A photosensitizer, its preparation method and application

Abstract

The application belongs to the technical field of photosensitizer preparation and biological medicine, and particularly relates to a photosensitizer, a preparation method and application thereof. The photosensitizer based on the fluorescent dye Nile Red can produce O2 ·‑ under light, has the potential of type I photodynamic therapy, has good active oxygen production capacity in tumor cells, and is helpful to the application of tumor photodynamic therapy. The application proves that the photosensitizer exhibits excellent antitumor effect in a mouse solid tumor, can be widely applied to the field of antitumor treatment, and has good practical application value due to the simple synthesis method, good biological safety and significant tumor photodynamic therapy effect of the type I photosensitizer.

Description

Technical Field

[0001] The present invention belongs to the technical field of photosensitizer preparation and biomedicine, and in particular relates to a photosensitizer and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Photodynamic therapy (PDT) is a promising treatment method with the advantages of low systemic toxicity, non-invasiveness and few side effects. During photodynamic therapy, photosensitizers can produce highly toxic reactive oxygen species (ROS) under appropriate light exposure, which oxidize biological molecules in cells, mainly nucleic acids, proteins and lipids, leading to serious changes in cell signaling cascades or gene expression regulation, thereby producing cytotoxicity. According to the mechanism of ROS generation, PDT can be divided into type I and type II. Type I photochemical reactions produce free radicals through an electron transfer mechanism, including superoxide anions (O2 ·- ) and hydroxyl radicals (OH·). In the process of type II PDT, molecular oxygen ( 3 O2) is converted into highly cytotoxic singlet oxygen ( 1 O2). Due to its low oxygen requirement, type I PDT is more beneficial for hypoxic tumors. However, most clinically used photosensitizers rely on type II mechanisms, and the development of more effective type I photosensitizers remains challenging.

[0004] Nile Red is a well-known oxazine organic dye, widely recognized for its intense color and lipophilicity. It also possesses numerous excellent photophysical and photochemical properties. Nile Red-based fluorophores exhibit long emission wavelengths, a wide operating wavelength range, excellent photophysical and chemical stability, an excellent extended conjugated structure, and high fluorescence quantum yields and molar extinction coefficients. However, Nile Red dyes also have certain drawbacks, such as strong hydrophobicity, difficulty in structural modification, and a quantum yield susceptible to solvent polarity, which have limited their further development and application in the field of fluorescent probes. Summary of the Invention

[0005] To address the above issues, the present invention provides a photosensitizer, its preparation method, and its application. Based on the fluorescent dye Nile Red, the present invention designed and synthesized a Type I photosensitizer, Nile Violet NV, through single-atom replacement and structural modification, and applied it to photodynamic therapy of tumors. Based on the above research results, the present invention was completed.

[0006] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0007] The present invention provides a photosensitizer, which is a compound having a structure shown in Formula I:

[0008]

[0009] The present invention also provides a method for preparing the above-mentioned photosensitizer, which comprises:

[0010]

[0011] Preferably, the molar ratio of 2-amino-5-bromobenzenethiol to 2-chloro-1,4-naphthoquinone is 1:0.5-5, more preferably 1:1;

[0012] Preferably, the synthesis method of compound 1 comprises: reacting 2-amino-5-bromobenzenethiol and 2-chloro-1,4-naphthoquinone in the presence of an alkaline reagent and an organic solvent; and further purifying to obtain the compound.

[0013] The alkaline reagent includes but is not limited to one or more of potassium hydroxide, sodium hydroxide, potassium carbonate, cesium carbonate, and triethylamine, more preferably potassium hydroxide;

[0014] The organic solvent is not limited to one or more of methanol, ethanol, DMF, and ethyl acetate, more preferably methanol. The above synthesis reaction can be carried out at room temperature;

[0015] The purification process comprises: vacuum drying the reaction solution and then purifying it by column chromatography.

[0016] Preferably, the molar ratio of compound 1 to 1-tert-butyloxycarbonylpiperazine is 2:1-10, more preferably 2:3;

[0017] Preferably, the synthesis method of the compound Boc-NV comprises: reacting the compound 1 with 1-tert-butyloxycarbonylpiperazine in the presence of a metal catalyst, a ligand, a base and a solvent; and further purifying to obtain the compound.

[0018] The metal catalyst includes but is not limited to one or more of palladium acetate, palladium chloride, and triphenylphosphine palladium dichloride, and palladium acetate is more preferred.

[0019] The ligand is 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene.

[0020] The base includes but is not limited to one or more of potassium carbonate, cesium carbonate, triethylamine, and sodium carbonate, and is more preferably cesium carbonate.

[0021] The solvent includes but is not limited to one or more of dioxane, toluene, water, and tetrahydrofuran, and dioxane is more preferred.

[0022] The purification process comprises: vacuum drying the reaction solution and then purifying it by column chromatography.

[0023] The synthesis method of the compound represented by Formula I comprises: dissolving the compound Boc-NV in a solvent, adding a strong acid, stirring, and then purifying the obtained compound. The stirring can be performed at room temperature.

[0024] The solvent includes but is not limited to one or more of dichloromethane, 1,2-dichloroethane, and toluene, and dichloromethane is more preferred.

[0025] The strong acid includes but is not limited to one or two of trifluoroacetic acid, a methanol solution of hydrogen chloride, and a dioxane solution of hydrogen chloride, and trifluoroacetic acid is more preferred.

[0026] The purification process comprises: vacuum drying the reaction solution, extracting the obtained crude product with dichloromethane and saturated sodium bicarbonate solution, drying the organic phase with anhydrous sodium sulfate, and purifying the obtained crude product after vacuum drying by column chromatography.

[0027] The use of the above-mentioned photosensitizer or the photosensitizer prepared by the above-mentioned preparation method in the preparation of a photosensitizer for photodynamic therapy has been demonstrated through experiments to have a maximum absorption wavelength of approximately 550 nm and a maximum emission wavelength of approximately 650 nm. It can generate reactive oxygen species under light irradiation. In particular, as a Type I photosensitizer, it has excellent therapeutic potential for hypoxic tumors, effectively overcoming the dependence of traditional photosensitizers on oxygen in the hypoxic tumor environment.

[0028] Therefore, the present invention also provides the use of the above photosensitizer or the photosensitizer prepared by the above preparation method in the preparation of drugs for treating (adjuvant treating) tumors.

[0029] It should be noted that the term "tumor" as used herein, as known to those skilled in the art, includes benign and / or malignant tumors. Benign tumors are defined as excessive cell proliferation that is incapable of forming aggressive, metastatic tumors in the body. Conversely, malignant tumors are defined as cells with multiple cellular and biochemical abnormalities that are capable of developing systemic diseases (e.g., metastasis to distant organs). The tumor may be a solid tumor or a hematological tumor, with solid tumors being preferred. Further, the tumor is selected from the group consisting of the following benign or malignant tumors: B-cell lymphoma, sarcoma, lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic lymphoma, anaplastic large cell lymphoma, acute myeloid leukemia, acute lymphocytic leukemia, acute granulocytic leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, chronic granulocytic leukemia, chronic neutrophilic leukemia, acute undifferentiated cell leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma / Waldenstrom's macroglobulinemia, adult T-cell acute lymphocytic leukemia, mixed lineage leukemia, splenic marginal zone lymphoma, plasma cell myeloid leukemia,

[00135] The present invention relates to a type of cancer comprising the following: a neoplastic disease, a type of cancer comprising the following: a leukemia, a type of cancer comprising the following: a leukemia, a type of cancer comprising the following: a leukemia, a type of cancer comprising the following: a leukemia, a type of cancer comprising the following: a leukemia, a type of cancer comprising the following: a leukemia, a type of cancer comprising the following: a leukemia, a type of cancer comprising the following: a leukemia, a type of cancer comprising the following: a leukemia, a type of cancer comprising the following: a leukemia, a type of cancer comprising the following: a leukemia, a type of cancer comprising the following:

[0030] The present invention also provides a pharmaceutical composition comprising the above photosensitizer.

[0031] The pharmaceutical composition of the present invention can be administered in any manner selected from the following: oral administration, spray inhalation, rectal administration, nasal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or by means of an explanted reservoir, among which oral, intramuscular, intraperitoneal or intravenous administration is preferred.

[0032] The present invention also provides a pharmaceutical preparation comprising the above-mentioned photosensitizer and pharmaceutically acceptable excipients and / or carriers.

[0033] The above-mentioned pharmaceutical preparations can be any pharmaceutical dosage form acceptable in pharmaceutical science, such as tablets, hard capsules, soft capsules, pills, micropills, granules, dry suspensions, powders, oral liquids, injections, etc.

[0034] The present invention also provides a treatment system, comprising:

[0035] a) the above-mentioned photosensitizer, pharmaceutical composition or pharmaceutical preparation, and;

[0036] b) Lighting device.

[0037] Wherein, the light source emitted by the illumination device may be 400-700nm.

[0038] The present invention also provides a method for (adjuvant) treating tumors, comprising administering a therapeutically effective dose of the above-mentioned photosensitizer, the above-mentioned pharmaceutical composition, the above-mentioned pharmaceutical preparation or the above-mentioned therapeutic system to a subject.

[0039] The term "subject" refers to an animal, preferably a mammal, and most preferably a human, that has been the subject of treatment, observation, or experiment. A "therapeutically effective dose" refers to an amount that results in an improvement in any parameter or clinical symptom. The actual dose may vary with each patient and does not necessarily refer to an amount that eliminates all disease symptoms. It can be determined using methods known in the art and will not be detailed here.

[0040] Beneficial technical effects of the above technical solution:

[0041] The photosensitizer based on Nile red provided by the above technical solution can produce O2 after illumination. ·- , with type I photodynamic therapy potential; the type I photosensitizer can efficiently generate ROS in solutions and cells, overcoming the shortcomings of poor photodynamic therapy effect in the hypoxic environment of tumors, and at the same time achieving strong photodynamic therapy ability for mouse solid tumors; at the same time, the above-mentioned type I photosensitizer has a simple synthesis method, good biosafety, and significant tumor photodynamic therapy effect, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0043] Figure 1 This is the H NMR spectrum of compound 1 in Example 1 of the present invention.

[0044] Figure 2 The NMR carbon spectrum of compound 1 in Example 1 of the present invention is

[0045] Figure 3 This is the H NMR spectrum of the compound Boc-NV in Example 1 of the present invention.

[0046] Figure 4 This is the C NMR spectrum of the compound Boc-NV in Example 1 of the present invention.

[0047] Figure 5 This is the H NMR spectrum of the Type I photosensitizer NV in Example 1 of the present invention.

[0048] Figure 6 This is the NMR carbon spectrum of the type I photosensitizer NV in Example 1 of the present invention.

[0049] Figure 7 These are the UV and fluorescence spectra of the type I photosensitizer NV in solvent in Example 2 of the present invention.

[0050] Figure 8 This is a fluorescence change curve of the type I photosensitizer NV after interacting with DHR123 in Example 3 of the present invention.

[0051] Figure 9 This is a graph showing the absorption change of the type I photosensitizer NV after reacting with ABDA in Example 4 of the present invention.

[0052] Figure 10 This is a graph showing the change in fluorescence intensity after the type I photosensitizer NV acts on DCFH-DA in MDA-MB-231 cells in Example 5 of the present invention.

[0053] Figure 11 This is a graph showing the cytotoxicity test results of the type I photosensitizer NV on MDA-MB-231 cells under normoxic and hypoxic conditions in Example 6 of the present invention.

[0054] Figure 12 This is a diagram showing the results of photodynamic therapy of mouse tumors using the type I photosensitizer NV in Example 7 of the present invention. DETAILED DESCRIPTION

[0055] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0056] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0057] The present invention is based on the fluorescent dye Nile red, and a type I photosensitizer Nile violet NV (i.e., the compound shown in formula I) is designed and synthesized through single-atom replacement and structural modification, and is applied to the photodynamic therapy of tumors.

[0058] The synthetic route of the type I photosensitizer Nile purple NV is as follows:

[0059]

[0060] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0061] Example 1

[0062] A method for preparing a type I photosensitizer NV based on Nile red, the specific synthesis steps are as follows:

[0063] (1) Synthesis of Compound 1: 2-Amino-5-bromobenzenethiol (4.08 g, 20.0 mmol) was dissolved in 150 mL of methanol at room temperature. Potassium hydroxide (1.23 g, 22.0 mmol) was added. After stirring for 30 min, 2-chloro-1,4-naphthoquinone (4.54 g, 20 mmol) was slowly added. Stirring was continued at room temperature for 6 h. After the reaction was completed, the reaction solution was vacuum dried by TLC monitoring, and the crude product was purified by column chromatography (petroleum ether / dichloromethane, 1:1, v / v). The product was collected to obtain 3.6 g of a brown solid with a yield of 53%. 1 H-NMR (400MHz, CDCl3) δ8.88-8.77(m,1H),8.34-8.23(m,1H),7.89-7.71(m,3H),7.57(dd,J=7.2,2.0Hz,2H),6.85(s,1H). 13 C-NMR (100MHz, DMSO) δ134.39,131.91,131.66,131.18,127.27,125.98,125.68,120.89.

[0064] (2) Synthesis of compound Boc-NV: Compound 1 (410.6 mg, 1.2 mmol), 1-tert-butyloxycarbonylpiperazine (335.3 mg, 1.8 mmol), palladium acetate (13.5 mg, 0.06 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (37.4 mg, 0.06 mmol) and cesium carbonate (586.5 mg, 1.8 mmol) were added to 12 mL of 1,4-dioxane, and the reaction solution was stirred at 100°C for 6 h. After the reaction was completed, the reaction solution was vacuum dried and the crude product was purified by column chromatography (petroleum ether / ethyl acetate, 1:1, v / v). The product was collected to obtain 467 mg of dark red solid with a yield of 87%. 1 H-NMR (400MHz, CDCl3) δ8.72 (dd, J=7.8, 1.5Hz, 1H), 8.24 (dd, J=7.6, 1.6Hz, 1H), 7.74-7 .64(m,3H),6.69(s,1H),3.59(dd,J=6.6,3.9Hz,4H),3.31(t,J=5.3Hz,4H),1.51(s,8H). 13 C-NMR (100MHz, CDCl3) δ180.09,154.59,150.90,140.19,137.78,134.79,134.64,132.29,131.44,1 31.17,130.09,125.86,125.58,124.96,118.79,114.64,107.87,80.36,77.27,53.46,47.04,28.44.

[0065] (3) Synthesis of Compound NV: Compound Boc-NV (160.0 mg, 0.36 mmol) was dissolved in 3 mL of dichloromethane, and 3 mL of trifluoroacetic acid was slowly added. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was vacuum dried. The crude product was extracted with 30 mL of dichloromethane and 30 mL of saturated sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate and vacuum dried. The crude product was purified by column chromatography (dichloromethane / methanol, 10:1, v / v). The product was collected to obtain 467 mg of a dark purple solid with a yield of 89%. 1H-NMR (400MHz, DMSO) δ8.73(dd,J=8.1,1.3Hz,1H),8.13(dd,J=7.8,1.5Hz,1H),7.83(d,J=1.5Hz,1H), 7.80-7.72(m,2H),7.23-7.16(m,2H),6.89(s,1H),3.43(d,J=5.0Hz,4H),2.89(dd,J=6.3,3.7Hz,4H). 13 C-NMR (100MHz, DMSO) δ179.10,151.99,138.13,134.92,134.81,132.15,131.86,130 .57,130.34,126.36,125.49,124.91,118.33,114.94,107.95,47.95,45.69,40.45.

[0066] Example 2

[0067] The absorption spectrum and fluorescence emission spectrum of the photosensitizer NV synthesized in Example 1 in DMSO solution were measured using an ultraviolet-visible light analyzer and a fluorescence spectrophotometer. Figure 7 , the maximum absorption wavelength of NV is around 550nm, and the maximum emission wavelength is around 650nm.

[0068] Example 3

[0069] Dihydrorhodamine (DHR123) was used as the superoxide anion (O2 ·- ) Monitoring probe detects NV produces O2 under light ·- DHR123 itself does not emit fluorescence, but can be detected by O2 ·- The photosensitizer NV and DHR123 probe were dissolved in PBS solution at a concentration of 5 μM. The mixture of NV and DHR123 was then exposed to a luminescent light source at 400-700 nm and 10 mW·cm -2 The solution was irradiated with LED light for 0, 1, 2, 3, 4, 5, 6, and 7 minutes. DHR123 without photosensitizer was used as a control, and the changes in fluorescence intensity after different illumination times were recorded. Figure 8 As shown in Figure 2, compared with the control group, the fluorescence of the mixed solution of NV and DHR123 under light conditions increased significantly with increasing irradiation time, indicating that compound NV effectively produced O2 under light. ·- .

[0070] Example 4

[0071] 9,10-Anthracenediyl-bis(methylene)dicarboxylic acid (ABDA) was used to determine singlet oxygen ( 1O2) generation. Rose Bengal (RB) was used as a standard reference. Under dark conditions, NV ethanol solution with an absorbance of about 0.2 was mixed with ABDA ethanol solution with an absorbance of about 1.0. Using 400-700 nm, 20 mW·cm -2 The LED light was irradiated for 0, 5, 10, 15, 20, 25, 30 and 35 minutes respectively. The absorption intensity changes of ABDA probe at 380 nm after different illumination times were recorded. Figure 9 As shown, RB can be efficiently generated under illumination 1 However, NV failed to cause significant changes in ABDA absorbance under light conditions, indicating that NV cannot effectively produce O2 under light. 1 O2.

[0072] Example 5

[0073] The ability of NV to generate ROS in MDA-MB-231 cells was detected using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Cells were incubated with NV (0.1 μM) for 2 h and then washed. The cells were incubated with 5 μM DCFH-DA for 30 min in the dark and then illuminated with 400-700 nm, 20 mW·cm -2 The cells were irradiated with LED light for 10 min, and then collected with trypsin and analyzed by flow cytometry. Figure 10 As shown in the figure, compared with the control group without drug treatment, NV basically did not affect the fluorescence intensity of DCFH-DA under dark conditions, while 400-700nm laser irradiation could significantly enhance the fluorescence intensity of DCFH-DA, indicating that NV can effectively produce reactive oxygen species in tumor cells under light irradiation.

[0074] Example 6

[0075] The CCK-8 method was used to evaluate the phototoxicity and dark toxicity of NV to MDA-MB-231 cells and 4T1 cells under normoxic (21% O2) and hypoxic (2% O2) conditions. Cells were incubated with different concentrations of NV for 2 h and then illuminated with 400-700 nm, 20 mW·cm -2 After 10 min of LED light irradiation, the cells were cultured for 24 h. To determine the cell viability, the cells were incubated with CCK-8 in the dark for 4 h, and the absorbance at 450 nm was measured by a microplate reader, and the IC 50 Value. Figure 11 As shown in Figure 2, NV has low dark toxicity but exhibits good phototoxicity under light conditions: IC 50 The value was 0.164±0.006μM, and the IC value for 4T1 cells was 50The value was 0.403±0.004μM. At the same time, NV also showed excellent phototoxicity under hypoxic conditions, with an IC of 0. 50 The value was 0.303±0.015μM, and the IC value for 4T1 cells was 50 The value was 0.693±0.042μM, which indicated that NV as a type I photosensitizer had good ability to treat hypoxic tumors, overcoming the dependence of traditional photosensitizers on oxygen in the hypoxic tumor environment.

[0076] Example 7

[0077] To further evaluate the therapeutic effect of NV on solid tumors in mice, 4T1 cells were subcutaneously inoculated into BALB / c mice to establish a 4T1 mammary tumor-bearing mouse model. In vivo treatment experiments were performed on 4T1 tumor-bearing mice. The mice were randomly divided into 4 groups (5 in each group): PBS group, PBS+Light group, photosensitizer NV group, and photosensitizer NV+Light group (400-700 nm, 300 mW·cm -2 , 5min, NV dose was 2.5mg / kg). Tumor volume changes were recorded every 2 days. After 14 days of treatment, all mice were euthanized, and tumors were collected and photographed. Figure 12 As shown, there was no significant change in the tumor volume of the NV group. In contrast, the tumor growth rate of the mice in the NV group after irradiation with 400-700 nm light was significantly slowed down, indicating that the photosensitizer NV has excellent photodynamic anti-tumor ability in vivo.

[0078] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent replacements for parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A photosensitizer, characterized in that A compound having the structure shown in Formula I:

2. A method for preparing a photosensitizer, characterized in that: The preparation method comprises:

3. The method for preparing a photosensitizer according to claim 2, wherein The molar ratio of 2-amino-5-bromobenzenethiol to 2-chloro-1,4-naphthoquinone is 1:0.5-5.

4. The method for preparing a photosensitizer according to claim 2, wherein The molar ratio of compound 1 to 1-tert-butyloxycarbonylpiperazine is 2:1-10.

5. Use of the photosensitizer according to claim 1 or the photosensitizer prepared by the preparation method according to any one of claims 2 to 4 in the preparation of a photosensitizer for photodynamic therapy.

6. Use of the photosensitizer according to claim 1 or the photosensitizer prepared by the preparation method according to any one of claims 2 to 4 in the preparation of drugs for treating tumors.

7. A pharmaceutical composition, characterized in that The photosensitizer comprises the photosensitizer according to claim 1 or the photosensitizer prepared by the preparation method according to any one of claims 2 to 4.

8. A pharmaceutical preparation, characterized in that The invention comprises the photosensitizer according to claim 1 or the photosensitizer prepared by the preparation method according to any one of claims 2 to 4 and pharmaceutically acceptable excipients and / or carriers.

9. A treatment system, characterized in that The treatment system comprises: a) the photosensitizer according to claim 1, the pharmaceutical composition according to claim 7, or the pharmaceutical preparation according to claim 8, and; b) Lighting device.

10. The treatment system according to claim 9, characterized in that The light source emitted by the illumination device is 400-700nm.

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