A molecular probe targeting stimulator of interferon genes, labeling method and application

Abstract

The present application provides a molecular probe targeting stimulator of interferon genes (STING). The probe has a structure of an interferon gene targeting group - acridone derivative, and different lengths of alkyl side chains are connected to the N atom site in the structure, or a short polyethylene glycol chain with 1, 2, 3 - triazole, or a side chain containing a chelating group with different numbers of alkyl groups. The small molecule targeting STING of the present application, as a STING agonist, can be used for STING - related immunotherapy. The fluorescence performance of the small molecule itself can be used for STING - targeted fluorescence imaging. After being labeled with radionuclides, the small molecule can be used as a radioactive tracer for in - vivo PET / SPECT imaging and treatment of STING - related diseases such as tumors and inflammation.

Description

Technical Field

[0001] The present application relates to the field of medical imaging technology, and in particular, to a molecular probe targeting stimulator of interferon genes (STING), a labeling method, and an application thereof. Background Art

[0002] Cancer seriously endangers human health. According to the global cancer statistics in 2020, there were 19.3 million new cases and nearly 10 million death cases in 2020. For decades, traditional anti-cancer treatment strategies have been surgery, chemotherapy, and radiotherapy. Although these therapies have provided substantial benefits in eradicating primary tumors, the recurrence of diseases caused by residual malignant cells or tumor metastasis remains a common problem. Cancer immunotherapy is becoming an attractive strategy among different treatment options. For example, chimeric antigen receptor T cell (CAR-T) therapy and immune checkpoint inhibitor therapy (ICIs) have shown great therapeutic potential in many clinical trial studies. Although various immunotherapy strategies have achieved remarkable effects clinically, efficacy is only observed in a small subset of patients. Increasing evidence indicates that the tumor immune microenvironment plays a more important role in tumor immune surveillance and immune evasion than immune checkpoints. The agonist immunotherapy of stimulator of interferon genes (STING) can reshape the immunosuppressive tumor microenvironment, enhance the infiltration of immune cells such as CD4 and CD8 T cells, turn the tumor from "cold" to "hot", thereby enhancing the effect of anti-tumor immunotherapy and inducing a sustained anti-tumor immune response. In addition, the combination of STING agonists with chemotherapy, radiotherapy, and ICIs for immunotherapy can achieve an effect of 1 + 1 > 2.

[0003] STING is also known as transmembrane protein 173, regulatory activator of interferon regulatory factor 3, and endoplasmic reticulum interferon-stimulated protein, etc., and is mainly expressed on the rough endoplasmic reticulum, mitochondria, and outer membranes of microsomes of human macrophages, T lymphocytes, dendritic cells, endothelial cells, epithelial cells, and fibroblasts, etc. As a key protein in the innate immune response, STING plays an important role in the process of stimulating the production of type I interferon (IFN). Type I IFN plays a key role in eliminating pathogens, activating immune responses, anti-tumor immunotherapy, etc. The expression of STING is closely related to the prognosis of cancer patients. Tumors with high STING expression are related to higher survival rates and earlier cancer stages. Currently, most detection methods for STING are puncture, immunohistochemistry of ex vivo tumors, etc. Due to the high heterogeneity of tumors and the dynamic changes in the expression of STING at all times, it is particularly crucial and important to develop a scheme for in vivo detection of STING.

[0004] PET and SPECT are the two main imaging modalities in nuclear medicine and are widely used in clinical practice. Among them, 97% of PET is used for therapeutic imaging clinically and plays an important role in the early diagnosis of tumors, multiple metastases, tumor staging, etc. At present, there are only CN112920172A and CN113429384A for STING-targeted tumor imaging probes. It is very urgent and necessary to develop new molecular imaging probes for visualizing STING expression in tumors. Summary of the Invention

[0005] This application is made in view of the above problems, and its purpose is to provide a molecular probe targeting stimulator of interferon genes with excellent imaging performance, including three categories.

[0006] Another object of the present invention is to provide a method for preparing the above STING molecular targeting probe.

[0007] Another object of the present invention is to provide a labeling method for the above STING molecular targeting probe.

[0008] Another object of the present invention is to provide the application of the above STING molecular targeting probe in fluorescence imaging.

[0009] Another object of the present invention is to provide the application of the above STING molecular targeting probe in the diagnosis and treatment of inflammatory diseases and tumor immunity.

[0010] To achieve the above object, the specific solutions of this application are as follows:

[0011] The first aspect of this application provides a class of molecular probes targeting stimulator of interferon genes (STING). This probe has an interferon gene targeting group - acridone derivative structure, and different lengths of alkyl side chains are connected to the N atom site in the structure. Its chemical synthesis process is as follows:

[0012]

[0013] Formula A, n = 1 - 10.

[0014] A class of molecular probes targeting stimulator of interferon genes (STING). This probe has an interferon gene targeting group - acridone derivative structure, and a polyethylene glycol short chain of 1, 2, 3 - triazole is connected to the N atom site in the structure. Its chemical synthesis process is as follows:

[0015]

[0016] Formula B, n = 1 - 10.

[0017] A class of molecular probes targeting stimulator of interferon genes (STING), the probe has a structure of an interferon gene targeting group - acridone derivative, and a side chain containing a chelating group with different numbers of alkyl groups is connected to the N atom in the structure. The chemical synthesis process is as follows:

[0018]

[0019] Formula C, where: R1 is a radionuclide labeling group; n is an integer from 1 to 10.

[0020] The radionuclide labeling group R1 is selected from any one of the following structures:

[0021]

[0022] The radionuclide is selected from 177 Lu, 90 Y, 18 F, 68 Ga, 99m Tc, 225 at least one of Ac.

[0023] The second aspect of the present application also provides a novel positron emission tomography (PET) or single photon emission computed tomography (SPECT) diagnostic and therapeutic agent using the above-mentioned molecular probe labeled with a radionuclide for targeting stimulator of interferon proteins.

[0024] The third aspect of the present application provides a method for 18 F-labeling a class of imaging agents targeting stimulator of interferon genes, and the specific labeling route is as follows:

[0025]

[0026] Formula A, n = 1 - 10.

[0027] A method for 18 F-labeling a class of imaging agents targeting stimulator of interferon genes, and the specific labeling route is as follows:

[0028]

[0029] Formula B, n = 1 - 10.

[0030] A method for radionuclide labeling of a class of imaging agents targeting stimulator of interferon genes, and the specific labeling method is as follows:

[0031] The labeling precursor 11 containing a radionuclide labeling group is chelated with a radionuclide to achieve radioactive labeling.

[0032] A preparation method of a molecular probe for a class of stimulator of interferon genes (STING) imaging agents. The radioactive compounds are named with a single prime (') superscript in the upper right corner of the number. For example, the radioactive forms of compounds 3 and 4 are named 3' and 4' respectively. Taking the structure with n = 1 in formula A as an example, during the reaction process, compounds 2, 3, and 4 are named 2-1, 3-1, and 4-1 respectively, and the radioactive compounds 3' and 4' are named 3'-1 and 4'-1 respectively. Taking the structure with n = 1 in formula B as an example, during the reaction process, compounds 5, 6, and 8 are named 5-1, 6-1, and 8-1 respectively, and the radioactive compounds 6' and 8' are named 6'-1 and 8'-1 respectively. Taking the structure with n = 3 in formula C as an example, when R1 is at this time, during the reaction process, compounds 9, 10, and 11 are named 9-3, 10-3, and 11-3 respectively. When the chelating radionuclide 68 is Ga, it is named 12'. The following are the chemical structural formulas and names of the example compounds:

[0033]

[0034] A preparation method of a molecular probe for a class of stimulator of interferon genes (STING) imaging agents. Synthesis of compound 1: Place 1 - 3 g of 2-bromo-5-methoxybenzoic acid and 0.5 - 1.5 g of 2,3-dimethylaniline in a clean reaction flask, add 4 - 8 mL of DMF to dissolve them thoroughly. Then add 0.02 - 0.07 g of copper powder, 0.02 - 0.07 g of Cu 2 O, 0.5 - 1.0 g of K 2 CO 3 and react at 100 - 120 °C for 10 - 14 h. After the reaction is completed, remove the solvent by a rotary evaporator, re-add NaOH (1 M, 20 - 30 mL) to dissolve it thoroughly, then filter and collect the filtrate. Dropwise add HCl to the filtrate to obtain a precipitate, filter and collect the precipitate and dry it. After thorough drying, add Eaton's reagent (4 - 8 mL) to the above precipitate and react at 80 - 100 °C for 0.5 - 2 h. Cool to room temperature, dropwise add the above reaction solution to saturated NaHCO 3 solution, and filter to obtain a military green solid. Dry and weigh it, and the yield is 58 - 68%.

[0035] Method for preparing molecular probe of a class of stimulator of interferon genes (STING) imaging agent, chemical synthesis of compound 4-1: Weigh 10 - 40 mg of compound 1 and 1 - 4 mg of sodium hydride (NaH), dissolve them in 1 - 3 mL of DMF solution, and stir at room temperature for 20 - 40 min. Then add dropwise 20 - 50 mg of compound 3-1, and stir at room temperature for reaction. Monitor the reaction process by TLC. After the reaction is completed, dilute the reaction solution with dichloromethane, extract with water, dry the organic phase with anhydrous magnesium sulfate, and purify by thin-layer chromatography. The developing agent is PE:EA = 2 - 4:1. Obtain the product 4-1 as a yellow solid.

[0036] Method for preparing a class of stimulator of interferon genes (STING) imaging agent 18 18F-labeling method, 18F-labeling process of radioactive compound 4'-1: ① QMA activation: 3 - 8 mL of K 18 2CO3 solution + 8 - 12 mL of ultrapure water; ② Preparation of eluent: Dissolve 13 - 18 mg of K 2 2CO3 in 0.6 - 1.0 mL of anhydrous acetonitrile, dissolve 3 - 8 mg of K 3 2CO3 in 0.05 - 0.4 mL of ultrapure water; ③ 18F treatment: Draw the corresponding activity of fluorine water with a syringe, hang it on the QMA column, and elute it with the eluent into a clean vial, and dry it by nitrogen blowing at 100 - 130 °C to remove water; then use 0.5 - 2 mL of anhydrous acetonitrile and repeat nitrogen blowing to dry three times; ④ After cooling to room temperature, add compound 2-1 (4 - 5 mg, dissolved in 0.4 mL of anhydrous acetonitrile) to it, and react with the cap pressed at 95 °C for 10 - 20 min; ⑤ After the reaction is completed, take a part for HPLC identification and a part for the second reaction; add compound 1 (2 - 3 mg) and potassium tert-butoxide (1.0 - 1.3 mg) to the above reaction flask, and react with the cap pressed at 80 - 95 °C for 15 - 30 min; after the reaction is completed, analyze and purify by HPLC to obtain the 222 18F-labeled radioactive product 4'-1. 2 2CO3 3 ; ③ 18 18F treatment: Draw the corresponding activity of fluorine water with a syringe, hang it on the QMA column, and elute it with the eluent into a clean vial, and dry it by nitrogen blowing at 100 - 130 °C to remove water; then use 0.5 - 2 mL of anhydrous acetonitrile and repeat nitrogen blowing to dry three times; ④ After cooling to room temperature, add compound 2-1 (4 - 5 mg, dissolved in 0.4 mL of anhydrous acetonitrile) to it, and react with the cap pressed at 95 °C for 10 - 20 min; ⑤ After the reaction is completed, take a part for HPLC identification and a part for the second reaction; add compound 1 (2 - 3 mg) and potassium tert-butoxide (1.0 - 1.3 mg) to the above reaction flask, and react with the cap pressed at 80 - 95 °C for 15 - 30 min; after the reaction is completed, analyze and purify by HPLC to obtain the 18 18F-labeled radioactive product 4'-1.

[0037] Method for preparing molecular probe of a class of stimulator of interferon genes (STING) imaging agent, chemical synthesis of compound 7: Dissolve compound 1 in DMSO solution, add potassium tert-butoxide, and stir at room temperature for 25 - 40 min; add dropwise propargyl bromide dissolved in 3 - 8 mL of DMSO, and stir at room temperature for reaction for 2 - 4 h; after monitoring the reaction by TLC and the reaction is completed, place the reaction solution on ice, extract with dichloromethane, and dry with anhydrous magnesium sulfate; then separate and purify by column chromatography, and the polarity of the developing agent is: PE:EA = 3 - 7:1, to obtain a yellow solid.

[0038] Preparation method of a molecular probe for a class of stimulator of interferon genes (STING)-targeted imaging agents, chemical synthesis process of compound 8-1: Weigh compound 2-(2-azidoethoxy)ethyl 4-methylbenzenesulfonate (5-1) and tetrabutylammonium fluoride in a clean vial, add a magnetic stir bar and react at room temperature for 2-4 h; after monitoring the reaction completion by TLC, add compound 3, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), sodium ascorbate and copper sulfate (CuSO 4 ), and react overnight at room temperature; after the reaction is completed, dilute the reaction solution with dichloromethane and extract with water; dry the organic layer with anhydrous MgSO 4 , purify the obtained product by HPLC using a semi-preparative column to obtain a yellow-green solid of compound 8-1.

[0039] A class of stimulator of interferon genes (STING)-targeted imaging agents 18 18F-labeling method, 18F-labeling process of the radioactive compound 8'-1: ① QMA activation: 3-8 mL of K 18 2CO 2 3 solution + 8-12 mL of ultrapure water; ② eluent preparation: 13-18 mg of K 3 2CO 222 3 dissolved in 0.5-1.0 mL of anhydrous acetonitrile, 3-8 mg of K 2 2CO 3 3 dissolved in 0.1-0.3 mL of ultrapure water; ③ 18 18F treatment: Withdraw the corresponding activity of fluorine water with a syringe, hang it on the QMA column, and elute it with the eluent into a clean vial, and dry it under nitrogen blowing at 110 °C to remove water; then use 1 mL of anhydrous acetonitrile and repeat nitrogen blowing and drying three times; ④ After cooling to room temperature, add compound 5-1 (1-3 mg, dissolved in 0.4 mL of anhydrous acetonitrile) to it, and react under capping at 80-95 °C for 10-20 min. ⑤ After the reaction is completed, take a part for HPLC identification and a part for the second reaction. Add compound 7 (1-3 mg), TBTA (0.05-0.2 M, 50-200 μL), CuSO 4 (0.05-0.2 M, 50-200 μL), sodium ascorbate (0.2-0.4 M, 50-200 μL) to the above reaction vial, cap it and react with shaking at 60-80 °C for 15-30 min. After the reaction is completed, cool to room temperature, analyze and purify the product by HPLC to obtain 18 18F-labeled radioactive product 8'-1.

[0040] A method for preparing a molecular probe of a class of stimulator of interferon genes (STING)-targeted imaging agents. The chemical synthesis process of compound 9-3: 200-350 mg of compound 1 and 100-150 mg of potassium tert-butoxide are dissolved in 3-5 mL of DMSO solution and stirred at room temperature for 25-40 min. 300-450 mg of 6-(Boc-amino)hexyl bromide is added dropwise, and the reaction is stirred at room temperature for 2-5 h. After the reaction is completed, the reaction solution is diluted with EA, extracted with water, and the organic phase is collected and dried over anhydrous magnesium sulfate. Purification by column chromatography, with the eluent being PE:EA = 4-7:1. The product 9-3 is obtained.

[0041] A method for preparing a molecular probe of a class of stimulator of interferon genes (STING)-targeted imaging agents. The chemical synthesis process of compound 10-3: Weigh 4-6 mg of compound 9-3 into a clean vial, add 500-600 μL of an equal-volume mixed solution of trifluoroacetic acid and dichloromethane, and shake the reaction at room temperature for about 15-30 min. Monitor the reaction process by HPLC. It is found that the deprotection process is fast and relatively complete, with a conversion rate above 93-98%, and the product peak is identified by mass spectrometry.

[0042] A method for preparing a molecular probe of a class of stimulator of interferon genes (STING)-targeted imaging agents. The chemical synthesis process of compound 11-3: Dissolve 3-6 mg of compound 10-3 in about 400-600 μL of DMF, add 5-6 mg of NOTA-NHS and 15-30 μL of N,N-diisopropylethylamine (DIPEA), and shake the reaction at room temperature for 2-4 h. After the reaction is completed, analyze and purify by HPLC to obtain compound 11-3, and the product peak is identified by mass spectrometry.

[0043] A method for preparing a molecular probe of a class of stimulator of interferon genes (STING)-targeted imaging agents. The 68 Ga-labeling process of imaging agent 12': 40-60 μg of precursor 11-3 is dissolved in 200-400 μL of acetonitrile), add 200-400 μL of 0.20-0.30 M sodium acetate solution, and react at 45-60 °C for 10-20 min. Analyze and purify the reaction solution by radio-HPLC to obtain 68 the Ga-labeled product 12'.

[0044] Advantages of this application: The STING-targeted imaging agent containing acridone in this application has a high radiochemical yield and specific activity, good chemical stability, high affinity for STING, and the preparation method is simple and feasible, and can be used for PET imaging of STING in tumor immune responses.

[0045] The stimulator of interferon genes (STING)-targeted imaging agent of the present application has excellent biological properties. It has high uptake and specificity in tumors with high STING expression, meeting the conditions for use as a STING imaging agent. In addition, considering that STING plays a key role in various diseases, such as various disease models including infection, cancer, aging, autophagy, and autoimmune diseases, this STING imaging agent can provide imaging guidance for various diseases. For example, it can provide new ideas for the early diagnosis and prognosis evaluation of inflammatory diseases and feasible solutions for the visualization evaluation of STING in tumors. Additionally, the probe itself is also an agonist targeting STING and is expected to be used in the immunotherapy of tumors. The optical properties of the probe itself are expected to expand its applications in STING-related optical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 HPLC identification of compounds 4-1 and 8-1 in the examples of the present invention and the corresponding 18 radio-HPLC identification diagram of the radiolabeled probe after

[0047] Figure 2 Diagram of the stability analysis of the radioactive probe 4'-1 in vitro in normal saline and serum.

[0048] Figure 3 Diagram of the stability analysis of the radioactive probe 8'-1 in vitro in normal saline and serum.

[0049] Figure 4 Diagram of the uptake and blocking experiment results of the radioactive probe 8'-1 in Raw264.7 cells.

[0050] Figure 5 Diagram of the saturation binding experiment results of the radioactive probe 8'-1 in Raw264.7 cells.

[0051] Figure 6 UV absorption and fluorescence emission spectra of the small molecule compound 8-1.

[0052] Figure 7 PET imaging diagrams of the radioactive probe 8'-1 in CT26, B16F10, MC38, and Panc02 tumor-bearing mice. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0053] Hereinafter, embodiments of a molecular probe, a labeling method, and an application thereof that specifically disclose the present application targeting stimulator of interferon genes (STING) will be described in detail with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary details are omitted. For example, there may be cases where details of well-known matters are omitted and repeated descriptions of actually identical structures are omitted. This is to prevent the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the accompanying drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter recited in the claims.

[0054] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a specific range. The range defined in this way can include or exclude the end values and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, ranges of 60-110 and 80-120 are also contemplated. In addition, if the minimum range values of 1 and 2 are listed, and if the maximum range values of 3, 4, and 5 are listed, the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In the present application, unless otherwise stated, the numerical range "a-b" represents an abbreviated representation of any real number combination between a and b, where both a and b are real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been fully listed herein, and "0-5" is only an abbreviated representation of these numerical combinations. Additionally, when it is stated that a certain parameter is an integer ≥2, it is equivalent to disclosing that the parameter is, for example, the integers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0055] If there is no special instruction, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0056] If there is no special instruction, all technical features and optional technical features of the present application can be combined with each other to form a new technical solution.

[0057] Unless otherwise specified, all steps of this application can be carried out sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method can include steps (a) and (b) carried out sequentially, or can also include steps (b) and (a) carried out sequentially. For example, when it is mentioned that the method further includes step (c), it means that step (c) can be added to the method in any order. For example, the method can include steps (a), (b) and (c), or can also include steps (a), (c) and (b), or can also include steps (c), (a) and (b), etc.

[0058] Unless otherwise specified, the terms "comprising" and "including" mentioned in this application mean open-ended, and can also be closed-ended. For example, the "comprising" and "including" can mean that other components not listed can also be included or contained, or can only include or contain the listed components.

[0059] Unless otherwise specified, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) while B is true (or exists); or both A and B are true (or exist).

[0060] Examples

[0061] Hereinafter, examples of this application will be described. The examples described below are exemplary and are only used to explain this application, and should not be construed as a limitation to this application. For those without specific technologies or conditions noted in the examples, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0062] A molecular probe targeting stimulator of interferon genes (STING), characterized in that: the imaging agent has an interferon gene targeting group - acridone derivative structure, and different lengths of alkyl side chains are connected to the N atom site in the structure, or a short polyethylene glycol chain with 1, 2, 3 - triazole, or a side chain containing a chelating group with different numbers of alkyl groups. Its structural general formula is as follows:

[0063]

[0064] For formula A, n = 1 - 10; for formula B, n = 1 - 10; for formula C, where: R1 is a radionuclide labeling group; n is an integer from 1 to 10.

[0065] The radionuclide labeling group R1 is selected from any of the following structures:

[0066]

[0067] The radionuclide is selected from 177 Lu, 90 Y, 18 F, 68 Ga, 99m Tc, 225 at least one of Ac.

[0068] The above three types of molecular probes targeting interferon gene stimulators are characterized in that: the molecule is 18 F, 177 Lu, 90 Y, 18 F, 68 Ga, 99m Tc, 225 a novel positron emission tomography (PET) diagnostic and therapeutic agent or single photon emission computed tomography (SPECT) diagnostic and therapeutic agent labeled with Ac targeting interferon gene stimulatory protein.

[0069] The synthesis and labeling method of the above three types of imaging agents targeting interferon gene stimulators is characterized by the following steps ( 19 F] represents a non-radioactive compound, 18 F] represents a radioactive compound):

[0070]

[0071] Chemical synthesis of formula A and 18 F labeling step.

[0072]

[0073] Chemical synthesis of formula B and 18 F labeling step.

[0074]

[0075] Chemical synthesis step of the labeling precursor of formula C.

[0076] The specific chemical synthesis process involved in the reaction is as follows:

[0077] The naming of radioactive compounds is represented by adding a single quote (') to the upper right corner of the number. For example, the radioactive forms of compounds 3 and 4 are named 3' and 4' respectively. Taking the structure with n = 1 in formula A as an example, during the reaction process, the compounds 2, 3, and 4 are named 2-1, 3-1, and 4-1 respectively, and the radioactive compounds 3' and 4' are named 3'-1 and 4'-1 respectively. Taking the structure with n = 1 in formula B as an example, during the reaction process, the compounds 5, 6, and 8 are named 5-1, 6-1, and 8-1 respectively, and the radioactive compounds 6' and 8' are named 6'-1 and 8'-1 respectively. Taking the structure with n = 3 in formula C as an example, when R1 is at the time, during the reaction process, the compounds 9, 10, and 11 are named 9-3, 10-3, and 11-3 respectively. When the chelating radionuclide 68 is Ga, it is named 12'.

[0078] 1) Synthesis of compound 1: Place 2-bromo-5-methoxybenzoic acid (1.48 g, 6.4 mmol) and 2,3-dimethylaniline (0.775 g, 6.4 mmol) in a clean reaction flask, and add 6 mL of DMF to dissolve them thoroughly. Then add copper powder (0.05 g, 0.78 mmol), Cu 2 O (0.05 g, 0.35 mmol), K 2 CO 3 (0.71 g, 5.14 mmol), and react at 110 °C for 12 h. After the reaction is completed, remove the solvent by a rotary evaporator, add NaOH (1 M, 25 mL) again to dissolve it thoroughly, then filter and collect the filtrate. Add HCl dropwise to the filtrate to obtain a precipitate, filter and collect the precipitate and dry it. After thorough drying, add Eaton's reagent (6 mL) to the above precipitate and react at 90 °C for 1 h. Cool to room temperature, add the above reaction solution dropwise to the saturated NaHCO 3 solution, and filter to obtain a military green solid. Weigh it after drying, 1.04 g, with a yield of 64%. 1 1H NMR (400 MHz, DMSO-d 6 ) δ 10.47 (s, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 9.1 Hz, 1H), 7.62 (d, J = 2.5 Hz, 1H), 7.41 (dd, J = 9.1, 2.7 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 3.88 (s, 3H), 2.50 (s, 3H), 2.44 (s, 3H).

[0079] 2) Chemical synthesis of compound 4-1: Weigh compound 1 (25.33 mg, 0.1 mmol) and NaH (2.879 mg, 0.12 mmol), dissolve them in 2 mL of DMF solution, and stir at room temperature for 30 min. Then add dropwise compound 3-1 (33.49 mg, 0.12 mmol), and stir the reaction at room temperature. Monitor the reaction progress by TLC. After the reaction is completed, dilute the reaction solution with dichloromethane, extract with water, dry the organic phase with anhydrous magnesium sulfate, and purify by thin-layer chromatography. The developing solvent is PE:EA = 3:1. A total of 7 mg of the yellow solid product 4-1 is obtained, with a yield of 16%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.06 (d, J = 9.3 Hz, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.85 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.4 Hz, 3H), 7.46 (s, 1H), 7.39 (d, J = 8.8 Hz, 1H), 4.51 (dd, J = 24.2, 2.7 Hz, 4H), 3.96 (s, 3H), 2.78 (s, 3H), 2.51 (s, 3H), 2.43 (s, 3H).

[0080] 3) 18 F labeling process of radioactive compound 4'-1: ① QMA activation: 5 mL of K 2 CO 3 solution + 10 mL of ultrapure water; ② Preparation of eluent: Dissolve 15 mg of K 222 in 0.8 mL of anhydrous acetonitrile, and dissolve 5 mg of K 2 CO 3 in 0.2 mL of ultrapure water. ③ 18 F treatment: According to the experimental requirements, draw the corresponding activity of fluorine water with a syringe, hang it on the QMA column, and elute it into a clean vial with the eluent, and dry it under nitrogen blowing at 110 °C to remove water. Then use 1 mL of anhydrous acetonitrile and repeat nitrogen blowing and drying three times. ④ After cooling to room temperature, add compound 2-1 (4 - 5 mg, dissolved in 0.4 mL of anhydrous acetonitrile) to it, and react under capping at 95 °C for 15 min. ⑤ After the reaction is completed, take a part for HPLC identification and a part for the second reaction. Add compound 1 (2 - 3 mg) and potassium tert-butoxide (1.0 - 1.3 mg) to the above reaction flask, and react under capping at 85 °C for 20 min. After the reaction is completed, analyze and purify by HPLC. The HPLC mobile phase conditions are: 70% CH 3 CN + 30% H 2 O or 0 - 10 min: 50 - 95% CH 3 CN + 50 - 5% H 2 O, 10 - 15 min: 95% CH 3 CN + 5% H2 O, 15 - 20 min: 50% CH 3 CN + 50% H 2 O, 254 nm, 3 mL / min.

[0081] 4) Synthesis of Compound 7: Dissolve Compound 2 (65.5 mg, 0.25 mmol) in about 2 mL of DMSO solution, add potassium tert - butoxide (40.4 mg, 0.36 mmol), and stir at room temperature for 30 min. Dropwise add propargyl bromide (34 mg, 0.34 mmol) dissolved in 5 mL of DMSO, and stir at room temperature for 3 h. After monitoring the reaction by TLC until completion, place the reaction solution on ice, extract with dichloromethane, and dry with anhydrous magnesium sulfate. Then separate and purify by column chromatography, with the eluent polarity: PE:EA = 5:1. Obtain 63.3 mg of yellow solid, with a yield of 87%. 1 H NMR (400 MHz, DMSO - d 6 ) δ 8.03 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 9.2 Hz, 1H), 7.61 (d, J = 2.9 Hz, 1H), 7.48 (dd, J = 9.2, 2.9 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 4.94 (d, J = 1.4 Hz, 2H), 3.89 (s, 3H), 3.41 (s, 1H), 2.58 (s, 3H), 2.44 (s, 3H).

[0082] 5) Chemical synthesis process of Compound 8 - 1: Weigh 2 - (2 - azidoethoxy)ethyl 4 - methylbenzenesulfonate (5 - 1) (0.07 g, 0.25 mmol) and tetrabutylammonium fluoride (TBAF, 1.0 mol / L in THF, 1.23 mL) into a clean vial, add a magnetic stir bar, and react at room temperature for 3 h. After monitoring the reaction by TLC until completion, add Compound 3 (0.07 g, 0.24 mmol), tris[(1 - benzyl - 1H - 1,2,3 - triazol - 4 - yl)methyl]amine (TBTA, 0.13 g, 0.24 mmol), sodium ascorbate (0.048 g, 0.24 mmol), and CuSO 4 (0.039 g, 0.24 mmol), and react at room temperature overnight. After the reaction is complete, dilute the reaction solution with dichloromethane and extract with water. Dry the organic layer with anhydrous MgSO 4 The obtained product is purified by HPLC, using a semi - preparative column (Gemini 5μm C18 LC Column 250×10 mm), and the mobile phase conditions: isocratic 60% CH 3 CN + 40% H 2O, 3 mL / min, 254 nm. A total of 0.03 g of yellow-green solid 8-1 was obtained, with a yield of 29%. 1 H NMR (400 MHz, CDCl 3 ) δ 8.17 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 2.9 Hz, 1H), 7.56 (d, J = 9.2 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 6.73 (s, 1H), 5.53 (s, 2H), 4.48–4.42 (m, 1H), 4.35–4.31 (m, 1H), 4.28 (t, J = 4.9 Hz, 2H), 3.88 (s, 3H), 3.67–3.63 (m, 2H), 3.53–3.48 (m, 1H), 3.46–3.40 (m, 1H), 2.55 (s, 3H), 2.43 (s, 3H).

[0083] 6) 18 F-labeling process of radioactive compound 8'-1: ①–③ are the same as the 18 F-labeling process of compound 6. ④ After cooling to room temperature, compound 5-1 (2 mg, dissolved in 0.4 mL of anhydrous acetonitrile) was added thereto, and the reaction was carried out under a sealed cap at 85 °C for 15 min. ⑤ After the reaction was completed, a part was used for HPLC identification, and a part was used for the second reaction. Compound 7 (2 mg), TBTA (0.1 M, 100 μL), CuSO 4 (0.1 M, 100 μL), sodium ascorbate (0.3 M, 100 μL) were added to the above reaction flask, and after sealing the cap, the reaction was shaken at 70 °C for 20 min. After the reaction was completed, it was cooled to room temperature, and the product 8'-1 was obtained by HPLC analysis and purification. The HPLC mobile phase conditions for probe purification were: 0–20 min: 40-95% CH 3 CN + 60-5% H 2 O, 254 nm, 3 mL / min. The HPLC mobile phase conditions for probe analysis were: 50% CH 3 CN + 50% H 2 O, 254 nm, 1 mL / min.

[0084] 7) Chemical synthesis process of compound 9-3: Compound 1 (253.3 mg, 1 mmol) and potassium tert-butoxide (161.58 mg, 1.44 mmol) were dissolved in 4 mL of DMSO solution and stirred at room temperature for 30 min. 6-(Boc-amino) bromohexane (381.08 mg, 1.36 mmol) was added dropwise, and the reaction was stirred at room temperature for 3 h. After the reaction was completed, the reaction solution was diluted with EA, extracted with water, and the organic phase was collected and dried over anhydrous magnesium sulfate. Purification by column chromatography, the eluent was PE:EA = 5:1. A total of 281 mg of product 9-3 was obtained, with a yield of 62%.1 HNMR(400MHz,CDCl 3 ) δ 8.12 (d, J = 9.3 Hz, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.41 (dd, J = 9.3 Hz, 1H), 7.37 (d, J = 2.3 Hz, 1H), 7.33 (d, J = 8.8 Hz, 1H), 4.25 (t, J = 6.5 Hz, 2H), 3.98 (s, 3H), 3.15 (d, J = 6.3 Hz, 2H), 2.87 (s, 3H), 2.54 (s, 3H), 2.29 (d, J = 20.3 Hz, 2H), 2.05 - 1.98 (m, 2H), 1.71 - 1.64 (m, 2H), 1.58 - 1.51 (m, 2H), 1.44 (s, 12H). 13 C NMR(101MHz,CDCl 3 ) δ 184.05, 164.21, 158.71, 156.55, 148.42, 146.87, 136.06, 134.09, 131.92, 128.88, 124.46, 120.13, 119.15, 118.75, 97.66, 75.97, 55.45, 30.60, 30.12, 28.41, 26.72, 25.95, 20.72, 13.56.

[0085] 8) Chemical synthesis process of compound 10 - 3: Weigh compound 9 - 3 (~5 mg, 0.011 mmol) into a clean vial, add an equal - volume mixed solution of 500 - 600 μL trifluoroacetic acid and dichloromethane, and shake the reaction at room temperature for about 20 min. Monitor the reaction process by HPLC. It is found that the de - protection process is very fast and relatively complete, with a conversion rate above 95%. The product peak is identified by mass spectrometry. HRMS(ESI) m / z: calculated for C 22 H 29 N 2 O 2 + [M + H] + , 353.2224, found 353.1475.

[0086] 9) Chemical synthesis process of Compound 11-3: Dissolve Compound 10-3 (~4 mg, 0.011 mmol) in about 500 μL of DMF, add 5-6 mg of NOTA-NHS and 20 μL of N,N-diisopropylethylamine (DIPEA), and react with shaking at room temperature for 3 h. After the reaction, analyze and purify by HPLC to obtain about 5 mg of Compound 11-3 with a yield of 69%. The product peak was identified by mass spectrometry, HRMS(ESI) m / z: calculated for C 34 H 48 N 5 O 7 +[M+H] + , 638.3548, found 638.2251.

[0087] 10) 68 Ga labeling process of the radioactive compound 12': Add the precursor 11-3 (50 μg dissolved in 300 μL of acetonitrile) to 300 μL of 0.25 M sodium acetate solution and react at 50 °C for 15 min. Analyze and purify the reaction solution by radio-HPLC to obtain 68 the Ga-labeled product 12'.

[0088] Experimental Example 1

[0089] Determination of the lipophilic-hydrophilic distribution coefficient (logD) of Probes 4'-1, 8'-1, and 12':

[0090] The calculation formula for the lipophilic-hydrophilic distribution coefficient is: logD = Ao / Aw, where Ao represents the radioactivity count in the n-octanol phase and Aw represents the radioactivity count in the PBS phase. The specific process is as follows: Take the prepared radioactive probe (0.37 MBq, 10 μL) and place it in a mixed solution of n-octanol (990 μL) and PBS (1 mL, pH = 7.4). After vortexing for 2 min, centrifuge at 13000 r / min for 5 min to separate the two phases. Take 4 samples of 100 μL each from the aqueous phase and the lipid phase and label them. This is the result of the first distribution. Take 100 μL of the centrifuged n-octanol phase and re-distribute it in a solution of n-octanol / PBS (v / v) = 0.9:1 mL. After vortexing for 2 min, centrifuge at 13000 r / min for 5 min. Repeat this operation twice to obtain the results of the second and third distributions. Take the results of the last distribution and calculate the average value of logD. The specific values of the lipophilic-hydrophilic distribution coefficient of the probe are as follows:

[0091] probe lipid-water partition coefficient (logD) 4'-1 2.08±0.24 8'-1 1.50±0.29 12' 1.04±0.44

[0092] Experimental Example 2

[0093] In vitro stability

[0094] Take the prepared probe (3.7 MBq, 100 μL) and incubate it with 900 μL of normal saline or serum at 37 °C. At the corresponding time points, 10 μL was directly taken with a syringe for HPLC analysis in the normal saline group. In the serum group, it was first precipitated with an equal volume of acetonitrile (+0.1% TFA), vortexed for 2 min, and then centrifuged at 13,000 r / min for 5 min to fully precipitate components such as proteins in the serum sample. Then, the supernatant was filtered through a 0.22 μm filter membrane and analyzed for the radiochemical purity of the product by radio-HPLC for stability analysis.

[0095] It can be seen from Figure 2 and Figure 3 that after the probes 4'-1 and 8'-1 were incubated in normal saline and serum in vitro for 2 h, >99% of the radioactive products existed in the form of probes, indicating that the probes had good stability in both normal saline and serum in vitro.

[0096] Experimental Example 3

[0097] Cell uptake and blocking experiment of the probe

[0098] Seed Raw 264.7 cells in a 24-well plate and let them adhere overnight. Then take out the well plate with monolayer adherent cells, carefully aspirate all the culture medium, and then add 0.5 mL of culture medium to each well plate, gently shake and aspirate to wash away the dead or detached cells on the surface. Take an appropriate amount of the probe solution purified by HPLC and add it to the culture medium, and dilute the radioactivity to 1 - 3 μCi / mL. First, add 10 μL of DMXAA solution (5 mg / mL) to the wells in the inhibition group, then add 100 μL of the culture medium containing the label to all the well plates, and finally add 200 μL of culture medium. Incubate in a 37 °C incubator for 30 min, 60 min, and 120 min respectively. After the corresponding incubation time, aspirate the culture medium, add 0.5 mL of ice-cold PBS (containing 0.2% BSA) and wash twice, then add 1 mL of NaOH to lyse the cells, and let it stand at room temperature for 5 min. Gently scrape the bottom of the well with a pipette tip to completely detach the cells, aspirate all the solutions and cells in the well plate into a marked plastic hose, and count the samples with an automatic gamma counter. Note: DMXAA is a widely recognized small molecule agonist targeting STING, and DMXAA blocking is used to confirm the specificity of probe cell uptake.

[0099] It can be seen from Figure 4 that the uptake of the probe 8'-1 in cells decreased significantly after DMXAA blocking, indicating that the uptake of the probe in cells is specific uptake based on STING. At the time points of 30 min, 60 min, and 120 min, the uptake of the probe decreased by 65%, 65%, and 75% respectively after DMXAA blocking.

[0100] Experimental Example 4

[0101] Cell saturation binding experiment of the probe

[0102] Seed Raw 264.7 cells in a 24-well plate and let them adhere and grow overnight. Then take out the plate with monolayer adherent cells, carefully aspirate all the culture medium, and then add 0.5 mL of culture medium to each well, gently shake and aspirate to wash away the dead or detached cells on the surface. After preparing the probe at a concentration of 12 - 235 nM, add it to the corresponding wells and incubate for 1 h. Then aspirate the culture medium in all the wells, add 0.5 mL of ice-cold PBS (containing 0.2% BSA) and wash twice. After completely washing away the unbound radioactive probe, add 1 mL of NaOH and place it at room temperature for 5 min. Gently scrape the bottom of the well with a pipette tip, aspirate all the solution and cells in the well into a marked plastic hose, and count the samples with an automatic gamma counter.

[0103] It can be seen from Figure 5 that the uptake of probe 8'-1 in cells shows a gradually saturating trend with the increase of probe concentration. The measured affinity of probe 8'-1 for the STING target is Kd = 40.62 ± 13.63 nM, Bmax = 5.61×10 3 , indicating that the probe has a high affinity for the target.

[0104] Experimental Example 5

[0105] As Figure 6 shown, the ultraviolet absorption of compound 8-1 in ethanol solution measured by an enzyme-linked immunosorbent assay (ELISA) reader and a fluorescence spectrophotometer is 401 nm, and the fluorescence emission is 480 nm. The unique optical properties of the molecule itself enable it to be used in fluorescence-related experiments such as laser confocal microscopy, flow cytometry sorting, and fluorescence imaging, providing a research tool with optical means for the study of STING.

[0106] Application Example 6

[0107] In vivo PET imaging of the probe in several tumor-bearing mice

[0108] Select 4 - 6-week-old Balb / c mice. After depilation, inoculate CT26 cells on the right upper limb to construct a CT tumor mouse model; select 4 - 6-week-old C57BL / 6 mice. After depilation, inoculate B16F10, MC38, and Panc02 cells on the right upper limb to construct B16F10, MC38, and Panc02 tumor mouse models. When the tumor volume of the tumor-bearing mice grows to an appropriate size (100 - 250 mm 3) Inject the prepared probe (3.7 - 7.4 MBq, 100 μL) via the tail vein. After the corresponding time points, anesthetize the mice with isoflurane, and collect PET imaging maps using MicroPET / CT. Draw regions of interest (ROIs) at sites such as tumors and major organs, and calculate the corresponding target-to-non-target ratios. Explore the tumor imaging and imaging quality of the molecular probe in the tumor model, the in vivo metabolism in major metabolic organs such as the liver and kidneys, etc., and calculate the corresponding target-to-non-target ratios.

[0109] As Figure 7 , from the in vivo PET imaging results of probe 8'-1 in multiple tumor models, the probe has excellent tumor imaging ability, can completely display the outline of the tumor, and the background uptake of the probe in other parts is very clean. Except for tumor uptake, it mainly exists in metabolic organs such as the liver and intestine in the body.

[0110] It should be noted that this application is not limited to the above embodiments. The above embodiments are only examples, and embodiments with the same technical idea essence and the same function and effect within the technical solution scope of this application are all included in the technical scope of this application. In addition, within the scope of not departing from the main idea of this application, various deformations that those skilled in the art can think of imposed on the embodiments, and other ways constructed by combining some constituent elements in the embodiments are also included in the scope of this application.

Claims

1. A class of molecular probes targeting stimulator of interferon genes, characterized in that, the probe has a structure of an interferon gene targeting group - acridone derivative, and different lengths of alkyl side chains are connected to the N atom part in the structure, and its structural general formula is as follows: Formula A, n = 1 - 10.

2. A class of molecular probes targeting stimulator of interferon genes, characterized in that, the probe has a structure of an interferon gene targeting group - acridone derivative, and a short chain of polyethylene glycol of 1,2,3 - triazole is connected to the N atom part in the structure, and its structural general formula is as follows: Formula B, n = 1 - 10.

3. A class of molecular probes targeting stimulator of interferon genes, characterized in that, the probe has a structure of an interferon gene targeting group - acridone derivative, and a side chain containing a chelating group with different numbers of alkyl groups is connected to the N atom part in the structure, and its structural general formula is as follows: Formula C, wherein: R1 is a radionuclide labeling group; n is an integer from 1 to 10; the radionuclide labeling group R1 is selected from any one of the following structures: The radionuclide is selected from 177 Lu, 90 Y, 18 F, 68 Ga, 99m Tc, 225 at least one of Ac.

4. The class of molecular probes targeting stimulator of interferon genes (STING) according to any one of claims 1 - 3, characterized in that, the application of the molecular probe in the preparation of a novel positron emission tomography (PET) diagnostic and therapeutic agent or a single photon emission computed tomography (SPECT) diagnostic and therapeutic agent targeting stimulator of interferon proteins.

5. The labeling method of the class of molecular probes targeting stimulator of interferon genes according to claim 1, and the specific labeling route is as follows: 。 6. The labeling method of the class of molecular probes targeting stimulator of interferon genes according to claim 2, and the specific labeling route is as follows: 。 7. The labeling method of the class of molecular probes targeting stimulator of interferon genes according to claim 3, and the specific labeling method is as follows: The labeling precursor 11 containing the R1 nuclide-labeled group is radioactively labeled by chelating with a radionuclide selected from one of 177 Lu, 90 Y, 18 F, 68 Ga, 99m Tc, 225 Ac; The chemical synthesis process is as follows: ， Formula 11, wherein: R1 is a radionuclide labeling group; n is an integer from 1 to 10.

8. The application of the class of molecular probes targeting stimulator of interferon genes according to any one of claims 1 - 3 in the preparation of medicaments for tumor diagnosis and treatment, or in the preparation of drugs for tumor immunotherapy.

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