阅读说明：本技术 一种检测环氧合酶2的光动力治疗探针及其制备 (Photodynamic therapy probe for detecting cyclooxygenase 2 and preparation thereof ) 是由 颜梅 卫先哲 张晶 杨小凤 李增军 刘海云 于京华 于 2020-07-20 设计创作，主要内容包括：本发明公开了一种检测环氧合酶2的光动力治疗探针及其制备方法,所述的探针化合物结构如式Ⅰ所示。该前药分子将癌症标志物环氧合酶2的有效抑制剂消炎痛,利用柔性丁烷与双光子萘酰胺荧光团连接,并且引入Ce6光敏剂用于光动力治疗。探针在缓冲溶液和正常细胞中无明显荧光,而在过表达环氧合酶2的溶液和癌细胞中荧光和单线态氧量子产率剧烈增加,实现荧光成像协同光动力治疗。优势在于,探针兼具分子靶向作用和光动力疗效,是一种鉴定肿瘤并在光激发下生成单线态氧杀死肿瘤的有效工具。(The invention discloses a photodynamic therapy probe for detecting cyclooxygenase 2 and a preparation method thereof, wherein the structure of a probe compound is shown as a formula I. The prodrug molecule links the potent inhibitor of the cancer marker cyclooxygenase 2 indomethacin, utilizes flexible butane to a two-photon naphthamide fluorophore, and incorporates the Ce6 photosensitizer for photodynamic therapy. The probe has no obvious fluorescence in a buffer solution and normal cells, and the fluorescence and singlet oxygen quantum yield is increased dramatically in a solution for over-expressing cyclooxygenase 2 and cancer cells, so that the fluorescence imaging synergistic photodynamic therapy is realized. The probe has the advantages of having molecular targeting effect and photodynamic curative effect, and being an effective tool for identifying the tumor and generating singlet oxygen to kill the tumor under the excitation of light.)

1. A preparation method of a photodynamic therapy probe for detecting cyclooxygenase 2 is characterized in that the structure of the compound is shown as a formula I:

formula I;

the preparation method comprises the following steps:

(1) dissolving 4-bromo-1, 8-naphthalic anhydride and 1, 4-butanediamine in an anhydrous dioxane solution, purging the mixture with argon, adding triethylamine, refluxing in argon for 8-12 hours, cooling, stirring at room temperature for 10-14 hours, precipitating a product with cold water, and filtering in vacuum to obtain a solid compound 1; (b) dissolving the compound 1 in a DMF solution, adding indomethacin, EDCl, HOBt and DMAP into the DMF solution, stirring the mixed solution for 20 to 24 hours at room temperature in nitrogen, and carrying out column purification to obtain a solid compound 2;

(2) (c) dissolving the compound 2 in a 2-methoxyethanol solution, adding 1, 4-butanediol thereto, refluxing the mixture in the dark for 12 to 16 hours, cooling to room temperature, and purifying with a column to obtain a solid compound 3; (d) dissolving compound 3 in DCM solution, adding Ce6, DCC and DMAP, stirring at room temperature for 20-24 hr after the mixture is completely dissolved, purifying the reaction mixture by precipitation in ether solution three times, and vacuum drying to obtain compound Npco-Ce6

。

2. The method for preparing a probe for photodynamic therapy for detecting cyclooxygenase 2 according to claim 1, wherein: the above-mentionedThe molar ratio of the 4-bromo-1, 8-naphthalic anhydride to the 1, 4-butanediamine in step (1) (a) is in the range of 1: (3-3.5); the molar concentration range of 4-bromine-1, 8-naphthalic anhydride dissolved in dioxane solution is 0.03-0.04 mol.L^-1(ii) a The volume of triethylamine is 7-10 mL; (b) the molar ratio of the compound 1 to the indomethacin is 1: (1-1.5); EDCl, HOBt and DMAP molar ratio range 1: (1.5-2): (1-1.5); the molar concentration range of the compound 1 dissolved in DMF is 0.03-0.035 mol.L^-1(ii) a The volume ratio of the dichloromethane to the ethanol in the column purification is (50-25): 1.

3. the method for preparing a probe for photodynamic therapy for detecting cyclooxygenase 2 according to claim 1, wherein: the mole ratio of the compound 2 to the 1, 4-butanediol in the step (2) (c) is in the range of 1: (1-1.5); the molar concentration range of the compound 2 dissolved in the 2-methoxy ethanol is 0.2-0.25 mol.L^-1(ii) a The volume ratio of ethanol to dichloromethane in column purification is (10-5): 1; (d) the molar ratio of the compound 3 to Ce6 to DCC to DMAP is 1: (1.1-1.5): (1.3-1.8): (1.3-1.8); the molar concentration range of the compound 3 dissolved in DCM is 4-5 mmol.L^-1。

Technical Field

The invention relates to a two-photon fluorescent probe compound combining a tumor marker inhibitor and a photosensitizer, in particular to a preparation method of a fluorescent probe compound based on the combination of cyclooxygenase 2 inhibitor indomethacin and a photosensitizer Ce6, and application thereof in the fluorescent imaging synergistic photodynamic tumor treatment, belonging to the field of biological medicine.

Background

Cancer is a major disease worldwide, and one important factor affecting cancer mortality is the difficulty in obtaining an accurate early diagnosis. In addition, tumor recurrence due to incomplete resection during surgery is another challenge for cancer treatment. Therefore, there is an urgent need for early and accurate diagnosis of cancer to allow visualization of the localization of tumors and disseminated cancerous tissue. Cyclooxygenase 2 is an enzyme that is overexpressed in cancer cells and rarely expressed in normal cells, and clinical data indicate that overexpression of cyclooxygenase 2 occurs at all stages from early cancer to metastasis and spread, and its content increases as cancer progresses. Therefore, cyclooxygenase 2 is expected to be a specific marker for cancer imaging, especially in tumors such as gastric cancer, pancreatic cancer, and colon cancer.

Notably, laparoscopic endoscopic surgery has made significant progress in the treatment of many gastrointestinal disorders, with the advantages of reduced surgical trauma and complications, rapid post-operative recovery, etc. Recently, tumor-specific imaging agents have been developed to image-guide more effective treatment of gastrointestinal cancer. In particular, laparoscopes allow light to be easily delivered to the diseased site and use photodynamic therapy (PDT) to treat gastric cancer using Photosensitizers (PSs) to generate Reactive Oxygen Species (ROS) through light of a range of wavelengths. Ideally, the photosensitizer should be readily soluble in water and be able to accurately target the disease site. Meanwhile, the fluorescence imaging of the active probe can visualize malignant tumors, has high selectivity, high resolution and non-invasiveness, and effectively avoids radiation hazards caused by the current imaging technology (such as positron emission tomography, single photon emission computed tomography and X-ray imaging).

The invention discloses a photodynamic therapy probe for detecting cyclooxygenase 2 and a preparation method thereof, wherein the structure of a probe compound is shown as a formula I. The prodrug molecule links the potent inhibitor of the cancer marker cyclooxygenase 2 indomethacin, utilizes flexible butane to a two-photon naphthamide fluorophore, and incorporates the Ce6 photosensitizer for photodynamic therapy. The probe has no obvious fluorescence in a buffer solution and normal cells, and the fluorescence and singlet oxygen quantum yield is increased dramatically in a solution for over-expressing cyclooxygenase 2 and cancer cells, so that the fluorescence imaging synergistic photodynamic therapy is realized. The probe has the advantages of having molecular targeting effect and photodynamic curative effect, and being an effective tool for identifying the tumor and generating singlet oxygen to kill the tumor under the excitation of light.

Disclosure of Invention

The technical problems to be solved by the invention are as follows: the prodrug compound has the advantage of performing two-photon fluorescence imaging on cancer. Secondly, a fluorescent diagnostic reagent is provided to effectively, accurately and in-situ respond to the cyclooxygenase 2. Thirdly, a photosensitizer compound is provided, which has the advantage of photodynamic therapy for cancer. Fourthly, the application of guiding the abdominal cavity endoscope operation by the fluorescence imaging and the photodynamic therapy in the gastric cancer mouse model is provided.

In order to solve the technical problems, the technical scheme is as follows:

the invention provides a photodynamic therapy probe for detecting cyclooxygenase 2, which has the following molecular structural formula:

compound Npco-Ce6

The invention also provides a preparation method of the photodynamic therapy probe for detecting the cyclooxygenase 2, which comprises the following steps:

(1) (a) dissolving 4-bromo-1, 8-naphthalic anhydride (1 eq) and 1, 4-butanediamine (3-3.5 eq) in an anhydrous dioxane solution, purging the mixture with argon and adding triethylamine (7-10 mL), refluxing for 8-12 hours in argon, cooling, stirring at room temperature for 10-14 hours, precipitating the product with cold water and vacuum filtering to obtain a solid compound 1; (b) dissolving compound 1 (1 eq) in DMF solution, adding indomethacin (1-1.5 eq), EDCl (1 eq), HOBt (1.5-2 eq) and DMAP (1-1.5 eq) thereto, stirring the mixed solution at room temperature under nitrogen for 20-24 hours, and column-purifying (dichloromethane/ethanol) to obtain solid compound 2;

(2) (c) dissolving the compound 2 (1 eq) in a 2-methoxyethanol solution, and adding 1, 4-butanediol (1-1.5 eq) thereto, refluxing the mixture in the dark for 12-16 hours, cooling to room temperature, and then column-purifying (dichloromethane/ethanol) to obtain a solid compound 3; (d) the compound 3 (1 eq) was dissolved in DCM solution, Ce6 (1.1-1.5 eq), DCC (1.3-1.8 eq) and DMAP (1.3-1.8 eq) were added thereto, and after the mixture was completely dissolved, it was stirred at room temperature for 20-24 hours, and the reaction mixture was purified by precipitation in ether solution three times, and then dried under vacuum to obtain the compound Npco-Ce 6.

The invention has the advantages that:

the prodrug molecule has the advantages that the cyclooxygenase 2 inhibitor targets gastric cancer cells, and the photosensitizer probe is accurately delivered to a tumor part.

The prodrug molecules of the present invention, through the binding of the indomethacin moiety to the cyclooxygenase-2, create a folded conformation that allows for the effective accumulation of the photosensitizer at the tumor site.

The prodrug molecule of the invention overcomes the defect that the prior photosensitizer exciting light is visible light and can not carry out photodynamic therapy on deep tumors.

The prodrug molecule of the invention, as a novel cyclooxygenase 2 inhibitor combined photosensitizer fluorescent probe, can enhance the yield of singlet oxygen quanta and generate a synergistic antitumor effect.

Therefore, the invention is an effective tool for realizing the continuous release of singlet oxygen after targeting tumor cells in a non-invasive way and cooperating with fluorescence imaging and photodynamic therapy. Has wide application prospect in the field of biomedical analysis and detection.

Detailed Description

The present invention will be described in further detail with reference to specific examples.