novel selenium-containing photosensitizer with anti-tumor angiogenesis effect for the first time. It is used in the treatment of skin squamous cell carcinoma, and the effect is remarkable. It is much lower than the 5-ALA used in the market, and it does not contain antibiotics, does not produce drug resistance, has few side effects, is safe and non-toxic, and is economical and practical; and, due to the limited penetration of light and some other factors, even without light The dynamic response of tumor tissues can also achieve the effect of treating tumors by inhibiting tumor angiogenesis.

Description

A kind of selenium-containing photosensitizer and its preparation method and application

technical field

The invention relates to a selenium-containing photosensitizer and its preparation method and application, belonging to the field of biomedicine.

Background technique

Epithelial cancer is a kind of skin cancer, and its main features are: it starts as a blemish-like raised mass, with a hard base, a rough surface like a cauliflower, visible telangiectasia, and spike-like cutin at the top.

Photodynamic therapy (PDT) is a new non-invasive or minimally invasive treatment technology that combines light, photosensitizers and oxygen molecules in tissues to selectively destroy diseased tissues through photodynamic reactions to achieve therapeutic purposes. PDT is an emerging medical discipline that is researching and developing the treatment of tumors and other benign diseases. It is another new tumor therapy after traditional tumor therapies such as surgery, radiotherapy, chemotherapy and immunotherapy. It is based on the difference in the concentration of photosensitizers in normal and target tissues, and the direct irradiation of light on the target tissue by means of spatial confinement and focusing. After injection, the photosensitizer accumulates in the target tissue. During the accumulation period, the target tissue is directly irradiated with light to activate the photosensitizer. The activated photosensitizer transfers energy to the ground state oxygen ( ³ O ₂ , ³ ∑g-) to form various forms of Reactive oxygen species damage the important structure and function of cells, leading to tissue destruction.

PDT relies heavily on phototherapeutic drugs (photosensitizers). The proposal, development and application of photodynamic therapy are gradually perfected along with the development of photosensitizers. An ideal photosensitizer should have the following characteristics: 1) stable properties and clear structure; 2) strong phototoxicity when exposed to light, and low toxicity to the body; 3) high selectivity in the target tissue without staying too long ; 4) strong photosensitizing power, triplet oxygen output and long life; 5) strong absorption in the phototherapy window (600-900nm) to facilitate the use of light sources that penetrate deeper into human tissue; 6) in the Soluble at physiological pH. However, the current photosensitizers still have many deficiencies, such as low molar absorbance, inability to fully convert light into cytotoxic substances, and limited enrichment effects. Such as the paper (Liu Minghui, Liu Junhong, Han Guiyan, Zhang Xingjie, Sheng Chunquan, Yao Jianzhong. Design and synthesis of chlorin p6-13,15-cycloimide photosensitizer[J]. Journal of Pharmaceutical Practice, 2017,35(01) :26-30+35.) Although the extracted chlorin p6-13,15-cyclic imide derivatives have a maximum absorption wavelength of 698nm and have a relatively stronger tissue penetration depth, they do not bind to organisms The metabolic characteristics were further designed and optimized.

Apoptosis is a physiological process involving multiple factors, involving immune response, gene regulation, signal transduction and other processes. Apoptosis can play a regulatory role in a variety of normal physiological processes, and if this function is abnormal or missing, it can lead to a series of pathological conditions. One of the characteristics of tumor cells is the lack of this programmed death process. The British scientist Hickman J A first proposed that the induction of tumor cell apoptosis can be used as the main goal and means of future tumor treatment research, and then the induction of tumor cell apoptosis has become a new way of tumor treatment research, and it has also become a new target for screening anticancer drugs , New hot spots.

The endoplasmic reticulum is an organelle with important functions, and it is the main place for protein synthesis, calcium ion storage and lipogenesis in eukaryotic cells. The functional state of the endoplasmic reticulum is critical for protein folding, maturation, and movement. Changes in the cell state can interfere with the normal function of the ER, causing ER dysfunction, protein misfolding and aggregation in the lumen with unfolded proteins, and disturbances in calcium balance will all cause ER stress. The ASK/JNK signaling pathway is one of the three important signals in endoplasmic reticulum stress. Activating the ASK/JNK signaling pathway causes cells to generate endoplasmic reticulum stress, increases the expression of Bip and Chop proteins, and increases the pro-apoptosis and oxidation Protein product, induces apoptosis. Therefore, it can induce endoplasmic reticulum stress by activating the ASK/JNK signaling pathway of tumor cells, thereby inducing cell apoptosis.

How to fight against tumor growth has become a current research hotspot, but there are still some problems, such as patent, application number: CN201611022732.6, publication number: CN106632195A, a research method of Vitex vitae anti-tumor compound, which mentioned The method of extracting anti-tumor compounds in Vitex vitae does not combine the specific metabolic characteristics of tumors to improve the precision of the compounds' action on tumors.

Using PDT to treat tumors through apoptosis has many advantages, such as: (1) less trauma; (2) less toxicity; (3) good selectivity; (4) good applicability; (5) good repeatability; (6) ) can be palliative treatment; (7) can eliminate recessive cancer lesions; (8) can protect appearance and important organ functions, etc. At present, PDT has gradually become one of the conventional treatment methods for tumors. It can achieve efficient treatment of tumors through various mechanisms such as directly killing tumor cells, destroying tumor vasculature, and activating the body's anti-tumor immunity; cell apoptosis can also be enhanced. Anti-tumor drugs targeting cell signal transduction, etc. Therefore, how to use photosensitizers to control apoptosis in the process of photodynamic therapy and improve the efficacy of photodynamic therapy is a subject worth studying.

Contents of the invention

The technical problem solved by the present invention is, aiming at the above technical problems, the purpose of the present invention is to provide a kind of active oxygen that can not only generate a large amount of reactive oxygen species to destroy tumor cells, but also inhibit tumor growth by inhibiting tumor angiogenesis, with few side effects and quick effect. new photosensitizer.

The technical scheme of the present invention is to provide a selenium-containing photosensitizer, the structural formula of which is:

In the above structural formula of the photosensitizer, the dotted lines between O atoms and O atoms, and between O atoms and N atoms represent hydrogen bonds.

The present invention also provides a preparation method of the above-mentioned selenium-containing photosensitizer, comprising the following steps:

(1) Dissolving acetic acid and α, β-tetrahydropyrrolecarboxylic acid acetic acid in ethanol (preferably commercially available 95% ethanol) and reacting at 50-70°C to obtain an intermediate product A, the structural formula of the intermediate product A is :

(2) Will Heating and reacting with intermediate product A in an organic solvent containing benzene for 6-12 hours to obtain intermediate product B, wherein the reaction temperature is 200°C; the structural formula of intermediate product B is:

(3) intermediate product B and Heat and stir in aniline, gradually add alkali to adjust the pH to 6.5-7.2, react for 5-7 hours, extract and purify the reaction mixture to obtain the selenium-containing photosensitizer.

Preferably, in step (1), the molar ratio of acetic acid to α,β-tetrahydropyrrolecarboxylic acid acetic acid is 1:1-1.5.

Preferably, in step (2), And the molar ratio of intermediate product A is 1:1.5-2.

Preferably, in step (3), intermediate product B and The molar ratio is 2.5-3:1.

Preferably, in step (3), the alkali is sodium hydroxide.

The invention also provides the application of the selenium-containing photosensitizer in the preparation of drugs for treating tumors.

Preferably, the tumor is skin cancer.

Preferably, the skin cancer is epithelial cancer.

The drug for treating tumors includes a photosensitizer and auxiliary additives; the weight percent content of the selenium-containing photosensitizer in the drug composition is 0.01-9.99%, preferably 0.01-0.25%.

The auxiliary additives include pharmaceutically acceptable excipients or auxiliary materials. The auxiliary materials or excipients include but are not limited to mannitol, sodium bisulfite, starch, dextrin powder, absolute ethanol, water for injection, powdered sugar, lactose, hypromellose, magnesium stearate, sucrose, Povidone K30.

The beneficial effect of the present invention is that the present invention is closely combined with the biological characteristics of the tumor, and synthesized for the first time a new type of selenium-containing photosensitizer with anti-tumor angiogenesis effect, which is used in the treatment of skin squamous cell carcinoma, and the effect is remarkable and the dose is much lower The 5-ALA used in the market does not contain antibiotics, does not produce drug resistance, has few side effects, is safe and non-toxic, and is economical and practical. Moreover, due to the limited penetration of light and some other factors, even tumor tissues that do not produce photodynamic responses can achieve the effect of treating tumors by inhibiting tumor angiogenesis.

Description of drawings

Figure 1 shows the chemical structural formula of a selenium-containing photosensitizer.

Figure 2 shows the hydrogen spectrum of the selenium-containing composite photosensitizer.

Figure 3 represents the carbon spectrum of the novel photosensitizer containing selenium.

Fig. 4 shows a flow cytogram of A431 squamous cell carcinoma cells.

Figure 5 shows the fluorescence results of apoptosis-related proteins in immune cells

Figure 6 shows the results of Western Blot of endoplasmic reticulum stress-related proteins.

Figure 7 shows the results of Western Blot of proteins related to the endoplasmic reticulum stress signaling pathway.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

Embodiment 1: the synthesis of photosensitizer EtNBSe-PDT

The synthetic method of EtNBSe-PDT comprises the following steps:

(1) Acetic acid and α, β-tetrahydropyrrolecarboxylic acid acetic acid are dissolved in 95% ethanol to obtain intermediate product A, and the reaction process is as follows:

The molar ratio of acetic acid to α, β-tetrahydropyrrolecarboxylic acid acetic acid is 1:1-1.5, and the reaction temperature is 50-70°C.

(2) Will And the intermediate product A that step (1) obtains obtains intermediate product B in the benzene-containing organic solvent sealed heating reaction 6-12 hour, and reaction temperature is about 200 ℃, and its reaction process is as follows:

(3) intermediate product B obtained by step (2) and Heat and stir in aniline, gradually add sodium hydroxide to adjust the pH to 6.5-7.2, react for 5-7 hours, sequentially extract with petroleum ether, ethyl acetate, n-butanol, and separate and purify by silica gel column chromatography to obtain the final product, Its reaction formula is:

The molar ratio of the front and rear reactants is 2.5-3:1.

The structural formula of the final product, that is, the selenium-containing photosensitizer is shown in Figure 1, and its H NMR and C NMR spectra are shown in Figure 2 and Figure 3, respectively.

Example 2 EtNBSe-PDT causes apoptosis in epithelial cancer Example

Annexin V is a reagent for detecting cell apoptosis. In normal cells, phosphatidylserine is only distributed on the inner side of the lipid bilayer of the cell membrane. In the early stage of cell apoptosis, the membrane phosphatidylserine (PS) is turned from the inner side of the lipid membrane to outside. Therefore, Annexin V is a sensitive indicator for detecting early cell apoptosis. PI (propidium iodide) is a nuclear staining reagent that can stain DNA. Although PI cannot pass through the living cell membrane, it can pass through the damaged cell membrane and stain the nucleus. Therefore, the combination of Annexin V and PI can stain live and dead cells at the same time. According to flow cytogram analysis, under the action of EtNBSe-PDT, the number of apoptotic cells increased significantly compared with the blank group, which proved that EtNBSe-PDT can induce tumor cell apoptosis to achieve the purpose of anti-tumor.

(1) Experimental method

The squamous cell carcinoma A-431 cell line was cultured in DMEM medium with 10% calf serum, added with 100IU/L penicillin and 100mg/L streptomycin, and placed in a constant temperature incubator at 37°C and 5% CO2. Cultivate, after the cells grow to the bottom of the bottle, discard the culture medium, wash twice with PBS solution, then digest with 0.25% trypsin and 0.02% EDTA for 5 minutes, then culture in separate bottles, passaging once every 2 to 3 days, and take Cells in logarithmic growth phase were used for experiments.

A-431 cells in the logarithmic growth phase were digested with trypsin and inoculated into six-well culture plates, placed in a 5% CO2 incubator, and cultured at 37°C. When they grew to 80% to 90% confluent, 400nM Distilled water, 400nM EtNBSe, incubated for 1h, irradiated with 20J/cm ² red light for 15min, stained with Annexin V-PI after 16h, observed cell apoptosis by FACSCalibur flowcytometer.

(2) Experimental results

The results of flow cytometry showed that the apoptosis of A-431 cells 16h after EtNBSe-PDT was increased by 42.3% compared with the blank group ( FIG. 4 ). 400nM EtNBSe-PDT can obviously induce cell apoptosis.

Example 3 EtNBSe-PDT Induces Cell Apoptosis and Endoplasmic Reticulum Stress Example

The ASK/JNK signaling pathway is one of the three important signals in the endoplasmic reticulum stress. Activating the ASK/JNK signal can significantly promote the generation of endoplasmic reticulum stress in cells, and increase the expression of GADD153 and GRP78, indicating that the endoplasmic reticulum stress Excited. Caspase-3 is an important indicator of cell apoptosis, and the increase of Caspase-3 expression is an important symbol of cell apoptosis. According to the fluorescent results of immune cells, under the action of EtNBSe-PDT, the expression of Caspase-3 and Bcl-2 changed accordingly, indicating that the apoptosis increased; according to the results of Western Blot, under the action of EtNBSe-PDT, ASK/JNK Signal-related proteins change accordingly, ASK/JNK signaling is activated, and ER stress occurs. It shows that EtNBSe-PDT can obviously activate ASK/JNK signal, and induce cells to produce endoplasmic reticulum stress and apoptosis through ASK/JNK signal.

(1) Experimental method

The squamous cell carcinoma A-431 cell line was cultured in DMEM medium with 10% calf serum, added with 100IU/L penicillin and 100mg/L streptomycin, and placed in a constant temperature incubator at 37°C and 5% CO2. Cultivate, after the cells grow to the bottom of the bottle, discard the culture medium, wash twice with PBS solution, then digest with 0.25% trypsin and 0.02% EDTA for 5 minutes, then culture in separate bottles, passaging once every 2 to 3 days, and take Cells in logarithmic growth phase were used for experiments.

A-431 cells in the logarithmic growth phase were digested with trypsin and inoculated into six-well culture plates, placed in a 5% CO2 incubator, and cultured at 37°C. When they grew to 80% to 90% confluent, they were added with Amount of distilled water and 400nM EtNBSe reagent were incubated for 1 hour, and then irradiated with 20 J/cm ² red light for 15 minutes. After 16 hours, the cells were collected to extract total protein, and the Western Blot method was used to detect GADD153 and GRP78 in the cells.

A-431 cells in the logarithmic growth phase were digested with trypsin and inoculated into six-well culture plates, placed in a 5% CO2 incubator, and cultured at 37°C. When they grew to 80% to 90% confluent, an equal amount of After incubation with distilled water and 400nM EtNBSe for 1 hour, the cells were irradiated with 20 J/cm ² red light for 15 minutes. After 16 hours, the cells were collected for immunofluorescence staining, and the expression of Caspase-3 in the cells was observed.

A-431 cells in the logarithmic growth phase were digested with trypsin and inoculated into six-well culture plates, placed in a 5% CO2 incubator, and cultured at 37°C. When they grew to 80% to 90% confluent, they were added with Amount of distilled water, 100nM EtNBSe, 200nM EtNBSe agent and 400nM EtNBSe were incubated for 1h, and then irradiated with 20J/ ^cm2 red light for 15min. After 0.5h, the cells were collected to extract the total protein, and Western Blot was used to detect p-ASK and p- Changes in JNK expression.

(2) Experimental results

Cell immunofluorescence experiments also clearly demonstrated that 400nM EtNBSe-PDT can significantly promote the increase of Caspase-3 expression (Figure 5), indicating that 400nM EtNBSe-PDT can significantly promote cell apoptosis. Western Blot results analysis found that 400nM EtNBSe-PDT can significantly increase the expression of GADD153 and GRP78 in cells (Figure 6), indicating that 400nM EtNBSe-PDT can significantly induce cells to produce endoplasmic reticulum stress.

The Western Blot results of the changes in the expression of p-ASK and p-JNK in cells treated with gradient EtNBSe-PDT showed that 0-400nM EtNBSe-PDT can significantly promote the increase of p-ASK, while the activation of p-JNK by EtNBSe-PDT requires The dose of EtNBSe reached 400nM before it became more obvious, but in general EtNBSe-PDT can significantly activate ASK/JNK signaling, and induce cells to produce endoplasmic reticulum stress and apoptosis through ASK/JNK signaling (Figure 7).

Claims (9)

1. a selenium-containing photosensitizer, is characterized in that, the structural formula of described photosensitizer is: 2. a preparation method of selenium-containing photosensitizer as claimed in claim 1, is characterized in that, comprises the following steps: (1) Dissolving acetic acid and α,β-tetrahydropyrrolecarboxylic acid acetic acid in ethanol, reacting at 50-70°C to obtain intermediate product A, the structural formula of the intermediate product A is: (2) Will The intermediate product A is sealed and heated in an organic solvent containing benzene for 6-12 hours to obtain the intermediate product B, wherein the reaction temperature is 200 ° C; the structural formula of the intermediate product B is: (3) intermediate product B and Heat and stir in aniline, gradually add alkali to adjust the pH to 6.5-7.2, react for 5-7 hours, extract and purify the reaction mixture to obtain the selenium-containing photosensitizer. 3. The preparation method according to claim 2, characterized in that, in step (1), the molar ratio of acetic acid to α,β-tetrahydropyrrolecarboxylic acid acetic acid is 1:1-1.5. 4. preparation method as claimed in claim 2 is characterized in that, in step (2), And the molar ratio of intermediate product A is: 1:1.5-2. 5. preparation method as claimed in claim 2 is characterized in that, in step (3), intermediate product B and The molar ratio is 2.5-3:1. 6. preparation method as claimed in claim 2 is characterized in that, in step (3), described alkali is sodium hydroxide. 7. The application of the selenium-containing photosensitizer according to claim 1 in the preparation of a drug for treating tumors. 8. The use according to claim 7, wherein the tumor is skin cancer. 9. The use according to claim 7, wherein the skin cancer is epithelial cancer.