Imidazolopyridone compounds binding to sigma-2 receptors, preparation method and application thereof

Abstract

The present invention belongs to the field of radiopharmaceutical chemistry and clinical nuclear medicine, and specifically relates to a preparation method and application of imidazopyridone compounds that bind to sigma-2 (σ2) receptors. The general structural formula of the compound is shown in formula (I), which has nanomolar affinity, high subtype selectivity and specificity for σ2 receptors / TMEM97. When the R group in the compound of formula (I) is 18 F, after 18 F-OTs-substituted labeled precursors undergo nucleophilic substitution to prepare corresponding tracers, which have high radiochemical yield and radiochemical purity, excellent biological properties, and can be used for PET imaging of tumor proliferation and atherosclerotic plaques, with broad clinical application prospects. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the technical field of radiopharmaceutical chemistry and clinical nuclear medicine, and in particular to an imidazopyridone compound binding to a sigma-2 (σ2) receptor, a preparation method and application thereof. Background Art

[0002] Sigma (σ) receptors are a new type of receptor that is different from opioid receptors and include two subtypes: sigma-1 (σ1) and sigma-2 (σ2). In 2017, the σ2 receptor was identified as transmembrane protein 97 (TMEM97) located in the endoplasmic reticulum. The σ2 receptor / TMEM97 is a regulatory gene for lipid and cholesterol metabolism. Its expression in tumor cells during the proliferative phase is 8-10 times that in the resting phase, and it is a biomarker for tumor proliferation. Therefore, the σ2 receptor / TMEM97 is a target related to lipid and cholesterol metabolism that reflects the proliferation state of tumor cells. Positron emission tomography (PET) imaging targeting the σ2 receptor / TMEM97 can help achieve early diagnosis of cancer, assist in clinical treatment planning, and evaluate patient treatment efficacy and prognosis.

[0003] Colorectal cancer (CRC) is the most common digestive system malignancy, accounting for 10.2% of all cancers and the second most common cause of cancer death. Dissemination of the disease to distant organs is considered the main cause of death in CRC patients. Approximately 20% of CRC patients are diagnosed with metastases, of which 10% to 15% are lung metastases, making it the second most common site of CRC metastasis after the liver and the most common site of extraperitoneal metastasis. Lung metastasis often indicates advanced disease, a poor prognosis, and a short overall survival. For patients with lung metastases, timely and effective treatment to delay and terminate the progression of metastases can significantly improve patient survival.

[0004] Although drug treatment for tumors has developed rapidly in recent years, surgical treatment is currently the only way for patients with colorectal cancer to achieve long-term survival. The treatment strategies for patients with metastatic colorectal cancer are completely different from those for patients with non-metastatic colorectal cancer. The surgical indications for patients with colorectal cancer with distant metastasis should be strictly controlled, especially when the metastatic lesions are in a period of rapid proliferation. After COVID-19, the detection rate of lung nodules has increased significantly compared with before. For patients with colorectal cancer, determining whether their lung nodules are tumor metastasis and the proliferation of these metastatic lesions play a decisive role in the patient's treatment strategy. Although pathological diagnosis is the gold standard for determining lung metastasis, it is somewhat traumatic. At the same time, there is a greater risk of sampling metastatic lesions around the heart. [ 18F]FDG imaging is currently the most widely used noninvasive method for diagnosing lung metastases. However, due to the high cardiac uptake of contrast agents, the heart has a certain "masking" effect on surrounding lesions. Furthermore, glucose uptake capacity does not fully represent tumor proliferation activity. Given that the σ2 receptor / TMEM97 is a biomarker that regulates cholesterol homeostasis and reflects tumor proliferation, in vivo imaging of colorectal cancer patients using σ2 receptor / TMEM97 tumor imaging agents could help distinguish metastatic from non-metastatic colorectal cancer patients, determine whether lung nodules in colorectal cancer patients are tumor metastases, and assess the proliferation of these metastatic lesions, thereby determining treatment strategies. Therefore, the design and development of σ2 receptor / TMEM97 tumor imaging agents will provide a highly effective tool for visualizing tumor proliferation.

[0005] σ2 receptor / TMEM97 has been very active in the field of tumor research. Its radionuclides (especially 99m Tc and 18 F) labeled σ2 receptor ligands are often used in tumor imaging studies. Although there are a variety of σ2 receptor radioligands reported for tumor imaging, such as PET imaging agents [ 11 C]RHM-1, 18 F]ISO-1 and [ 18 F]RHM-4, and SPECT imaging agents [ 123 I]RHM-4. However, currently only PET imaging agents [ 18 F]ISO-1 has entered Phase I clinical trials for multiple tumor studies (such as breast cancer, lymphoma, and head and neck cancer). 18 F]ISO-1 has entered human trials, but the probe still has some shortcomings, such as low affinity and specificity for σ2 receptor / TMEM97, poor selectivity for σ1 receptor subtype, and relatively high lipid solubility (logD 7.4 =3.06) leads to higher non-target tissue uptake, which interferes with the judgment of tumor-positive signals.

[0006] Research has shown that the deposition and oxidation of low-density lipoprotein (LDL) in arterial walls is one of the initiating factors of plaque formation. The σ2 receptor / TMEM97 is a cholesterol regulatory gene. It forms a ternary complex with progesterone receptor membrane component 1 (PGRMC1) and the low-density lipoprotein receptor (LDLR) to mediate LDL uptake. Therefore, σ2 receptor / TMEM97 is an ideal target for identifying rapidly progressing vulnerable plaques. Using this highly specific molecular probe, we quantitatively visualized σ2 receptor / TMEM97 expression in vivo during plaque formation and progression, further exploring the spatiotemporal patterns of σ2 receptor / TMEM97 expression in an atherosclerotic plaque model and the key role of σ2 receptor / TMEM97 in plaque formation and progression.

[0007] Therefore, the development of high-affinity and high-selectivity σ2 receptor / TMEM97 molecular probes can provide sensitive tools for visualizing tumor proliferation status and imaging atherosclerotic plaques.

[0008] Radionuclides 18 F has a suitable half-life (T 1 / 2 =109.8min), easy preparation, can be used for PET non-invasive imaging and data can be quantitatively analyzed. Therefore, the research has suitable affinity, high selectivity, and good metabolic properties in the body. 18 The F-labeled specific σ2 receptor / TMEM97 PET imaging agent has important clinical application prospects for the visualization of tumor proliferation status. Summary of the Invention

[0009] To address the challenges of existing σ2 receptor / TMEM97 molecular probes, the present invention provides an imidazopyridone compound that binds to the σ2 receptor / TMEM97, as well as its preparation method and application. This compound exhibits nanomolar affinity, high subtype selectivity, and specificity for the σ2 receptor / TMEM97, and can be used for PET imaging of tumor proliferation, with broad clinical application prospects.

[0010] In the first aspect, the imidazopyridone compound capable of binding to σ2 receptor / TMEM97 of the present invention has a structure represented by the general formula (I):

[0011]

[0012] Where:

[0013] R is F or 18 F.

[0014] Preferably, when R is 18F, the compound can be used for imaging.

[0015] In a second aspect, the present invention further provides a method for preparing the above-mentioned compound.

[0016] When R in the general formula (I) is F or 18 When F, the corresponding compound is prepared by the following synthetic route:

[0017]

[0018] The preparation steps corresponding to the above synthetic route are as follows:

[0019] (a) In tetrahydrofuran, compound 1 (2-amino-3-nitro-6-methoxypyridine) reacts with di-tert-butyl dicarbonate in the presence of 4-dimethylaminopyridine, followed by the addition of potassium carbonate and methanol to obtain compound 2; the reaction temperature is 55-65°C;

[0020] (b) Compound 2 was reacted with 10% palladium on carbon in methanol under a hydrogen atmosphere to obtain compound 3; the reaction temperature was 50°C;

[0021] (c) Compound 3 reacts with N,N'-carbonyldiimidazole in tetrahydrofuran in the presence of triethylamine to obtain compound 4;

[0022] (d) Compound 4 reacts with 1,4-dibromobutane in the presence of potassium carbonate and tetrabutylammonium iodide in N,N-dimethylformamide to obtain compound 5; the reaction temperature is 60°C;

[0023] (e) Compound 5 reacts with 5,6-dimethoxyisoindoline in the presence of potassium carbonate and triethylamine in acetonitrile to obtain compound 6;

[0024] (f) Compound 6 reacts with trifluoroacetic acid in dichloromethane at room temperature to obtain compound 7;

[0025] (g) Compound 7 was reacted with 1-bromo-2-fluoroethane in the presence of potassium carbonate and tetrabutylammonium iodide in N,N-dimethylformamide to obtain compound 8; the reaction temperature was 65°C;

[0026] (h) Compound 7 reacts with (2-bromoethoxy)-tert-butyldimethylsilane in the presence of potassium carbonate and tetrabutylammonium iodide in N,N-dimethylformamide to obtain compound 9; the reaction temperature is 60°C;

[0027] (i) Compound 9 reacts with 4 mol / L methanolic hydrochloric acid in methanol at room temperature to obtain compound 10;

[0028] (j) Compound 10 reacts with p-toluenesulfonyl chloride in the presence of triethylamine and 4-dimethylaminopyridine at room temperature in dichloromethane to obtain compound 11; p-toluenesulfonyl chloride is added at 0°C;

[0029] (k) Under sealed conditions, compound 11 and 4,7,13,16,21,24-hexaoxo-1,10-diazabicyclo[8.8.8]hexacosane (K 2.2.2 ) reacts under the action of potassium carbonate to obtain 18 F-labeled imidazopyridone σ2 receptor / TMEM97 compounds [ 18 F]8.

[0030] The specific conditions for each of the above steps are as follows:

[0031] The reaction reagents and conditions of each step are as follows:

[0032] (a) Di-tert-butyl dicarbonate, 4-dimethylaminopyridine, and tetrahydrofuran were reacted at 65°C for 1 hour; after cooling, potassium carbonate and methanol were added and the reaction was continued at 55°C for 2 hours;

[0033] (b) 10% palladium carbon, hydrogen, methanol, 50°C, reaction for 12 hours;

[0034] (c) N,N'-carbonyldiimidazole, triethylamine, tetrahydrofuran, 50°C for 2 hours;

[0035] (d) 1,4-dibromobutane, potassium carbonate, tetrabutylammonium iodide, N,N-dimethylformamide, reaction at 60°C for 2 hours;

[0036] (e) 5,6-dimethoxyisoindoline, potassium carbonate, triethylamine, acetonitrile, heated to reflux for 12 hours;

[0037] (f) Trifluoroacetic acid, dichloromethane, react at room temperature for 12 hours;

[0038] (g) 1-Bromo-2-fluoroethane, potassium carbonate, tetrabutylammonium iodide, N,N-dimethylformamide, reaction at 65°C for 12 hours;

[0039] (h) (2-Bromoethoxy)-tert-butyldimethylsilane, potassium carbonate, tetrabutylammonium iodide, N,N-dimethylformamide, reaction at 60°C for 2 hours;

[0040] (i) 4 mol / L methanolic hydrochloric acid, methanol, react at room temperature for 30 minutes;

[0041] (j) p-Toluenesulfonyl chloride, triethylamine, 4-dimethylaminopyridine, and dichloromethane were added at 0°C and reacted at room temperature for 12 hours;

[0042] (k) 4,7,13,16,21,24-hexaoxo-1,10-diazabicyclo[8.8.8]hexacosane (K 2.2.2 ), potassium carbonate, acetonitrile, 100°C, reaction for 8 minutes.

[0043] As one of the specific embodiments, when R in the general formula (I) is 18 When F, the above step (k) specifically includes the following steps:

[0044] S1. Activate a QMA column with 10 mL of NaHCO 3 (10%), 10 mL of H 2 O, and 10 mL of ethanol, respectively, to capture fluoride ions.

[0045] S2. Prepare 1mL K 2.2.2 / K2CO3 eluent will [ 18 F]F - Elute from the QMA column into a reaction flask, heat at 110°C under N2 to remove water, and then dry with anhydrous acetonitrile three times.

[0046] S3. After the water removal is completed, seal the reaction bottle, dissolve about 2 mg of the labeled precursor in 0.5 mL of anhydrous CH3CN, and transfer it to the reaction bottle containing [ 18 F]F - / K 2.2.2 The complex was fully mixed in a reaction bottle and heated at 100° C. for 8 min to obtain the product.

[0047] Among them, as described in S2 [ 18 F]F - / K 2.2.2 The complex is K containing 13 mg of 4,7,13,16,21,24-hexaoxo-1,10-diazabicyclo[8.8.8]hexacosane and 1.1 mg of potassium carbonate. + [ 18 F]F - Mixture, radioactivity 10-1000mCi.

[0048] In this scheme, the reaction product is separated and purified by HPLC. Preferably, the HPLC conditions are as follows: the mobile phase of the HPLC semi-preparative column (ReproSil-Pur Basic-C18 column, 250 mm × 10 mm, 5 μm) is preferably 50% acetonitrile in water containing 0.1% triethylamine, and the flow rate is 4 mL / min.

[0049] In this scheme, the obtained reaction product is identified by HPLC. Preferably, the HPLC conditions are as follows: an analytical column (Agela Venusil MP C18 column, 250 mm × 4.6 mm, 5 μm), and the HPLC analysis mobile phase is preferably 50% acetonitrile aqueous solution containing 0.1% triethylamine, with a flow rate of 1 mL / min.

[0050] Under the above conditions, a product with a radiochemical purity greater than 99% can be obtained.

[0051] In a third aspect, the present invention further provides a ligand targeting σ2 receptor / TMEM97, which comprises the compound represented by the above general formula (I).

[0052] In a fourth aspect, the present invention also provides a molecular probe for binding to σ2 receptor / TMEM97, comprising the general formula (I) wherein R is 18 F is the corresponding compound.

[0053] In a fifth aspect, the present invention further provides a tumor imaging agent targeting σ2 receptor / TMEM97, comprising the general formula (I) wherein R is 18 F is the corresponding compound.

[0054] In a sixth aspect, the present invention further provides the use of the above-mentioned molecular probes for binding to σ2 receptors / TMEM97, tumor imaging agents targeting σ2 receptors / TMEM97 in positron emission tomography imaging agents, or in the preparation of products for diagnosis, staging or efficacy evaluation of cancer patients.

[0055] The beneficial effects of the present invention are as follows:

[0056] The imidazopyridone σ2 receptor / TMEM97 compound provided by the present invention has nanomolar affinity, high subtype selectivity and specificity for σ2 receptor / TMEM97. 18 F, after 18 F performs nucleophilic substitution on -OTs-substituted labeled precursors to prepare corresponding tracers, which have high radiochemical yield and radiochemical purity, excellent biological properties, and can be used for PET imaging of tumor proliferation status, with broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 In Example 11 of the present invention 18 F-labeled compounds [ 18 F] HPLC analysis of 8 after incubation in normal saline for 30 min, 60 min, and 120 min.

[0058] Figure 2 The inhibitor CM398 is used to treat the 18 F-labeled compounds [ 18 F]8 uptake inhibition results in the brain and peripheral organs of normal mice.

[0059] Figure 3 In Example 11 of the present invention 18 F-labeled compounds [ 18 F]8 P-gp substrate experiment in normal mice.

[0060] Figure 4 In Example 11 of the present invention 18 F-labeled compounds [ 18 HPLC analysis of radioactive metabolites of F8 in nude mice bearing CT26 tumors.

[0061] Figure 5 In Example 11 of the present invention 18 F-labeled compounds [ 18 F]8 In vivo micro-PET / CT imaging and CM398 inhibition experimental results in nude mice bearing CT26 tumors. In the figure, A, E, C, and G are the imaging results at 30 and 90 minutes, respectively; B, F, D, and H are the imaging inhibition results at 30 and 90 minutes, respectively (A, B, C, and D are sagittal planes; E, F, G, and H are coronal planes).

[0062] Figure 6 In Example 11 of the present invention 18 F-labeled compounds [ 18 F]8 and [ 18 Comparison of micro-PET / CT imaging results of F]FDG in CT26 lung compression model mice. Figures A and C are [ 18 F]FDG imaging results, B and D are [ 18 F]8 Imaging results (A and B are sagittal planes, C and D are coronal planes).

[0063] Figure 7 The isolated aorta of atherosclerosis model mice and normal C57 mice was treated with oil red and the aorta of Example 11 of the present invention was treated with oil red. 18 F-labeled compounds [ 18 F]8 In vitro autoradiography comparison image (pink arrows indicate the plaque site)

[0064] Figure 8 In Example 11 of the present invention 18 F-labeled compounds [ 18 F]8 60 min in vivo imaging of normal C57 mice and atherosclerosis model mice with thymus removed. The yellow circle indicates the thymus site, and the pink circle indicates the aortic arch site. DETAILED DESCRIPTION

[0065] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0066] The synthetic routes of the compounds of Examples 1-10 of the present invention, as well as the radioactive nuclide fluorine-18 labeling route of Example 11, are as follows:

[0067]

[0068] In the present invention, the "%" mentioned about the solution concentration refers to the volume percentage concentration, and the calculation formula is: volume percentage concentration of the solution = solute volume / solution volume × 100%.

[0069] Example 1 Synthesis of Intermediate 2

[0070] Compound 1 (1.2 g, 10 mmol) was dissolved in 20 mL of tetrahydrofuran, and (Boc)2O (6.5 g, 30 mmol) and 4-dimethylaminopyridine (DMAP, 122 mg, 1 mmol) were added in sequence. The mixture was heated under reflux at 65°C for 1 h. After cooling to room temperature, K2CO3 (4.1 g, 30 mmol) was added, followed by 50 mL of methanol. The mixture was heated under reflux at 55°C for 2 h. After cooling to room temperature, the solvent was removed by rotary evaporation. 50 mL of water was added to the reaction flask, and the mixture was extracted with dichloromethane (3×50 mL). The organic phase was washed with saturated brine, then combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA=6:1) to obtain intermediate 2, the structure of which is shown below. The yield was 38.6%, and the product was light green crystals. The developing system was PE:EA=3:1 (R f =0.5). 1 H NMR (600MHz, DMSO-d6): δ10.19(s,1H),8.27(d,J=8.9Hz,1H),6.65(d,J=8.9Hz,1H),3.89(s,3H),1.39(s,9H).ESI-MS calcd.for C 11 H 15 N3NaO5[M+Na] + m / z 292.10; found m / z 292.09.

[0071]

[0072] Example 2 Synthesis of Intermediate 3

[0073] Intermediate 2 (1.7 g, 4.5 mmol) was dissolved in 10 mL of methanol, 10% Pd / C (46 mg, 0.45 mmol) was added, H2 was introduced, and the mixture was refluxed at 50°C overnight. The mixture was cooled to room temperature, filtered under reduced pressure, and the solvent was removed by rotary evaporation. The crude product was dissolved in methanol and then dry-coated. It was purified by silica gel column chromatography (PE:EA=3:1) to obtain intermediate 3, the structure of which is shown below. The yield was 92.4%, and the product was a light pink powder. The developing system was PE:EA=4:1 (R f =0.3). 1 H NMR (600MHz, DMSO-d6): δ8.83(s,1H),7.11(d,J=8.4Hz,1H),6.42(d,J=8.4Hz,1H),4.39(s,2H),3.66(s,3H),1.41(s,9H).ESI-MScalcd.for C 11 H 18 N3O3[M+H] + m / z 240.13; found m / z 240.13.

[0074]

[0075] Example 3 Synthesis of Intermediate 4

[0076] Intermediate 3 (500 mg, 2.1 mmol) was dissolved in 10 mL of tetrahydrofuran, and N, N'-carbonyldiimidazole (CDI, 374 mg, 2.3 mmol) and triethylamine (TEA, 608 μL, 4.2 mmol) were added in sequence. N2 was introduced and the mixture was heated under reflux at 50 ° C for 5 h. After cooling to room temperature, the solvent was removed by rotary evaporation. The crude product was dissolved in dichloromethane and methanol and then dry-coated. It was purified by silica gel column chromatography (PE: EA = 7:3) to obtain intermediate 4, the structure of which is as follows. Yield 36.4%, white solid, developing system PE: EA = 3:1 (R f =0.4). 1 H NMR (400MHz, DMSO-d6): δ11.80(s,1H),7.75(d,J=8.6Hz,1H),6.45(d,J=8.6Hz,1H),3.77(s,3H),1.53(s,9H).ESI-MS calcd for C 12 H 16 N3O4[M+H] + m / z 266.11; found m / z 266.11.

[0077]

[0078] Example 4 Synthesis of Intermediate 5

[0079] Intermediate 4 was dissolved in 5 mL of DMF, and K2CO3 (1 g, 7.5 mmol), TBAI (139 mg, 0.4 mmol), and 1,4-dibromobutane (271 μL, 2.3 mmol) were added in sequence. The mixture was heated under reflux at 65°C for 2 h. 50 mL of water was added to the reaction flask to quench the mixture, and the mixture was extracted with dichloromethane (3×50 mL). The organic phase was washed with saturated brine, then combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA=3:1) to obtain intermediate 5, the structure of which is shown below. The yield was 34.2%, and the product was a yellow oily liquid. The developing system was PE:EA=3:1 (R f =0.7). 1 H NMR (600MHz, DMSO-d6): δ7.80(d,J=8.5Hz,1H), 6.50(d,J=8.5Hz,1H), 3.83(s,3H), 3.80(t,J=6.2Hz,2H),3.55(t,J=6.1Hz,2H),1.85–1.74(m,4H),1.54(s,9H).ESI-MS calcd.for C 16 H 23 BrN3O4[M+H] + m / z 400.09; found m / z 400.09.

[0080]

[0081] Example 5 Synthesis of Intermediate 6

[0082] Intermediate 5 (380 mg, 0.9 mmol) was dissolved in 5 mL of acetonitrile, and K2CO3 (262 mg, 1.9 mmol), triethylamine (TEA, 138 μL, 0.9 mmol) and 5,6-dimethoxyisoindole (187 mg, 1 mmol) were added sequentially. The mixture was heated under reflux at 90°C overnight, cooled to room temperature, and the solvent was removed by rotary evaporation. 20 mL of water was added to the reaction flask to quench the mixture, and the mixture was extracted with dichloromethane (3×20 mL). The organic phase was washed with saturated brine, then combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA:MeOH:TEA=10:25:1:0.5) to obtain intermediate 6, the structure of which is shown below. The yield was 37.3%, and the product was a brown oily liquid. The developing system was PE:EA:MeOH:TEA=10:25:1:1 (R f =0.5). 1H NMR (600MHz, Chloroform-d): δ7.86 (d, J=8.5Hz, 1H), 6.71 (s, 2H), 6.43 (d, J=8.5Hz, 1H), 3.96 (q, J=7 .5Hz,3H),3.90(d,J=10.9Hz,6H),3.84(s,6H),2.79(s,2H),1.93–1.88(m,2H),1.65(s,11H).ESI-MS calcd.for C 26 H 35 N4O6[M+H] + m / z 499.25; found m / z499.25.

[0083]

[0084] Example 6 Synthesis of Intermediate 7

[0085] Intermediate 6 (176 mg, 0.35 mmol) was dissolved in 5 mL of dichloromethane, and 1 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 h. 20 mL of water was added to the reaction flask to quench the mixture, and a saturated solution of NaHCO3 was added dropwise to a pH of 9-10, and the mixture was extracted with dichloromethane (3×20 mL). The organic phase was washed with saturated brine, and then the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE: EA: MeOH: TEA = 10:25:1:0.5) to obtain intermediate 7, the structure of which is as follows. The yield was 78.2%, and the product was a white solid. The developing system was PE: EA: MeOH: TEA = 10:25:1:1 (R f =0.2). 1 H NMR (400MHz, DMSO-d6): δ10.77(s,1H),7.24(d,J=8.2Hz,1H),6.78(d,J=4.0Hz,2H),6.36(d,J=8.3Hz,1H),3.77(d ,J=3.7Hz,5H),3.67(d,J=7.6Hz,10H),2.63(t,J=6.9Hz,2H),1.76(t,J=7.1Hz,2H),1.44(t,J=7.4Hz,2H).ESI-MS calcd.for C 21 H 27 N4O4[M+H] + m / z 399.20; found m / z399.20.

[0086]

[0087] Example 7 Synthesis of Standard 8

[0088] Intermediate 7 (51 mg, 0.14 mmol) was dissolved in DMF (5 mL), and K2CO3 (58 mg, 0.42 mmol), phase transfer catalyst TBAI (10 mg, 0.03 mmol) and 1-bromo-2-fluoroethane (44 mg, 0.35 mmol) were added in sequence. The reaction was allowed to proceed at 60°C for 2 h. 20 mL of water was added to the reaction flask to quench the reaction, and the mixture was extracted with dichloromethane (3 × 20 mL). The organic phase was washed with saturated brine, then combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA:MeOH:TEA=10:20:1:0.5) to obtain standard product 8, the structure of which is shown below. The yield was 39%, and the product was a white solid. The developing system was PE:EA:MeOH:TEA=10:20:1:1 (R f =0.2). 1 H NMR (600MHz, Chloroform-d): δ7.25–7.22(m,1H),6.72(s,2H),6.42(d,J=8.4Hz,1H),4.69(dt,J=47.1,4.7Hz,2H),4.14(dt, J=27.4,4.7Hz,2H),4.00(t,J=7.1Hz,2H),3.91(s,7H),3.84(s,6H),2.85–2.73(m,2H),1.99–1.90(m,2H),1.70–1.64(m,2H). 13 C NMR (150MHz, Chloroform-d): δ160.10,153.66,148.48,141.05,131.66,118.29,117.78,105.96,10 1.37,82.78(d,J=170.7Hz),59.25,56.21,55.62,54.02,41.86(d,J=20.9Hz),40.00,26.30,26.14. 19 F NMR(565MHz,Chloroform-d):δ-219.90–-220.24(m).HR-MS calcd.for C 23 H 30 FN4O4[M+H] + m / z445.2245; found m / z 445.2248.

[0089]

[0090] Example 8 Synthesis of Intermediate 9

[0091] Intermediate 7 (50 mg, 0.125 mmol) was dissolved in DMF (5 mL), and K2CO3 (69 mg, 0.5 mmol), phase transfer catalyst TBAI (23 mg, 0.06 mmol) and (2-bromoethoxy)-tert-butyldimethylsilane (83 μL, 0.375 mmol) were added in sequence. The mixture was heated to reflux at 60°C for 2 h, cooled to room temperature, and the solvent was removed by rotary evaporation. 20 mL of water was added to the reaction flask and extracted with dichloromethane (3×20 mL). The organic phase was washed with saturated brine, then combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA:MeOH:TEA=10:20:1:1) to obtain intermediate 9, the structure of which is shown below. The yield was 84.8%, and the product was a green oily liquid. The developing system was PE:EA:MeOH:TEA=10:20:1:1 (R f =0.5). 1 H NMR (600MHz, Chloroform-d): δ7.25 (s, 1H), 6.71 (s, 2H), 6.39 (d, J = 8.3Hz, 1H), 3.99 (t, J = 7.1Hz, 2H), 3.94 (t, J = 5.3Hz, 2H), 3.91–3.85(m,9H),3.84(s,6H),2.78(s,2H),1.92(p,J=7.3Hz,2H),1.65(d,J=7.2Hz,2H),0.78(s,9H),-0.10(s,6H).ESI-MS calcd.forC 29 H 45 N4O5Si[M+H] + m / z 557.31; found m / z 557.31.

[0092]

[0093] Example 9 Synthesis of Intermediate 10

[0094] Intermediate 9 (50 mg, 0.09 mmol) was dissolved in 5 mL of methanol, and 1 mL of methanolic hydrochloric acid was added. The mixture was reacted at room temperature for 30 min, and the solvent was removed by rotary evaporation. 20 mL of water was added to the reaction flask to quench the mixture. A saturated solution of NaHCO3 was added dropwise until the pH was 9-10, and the mixture was extracted with dichloromethane (3×20 mL). The mixture was concentrated under reduced pressure to obtain intermediate 10, the structure of which is shown below. The crude product was a yellow solid, and the developing system was PE:EA:MeOH:TEA=10:20:1:1 (R f =0.2). 1H NMR (600MHz, DMSO-d6): δ7.48(d,J=8.3Hz,1H),6.86(s,2H),6.44(d,J=8.3Hz,1H),4.82(d,J=5.8Hz,1H),3.88–3 .71(m,9H),3.68(s,6H),3.59(q,J=5.4Hz,2H),3.03(d,J=7.5Hz,4H),1.78(p,J=7.0Hz,2H),1.56(s,2H).ESI-MS calcd.for C 23 H 31 N4O5[M+H] + m / z 443.22; found m / z 443.23.

[0095]

[0096] Example 10 Synthesis of Labeled Precursor 11

[0097] Under ice-cooling conditions, intermediate 9 (40 mg, 0.09 mmol) was dissolved in 5 mL of dichloromethane. Triethylamine (13 μL, 0.09 mmol), DMAP (2 mg, 0.018 mmol), and TsCl (34 mg, 0.18 mmol) were added sequentially and reacted at room temperature for 2 h. 20 mL of water was added to the reaction flask and extracted with dichloromethane (3×20 mL). The organic phase was washed with saturated brine, then combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA:MeOH:TEA=10:20:1.5:1) to obtain the labeled precursor 10, the structure of which is shown below. The yield was 53.7%, and the product was a yellow solid. The developing system was PE:EA:MeOH:TEA=10:20:2:1 (R f =0.4). 1 H NMR (400MHz, Chloroform-d): δ7.55(d,J=8.1Hz,2H),7.18(t,J=7.7Hz,3H),6.71(s,2H),6.40(d,J=8.3Hz,1H),4.30(t,J=5.0Hz,2H) ,4.07(t,J=5.1Hz,2H),3.96–3.85(m,9H),3.83(s,6H),2.78(t,J=7.5Hz,2H),2.38(s,3H),1.87(p,J=7.4Hz,2H),1.69–1.55(m,2H). 13C NMR(101MHz,Chloroform-d)δ160.05,153.33,148.54,145.07,140.77,132.39,129.78,127.74,118 .18,117.43,105.89,101.45,68.31,59.22,56.20,55.56,54.04,40.59,39.81,26.23,21.73.HR-MS calcd.for C 30 H 37 N4O7S[M+H] + m / z597.2377; found m / z 597.2381.

[0098]

[0099] Example 11 18 F-labeled imidazopyridone σ2 receptor / TMEM97 compounds [ 18 The preparation of F]8 comprises the following steps:

[0100] (1) A QMA column was activated with NaHCO3 (10%, 10 mL), H2O (10 mL), and ethanol (10 mL) for fluoride ion capture. A C-18 column was activated with ethanol (10 mL) and H2O for solid phase extraction.

[0101] (2) Use the prepared K 2.2.2 / K2CO3 eluent (1 mL) 18 F]F - Elute from the QMA column into a reaction flask, heat at 110°C under N2 to remove water, and then dry with anhydrous acetonitrile three times.

[0102] (3) After the water removal is completed, the reaction bottle is sealed and about 2 mg of the labeled precursor (Compound 11) is dissolved in 0.5 mL of anhydrous CH3CN and transferred to the reaction bottle containing [ 18 F]F- / K 2.2.2 The complex was fully mixed in a reaction bottle and heated at 100° C. for 8 min to obtain the product.

[0103] Among them, (3) [ 18 F]F - / K 2.2.2 The complex contains 13 mg of 4,7,13,16,21,24-hexaoxo-1,10-diazabicyclo[8.8.8]hexacosane (K 2.2.2 ) and 1.1 mg of potassium carbonate + [ 18 F]F -Mixture, radioactivity 10-1000mCi.

[0104] (4) After the reaction is completed, the reaction mixture is cooled to room temperature, the radioactivity is measured, and the product is isolated and purified by HPLC. In this protocol, the HPLC conditions are preferably as follows: a semi-preparative HPLC column (ReproSil-Pur Basic-C18 column, 250×10 mm, 5 μm) with a mobile phase of preferably 50% acetonitrile in water containing 0.1% triethylamine at a flow rate of 4 mL / min.

[0105] (5) Product Identification after Separation and Purification: In this protocol, the resulting reaction product is identified by HPLC. Preferably, the HPLC conditions are as follows: an analytical column (Agela Venusil MP C18 column, 250×4.6 mm, 5 μm), and the HPLC mobile phase is preferably a 50% acetonitrile aqueous solution containing 0.1% triethylamine at a flow rate of 1 mL / min. The HPLC separation and purification results in a radiochemical purity greater than 99% and a radiochemical yield of 8%-31%.

[0106] Example 12 Properties of Imidazolopyridone Compounds Binding to σ2 Receptor / TMEM97

[0107] The σ2 receptor / TMEM97 ligand and the corresponding 18 The F-labeled compound was prepared according to Example 1-12.

[0108] 1. Radioligand receptor competition binding assay (K i Value determination):

[0109] The in vitro affinity of sigma receptor ligand compounds for sigma receptors was determined using a radioligand receptor competition binding assay.

[0110] The affinity of σ1 receptor was determined using HEK293 cells transfected with human σ1 (Université de Nice Sophia-Antipolis, France) and (+)-[ 3 [H]pentazocine (PerkinElmer, 1050 GBq / mmol) was used as the receptor organizer and binding ligand.

[0111] The affinity of σ2 receptor / TMEM97 was determined using rat liver membrane protein and [ 3 [H]DTG (PerkinElmer, 1850 GBq / mmol, in the presence of 100 nM FTC-146) served as the receptor organizer and binding ligand.

[0112] The (+)-[ 3 H]pentazocine and [ 3 Nonspecific binding of (+)-[ 3 H]pentazocine and [ 3 The dissociation constant K of [H]DTG d 33nM and 50nM respectively.

[0113] (1) Rat liver was homogenized and rinsed with 50 mM Tris-HCl buffer (pH = 7.4, room temperature). The supernatant was discarded after centrifugation (15,000 rpm). The liver was rinsed with buffer and centrifuged again, and this process was repeated 5-6 times. The liver was frozen at a ratio of 1 g tissue / 10 mL buffer for later use. The homogenate of human sigma-1-transfected HEK293 cells used for the determination of σ1 receptor affinity was treated in the same manner as above.

[0114] (2) Take 5 mL of frozen membrane protein tissue, thaw and centrifuge, wash twice with buffer, and then add buffer to dilute to 12 mL of solution for use.

[0115] (3) Dissolve the compound in DMSO and prepare a buffer solution to a concentration of 10 -11 M-10 -5 M solution is reserved.

[0116] (4) The experiment was divided into a total binding group, a nonspecific binding group, and an experimental group. To the total binding group, 100 μL of the corresponding radioligand, 200 μL of tissue fluid, and 700 μL of binding buffer were added. To the nonspecific binding group, 100 μL of haloperidol was added, and to the experimental group, 200 μL of tissue fluid was replaced with the test compound solution. The total volume of all three groups was 1 mL.

[0117] (5) The solution was vortexed and incubated at room temperature for 60 min. The solution was then vacuum filtered through glass fiber filter paper (GF / B) pre-treated with 0.3% fresh PEI (90 min, 4°C). The membrane protein and free radioligand were collected using a cell harvester, and the filter was placed in a counting tube to determine the radioactivity count.

[0118] (6) The experiment was repeated three times, and the concentration of the compound required to reduce the amount of radioligand binding to the receptor by half (IC 50 value), applying the Cheng-Prusoff equation, by IC 50 The inhibition constant (K i value).

[0119]

[0120] In the above formula, [LT] is the concentration of radioligand, Kd K is the equilibrium dissociation constant for the binding of radioligand to receptor, d It can be obtained by Scatchard method, from the above K d and IC 50 The above formula can be used to calculate the K value of the ligand i The calculation results are shown in Table 1.

[0121] Table 1 Inhibition constants of compounds against σ1 receptor and σ2 receptor / TMEM97

[0122]

[0123] The results of the above competitive binding experiments show that the compound of Example 7 has high affinity (nanomolar order) and high subtype selectivity for σ2 receptor / TMEM97, and is one of the few reported σ2 receptor / TMEM97 ligands with nanomolar affinity and low lipid solubility for PET imaging. 18 F-labeled compounds have the potential to be developed into clinically applicable σ2 receptor / TMEM97 tumor imaging agents.

[0124] 2. Prepared in Example 11 18 F-labeled imidazopyridone σ2 receptor / TMEM97 compounds [ 18 Physicochemical properties of F]8

[0125] 2.1 Determination of lipid-water partition coefficient

[0126] Shake flask method: prepare equal volumes of PBS buffer (0.05M, pH=7.4) and n-octanol, mix them evenly and place them overnight for use. Take 3.0mL of PBS solution and 3.0mL of n-octanol in a centrifuge tube, add the purified product after HPLC separation 18 The F-labeled compound of Example 11 (3.0 MBq, 10 μL) was vortexed for 3 minutes and then centrifuged at 4000 rpm for 5 minutes. 100 μL of PBS buffer and 100 μL of n-octanol solution were taken and the radioactivity count was measured using a γ-counter to calculate the log D 7.4 Take the above 2.0mL PBS buffer and 2.0mL n-octanol in a new centrifuge tube, add 1.0mL PBS buffer and 1.0mL n-octanol, repeat the above steps twice, take 100μL PBS buffer and 100μL n-octanol solution, use γ-counter to measure the radioactivity count, and calculate logD 7.4 , three parallel experiments were performed, and the calculation formula of logD was:

[0127]

[0128] D = (radiotracer counts in 1 mL of n-octanol) / (radiotracer counts in 1 mL of PBS buffer)

[0129] The above experiment was repeated three times and the 18 log D of the F-labeled compound of Example 11 7.4 =1.62±0.03, indicating 18 The F-labeled compound of Example 11 has lower lipid solubility and is expected to effectively reduce nonspecific binding during biodistribution.

[0130] 2.2 In vitro salt stability

[0131] After HPLC separation and purification 18 F-labeled Example 11 compound (0.5 MBq, 100 μL) was prepared in a 7% ethanolic saline solution (at room temperature). HPLC analysis was performed to determine the percentage of the parent compound in the 7% ethanolic saline solution at 30, 60, and 120 minutes. An analytical column (Agela Venusil MP C18 column, 250×4.6 mm, 5 μm) was used, with the mobile phase preferably consisting of 50% acetonitrile in water containing 0.1% triethylamine at a flow rate of 1 mL / min. After 30, 60, and 120 minutes, HPLC analysis revealed radiochemical purities of >99% for the parent compound in saline. The results are shown in the attached figure. Figure 1 .

[0132] 3. 18 In vivo biological evaluation of F-labeled compound of Example 11

[0133] 3.1 18 Normal distribution of F-labeled compound of Example 11 in normal ICR mice

[0134] Experimental mice: 25 normal male ICR mice (18-22 g, 4-5 weeks old). Inject 0.1 mL of 18F-labeled compound of Example 11 (296-370 kBq) was injected at 2, 15, 30, 60, and 120 minutes after injection. Blood, whole brain, heart, liver, spleen, lung, kidney, pancreas, flesh, bone, stomach, small intestine, and tail were collected. Organs (except the stomach, small intestine, and tail) were weighed, and the radioactivity count (CPM) in each organ was determined using a γ-counter. Three replicates of 10% of the injected volume were used for data correction, along with the residual count in the tail. The %ID / organ or %ID / g (percentage of radioactivity per gram of organ) was calculated for each organ. The results are shown in Table 2.

[0135] Table 2 18 Distribution of F-labeled compound of Example 11 in normal ICR mice (% ID / g, mean ± SD, n = 4-5) a

[0136]

[0137]

[0138] a Data are means of%ID / g of tissue±SD, n=4-5.

[0139] b Percentage of injected dose per organ.

[0140] From the experimental results in Table 2, we can see that 18 Initial brain uptake of the F-labeled compound of Example 11 in normal ICR mice was low, reaching 1.94±0.19% ID / g at 2 minutes. Brain uptake was cleared over time, reaching 0.76±0.12% ID / g and 0.45±0.08% ID / g at 60 minutes and 120 minutes, respectively. The brain-to-blood ratio reached a maximum of 3.65±0.65 at 30 minutes. 18 The F-labeled compound of Example 11 had higher initial uptake values ​​in the heart, lung, and kidney (12.53±1.34% ID / g, 46.94±4.70% ID / g, and 20.44±2.67% ID / g, respectively), which decreased over time. The uptake value in the liver first increased and then decreased over time, while the uptake value in the small intestine increased from low to high, indicating that 18 The F-labeled compound of Example 11 is likely metabolized by the liver and small intestine. 18The bone uptake of the F-labeled compound of Example 11 did not increase over time from 2 min to 120 min, indicating that it had no obvious defluorination phenomenon in mice and had good in vivo stability.

[0141] 3.2 18 Inhibitory experiment of F-labeled compound of Example 11 in normal ICR mice

[0142] Experimental mice: 5 normal ICR mice (18-22g, 4-5 weeks old). 0.1 mL of CM398 solution (5 μmol / kg or 10 μmol / kg) was injected into the tail vein in advance, and 0.1 mL was injected 5 minutes later. 18 F-labeled compound from Example 11 (296-370 kBq) was administered. After 30 minutes, samples were collected from the blood, brain, heart, liver, spleen, lungs, kidneys, pancreas, meat, bones, stomach, small intestine, and tail. The assay was performed in the same manner as in 3.1, and the differences (p values) between the inhibition and normal groups were analyzed. The results are shown in Table 3.

[0143] Table 3 18 Inhibitory experiment of F-labeled compound of Example 11 in normal ICR mice (% ID / g, mean ± SD, n = 5) a

[0144]

[0145]

[0146] a Data are expressed as percentage of injected dose per gram,means±SD,n=4-5. b p values ​​for the control vs blocking group at 30min postinjectioncalculated by Student's t test.

[0147] According to the results of the inhibition experiment (Table 3 and Appendix Figure 2 ) It can be seen that CM398 (5 μmol / kg) has an effect on 18The F-labeled compound of Example 11 had a significant inhibitory effect on organs expressing σ2 receptors / TMEM97 in mice, such as the brain (-73%, p<0.001), heart (-64%, p<0.001), liver (-42%, p=0.001), spleen (-79%, p<0.001), lung (-84%, p<0.001), kidney (-83%, p<0.001), muscle (-50%, p=0.003), and bone (-69%, p=0.001). The brain / blood ratio inhibition rate reached 87% (p<0.001), and all the differences were significant. 18 The F-labeled compound of Example 11 binds to the σ2 receptor / TMEM97 in mice with high specificity. Furthermore, increasing CM398 concentrations did not significantly increase the inhibition of uptake in various organs, indicating that 5 μmol / kg of CM398 is sufficient to block the σ2 receptor / TMEM97 in mice.

[0148] 3.3 18 P-gp substrate assay of the F-labeled compound of Example 11

[0149] Prepare the P-gp inhibitor Tariquidar (1 mg / mL, 10% DMSO, 20% 1,2-propylene glycol, 30% PEG400, 40% saline) for use. Normal ICR male mice (18-22 g, 4-5 weeks) were injected with Tariquidar (5 mg / kg) via the tail vein 30 minutes before administration. 18 F-labeled Example 11 compound (296-370 kBq, 0.1 mL, in saline solution containing approximately 7% ethanol). After 2 minutes, blood, whole brain, and tail were collected and weighed separately (excluding the tail). Radioactivity counts (CPM) were determined using a γ-counter. 10% of the injection volume of three replicates was used as a correction along with the residual count in the tail to calculate the %ID / g or inhibition percentage for blood and whole brain. The experimental results are shown in the attached table. Figure 3 .

[0150] According to the experimental results ( Figure 3 ) It can be seen that injection 18 Two minutes after administration of the F-labeled compound of Example 11, the radioactive uptake in the brain and blood of normal mice was 1.94±0.19% ID / g and 1.58±0.27% ID / g, respectively, with a brain / blood ratio of 1.25±0.17. In the experimental group injected with Tariquidar in advance, the radioactive uptake in the brain and blood of mice was 7.51±0.83% ID / g (p<0.001) and 2.03±0.11% ID / g (p=0.09), respectively, with a brain / blood ratio of 3.69±0.37 (p<0.001). 18The brain uptake and brain / blood ratio of the F-labeled compound of Example 11 in mice increased by 287% and 195%, respectively, indicating that 18 The F-labeled compound of Example 11 is a P-gp substrate.

[0151] 3.4 18 Metabolism and stability evaluation of F-labeled compound of Example 11 in normal ICR mice

[0152] Nude mice bearing CT26 tumors (16-20 g, n = 3) were injected into the tail vein 18 The animals were anesthetized and sacrificed 30 minutes after administration of the F-labeled compound of Example 11 (0.8-1.0 mCi, 0.1 mL). Blood, whole brain, tumor, and liver were collected and placed in centrifuge tubes. Residual blood stains on the surface of the organs were washed with physiological saline. Acetonitrile (4°C, 0.5 mL) was added and the tissues were ground in a homogenizer. The radioactive substances were extracted with acetonitrile. The cells were vortexed and centrifuged. The supernatant was then transferred to a new centrifuge tube and acetonitrile was added to precipitate the protein. The cells were centrifuged and acetonitrile was added repeatedly until no new precipitate formed. The solution was filtered through a 0.22 μm organic filter membrane and then used for analysis. The resulting solution was analyzed by HPLC. Analytical conditions: Analytical column: Agela Venusil MP C18, 5 μm, 250×4.6 mm; mobile phase: 50% acetonitrile and 50% water (0.1% TEA); flow rate: 1 mL / min. The experimental results are shown in Table 4 and Appendix. Figure 4 .

[0153] Table 4 Injection 18 Percentage of radioactive metabolites and parent tracer in mouse plasma, brain, liver and tumor after 30 min of administration of F-labeled Example 11 compound (n=3) a

[0154]

[0155] a Data are expressed as percentage of total radioactivity,means±SD.

[0156] According to the results of the metabolic experiments (Table 4 and Appendix Figure 4 ) It can be seen that 18 The retention time of the F-labeled compound of Example 11 in HPLC was 12.49 min. In the brain and tumors, 18 The radioactive signal content of the parent compound of Example 11 labeled with F was greater than 90%, and the radioactive signal content of the parent compound in the tumor was as high as 98%. Only a small amount of hydrophilic radioactive metabolites were produced at 2.8 minutes and 3.5 minutes. In the blood and liver, 18The radioactive signal contents of the parent compound of Example 11 labeled with F were 41.79±4.07% and 67.48±0.52%, respectively. 18 The F-labeled compound of Example 11 has good stability in both mouse brain and tumors, and the results are consistent with the results of the biodistribution inhibition experiment in normal mice and the inhibition experiment in nude mice bearing CT26 tumors.

[0157] 3.5 18 Micro-PET / CT imaging of nude mice bearing CT26 tumors by the F-labeled compound of Example 11

[0158] Nude mice bearing CT26 tumors (18-20 g, n = 4-7) were injected with the drug via tail vein. 18 F-labeled Example 11 compound (6.5-10 MBq, 0.1 mL) was anesthetized with 1.5-2.5% isoflurane in an oxygen flow (1-2 L / min) 30 and 90 minutes after administration, respectively, and then weighed. The subject was then secured to the data acquisition bed and positioned in the center of the parallel scanner. The airflow was adjusted to 0.8-1.0 L / min (containing 1.5-2.0% isoflurane). PET data acquisition was performed continuously for 10 minutes, and CT scanning was performed for 10 minutes under the configured data acquisition program.

[0159] Inhibition experiment group: CM398 inhibition experiment was used to verify the tracer in the tumor 18 The specificity of the F-labeled compound of Example 11 for σ2 receptor / TMEM97 was determined by the inhibitor CM398 (5 μmol / kg, 0.1 mL) and 18 F-labeled compound of Example 11 was co-injected, and PET / CT data were collected at 30 and 90 minutes of the static phase. After the instrument completed data acquisition, the mouse's weight (g), initial radioactivity, residual activity (GBq), and specific acquisition time were input. The quantitative data were corrected and reconstructed, and the CT scan was used as the positioning basis. The reconstructed data was image and data processed by PMOD, and the radioactivity uptake SUV in the mouse tumor and lung was calculated. max The results of CT26 tumor imaging experiments are shown in Table 5 and Appendix Figure 5 .

[0160] Table 5 18 PET imaging results of the F-labeled compound of Example 11 in CT26 tumor mice a

[0161]

[0162] a Data are expressed as SUV max,means±SD.

[0163] b n=7

[0164] c n=4

[0165] b p values ​​for the control vs blocking group was calculated byStudent's t test(independent,two-tailed).

[0166] Quantitative analysis of PET imaging experiments by PMOD (Table 5 and Appendix Figure 5 ) It can be seen that injection 18 30 min after administration of the F-labeled compound of Example 11, there was obvious radioactive accumulation in the tumor area. Radioactive uptake SUV of the tumor in the normal group max was 1.05±0.24, the lung uptake value was low, and SUV max The radioactivity uptake SUV of the tumor in the CM398 inhibition group was 0.41±0.13, and the tumor / lung ratio was 2.39±0.97. max 0.37±0.10, lung SUV max The tumor / lung ratio was 0.20±0.07, the tumor / lung ratio was 2.01±0.71, and the tumor tracer uptake inhibition rate was 65% (p<0.001). 18 SUV of the tumor in the normal group was 90 min after the F-labeled compound of Example 11 was added. max was 1.13±0.22, lung SUV max The radioactivity uptake SUV of the tumor in the CM398 inhibition group was 0.28±0.07, and the tumor / lung ratio was 3.71±0.72. max 0.25±0.08, lung SUV max The tumor / lung ratio was 0.13±0.04, the tumor / lung ratio was 1.94±0.29, and the tumor tracer uptake inhibition rate was 78% (p<0.001). The results of PET imaging and inhibition experiments are shown in Table 5 and Appendix. Figure 5 The experimental results show that 8 The F-labeled compound of Example 11 has highly specific binding to the σ2 receptor / TMEM97, with a high tumor uptake value and a low lung uptake value. As time goes by, the tumor uptake value increases while the lung uptake value decreases, making the PET imaging effect clearer, as shown in FIG. Figure 5 shown.

[0167] 3.6 18 PET / CT imaging of F-labeled compound of Example 11 in CT26 lung compression model mice

[0168] Experimental control group: CT26 lung puncture model mice (17.3 g), fasted for 12 hours in advance, and injected [ 18 F]FDG (7.2 MBq, 0.1 mL) was administered 60 minutes after administration. The subjects were anesthetized with a 1.5-2.5% isoflurane-containing oxygen flow (1-2 L / min) and weighed. The subjects were then secured to the data acquisition bed and positioned in the center of the parallel scanner. The airflow was adjusted to 0.8-1.0 L / min (containing 1.5-2.0% isoflurane). Under the set PET data acquisition program, data were continuously acquired for 10 minutes and CT scans were performed for 10 minutes.

[0169] Experimental group: mice were fed with normal diet and injected with drugs via tail vein 18 F-labeled Example 11 compound (9.6 MBq, 0.1 mL) was administered, followed by anesthesia with a 1.5-2.5% isoflurane-infused oxygen flow (1-2 L / min), followed by weighing. The mouse was then secured to the data acquisition bed and positioned in the center of the parallel scanner. The airflow was adjusted to 0.8-1.0 L / min (containing 1.5-2.0% isoflurane). PET images were acquired for 0-60 minutes according to the program settings. After PET acquisition was complete, CT images were acquired for 10 minutes. After the instrument completed data acquisition, the mouse's weight (g), initial radioactivity, residual activity (GBq), and specific acquisition time were input. The quantitative data were calibrated and reconstructed, with the CT scan serving as the basis for positioning. 18 PET images of the F-labeled compound of Example 11 were divided into 10-min × 6 frames. Based on the CT images, regions of interest (ROIs) were drawn and integrated with the PET images. The standardized uptake values ​​(SUVs) of the ROIs were calculated and time-activity curves (TACs) were constructed. The reconstructed data of the experimental control group were processed by PMOD to calculate the radioactivity uptake SUVs in the mouse tumors and lungs. max . The experimental results show that [ 18 The uptake value of F]FDG in mouse heart was very high, while the uptake value in tumor was relatively low, and the imaging of lung tumor was not obvious. 18 Compared with F]FDG, 18 The F-labeled compound of Example 11 had a higher uptake value in CT26 tumors and a lower uptake value in the heart, with a higher tumor / lung ratio and tumor / heart ratio, and clear tumor imaging. Figure 6 .

[0170] 3.7 18 In vitro autoradiography and gross oil red staining of atherosclerotic mouse aorta by the F-labeled compound of Example 11

[0171] ApoE fed for 20 weeks- / - Mice (n=1) and wild-type mice (n=1) were injected into the tail vein 18 F-labeled compound from Example 11 (1.11 MBq, 0.1 mL). All mice were anesthetized and sacrificed 2 hours after injection. The heart and entire aorta were removed under a dissecting microscope, and the aorta was then removed. Periaortic adipose tissue was removed. The aorta was rinsed with normal saline, the surface droplets were blotted, and the aorta was exposed to a phosphorus storage screen (PerkinElmer, USA) for 10 minutes. Radioautographic images were then obtained using a phosphorus storage screen imaging system (Cyclone Plus, PerkinElmer, USA).

[0172] After autoradiography, the aorta was fixed in paraformaldehyde for at least 24 hours before removal. The peripheral fat was removed and the vessel was dissected longitudinally. The vessel was immersed in 60% isopropyl alcohol for 3 seconds, then in Oil Red O stain at 37°C in the dark for 60 minutes. The vessel was then removed and differentiated in 60% isopropyl alcohol until the intraluminal fatty plaques appeared orange-red or bright red, while the rest of the vessel was nearly colorless. Differentiation was then terminated by washing with distilled water. The aorta was removed, excess water was removed, and the vessel was placed on a black background with a ruler for photography.

[0173] The captured images were analyzed using Image J. Quantitative results were expressed as the percentage of lipid accumulation lesion area to the total aorta area. Under the dissecting microscope, atherosclerotic plaques were visible to the naked eye in the aortic arch of atherosclerosis model mice. Macroscopic Oil Red O staining showed that lipid droplets in this area were bright red, indicating that atherosclerotic plaques had indeed formed in this area. In contrast, the aorta of wild-type mice was almost colorless in macroscopic Oil Red O staining. The lipid content (56.1%) and the proportion of radioactive concentration-positive area (39.7%) were similar. More importantly, the radioactivity counts in the bright red areas of the aorta of the plaque model mice were significantly higher in the radioautograph than in the colorless areas in the Oil Red O staining. Radioautographs were analyzed using AIDA2.31 software. The red areas of the Oil Red O staining images were defined as lesions, and the colorless areas of the Oil Red O staining images were defined as background. Regions of interest were manually outlined in the radioautographs, and the software automatically calculated the radioactivity counts in the regions of interest. The calculated target / target ratio was 2.42, indicating that the tracer 18 The F-labeled compound of Example 11 accumulated in the atherosclerotic plaques in the aorta, and this tracer has the potential to be used for atherosclerotic plaque imaging. Figure 7 .

[0174] 3.8 18 Micro-PET / CT imaging of atherosclerosis in mice using the F-labeled compound of Example 11

[0175] After 20 weeks of feeding, ApoE- / - mice (n=4) or wild-type mice (n=1) were injected with tracer into the tail vein. 18 F-labeled compound from Example 11 (7.40-9.25 MBq, 0.1 mL) was administered, and in vivo static micro-PET / CT imaging was performed 60 minutes later. PET scanning time, anesthesia, CT scanning, and image reconstruction methods were the same as above. To eliminate thymus interference with aortic arch imaging, some mice (n=1) were immediately sacrificed after in vivo imaging, and the thymus was removed by thoracotomy. Micro-PET / CT static imaging was then performed using the same imaging methods as above.

[0176] The imaging results showed that 18 The uptake value of the F-labeled compound of Example 11 in the aortic arch region of plaque model mice (0.33±0.05) was significantly higher than that of wild-type mice (0.16). Imaging results indicate that the thymus is located near the aortic arch and has high uptake, which interferes with the delineation of the region of interest (VOI) in the aortic arch region. Therefore, in this study, mice were immediately anesthetized and sacrificed after 60 minutes of static imaging. The thymus was then removed by thoracotomy and whole-body imaging was performed to eliminate thymic interference. The results are shown in the attached figure. Figure 8 After thymus removal, significant radioactive uptake was still observed in the aortic arch of plaque model mice, indicating that 18 The F-labeled compound of Example 11 accumulated at the site of atherosclerotic plaques.

[0177] The above experimental results show that 18 F-labeled imidazopyridone σ2 receptor / TMEM97 ligand has high affinity, high subtype selectivity and high specificity for σ2 receptor / TMEM97. 18 F labeling has a high radiochemical yield and radiochemical purity, and has excellent in vitro and in vivo biological properties. In PET imaging of nude mice bearing CT26 tumors, 18 F-labeled imidazopyridone σ2 receptor / TMEM97 ligand can clearly perform PET imaging of CT26 tumors, with high specific tumor uptake and low lung uptake, resulting in a high tumor / lung ratio. As time goes by, the tumor uptake value increases while the lung uptake value decreases, making the PET imaging effect clearer. In PET imaging of CT26 lung compression model mice, 18 The F-labeled compound of Example 11 can clearly image lung tumors without interference from cardiac and pulmonary background, and [ 18 Compared with F]FDG, it has a greater advantage in imaging tumors around the lungs and heart.

[0178] therefore, 18F-labeled imidazopyridone σ2 receptor / TMEM97 ligand is an excellent specific CT26 tumor imaging agent, which is expected to be used for imaging studies of colorectal cancer lung metastasis and other tumors around the lungs and heart, providing a visualization tool for personalized cancer treatment in the future. 18 F-labeled imidazopyridone σ2 receptor / TMEM97 ligands have the potential to image atherosclerotic plaques. σ2 receptor / TMEM97 is a potential marker for atherosclerotic plaques. PET imaging targeting σ2 receptor / TMEM97 can be used to monitor the formation and progression of plaques and to evaluate the effectiveness of plaque treatment.

[0179] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A compound that binds to σ2 receptor / TMEM97, characterized in that It has the structure shown in general formula (I): Where: R is F or 18 F.

2. The method for preparing the compound according to claim 1, characterized in that When R in the general formula (I) is F or 18 When F, the corresponding compound is prepared by the following synthetic route: The preparation steps corresponding to the above synthetic route are as follows: (a) In tetrahydrofuran, compound 1 (2-amino-3-nitro-6-methoxypyridine) reacts with di-tert-butyl dicarbonate in the presence of 4-dimethylaminopyridine, followed by the addition of potassium carbonate and methanol to obtain compound 2; the reaction temperature is 55-65°C; (b) Compound 2 was reacted with 10% palladium on carbon in methanol under a hydrogen atmosphere to obtain compound 3; the reaction temperature was 50°C; (c) Compound 3 reacts with N,N'-carbonyldiimidazole in tetrahydrofuran in the presence of triethylamine to obtain compound 4; (d) Compound 4 reacts with 1,4-dibromobutane in the presence of potassium carbonate and tetrabutylammonium iodide in N,N-dimethylformamide to obtain compound 5; the reaction temperature is 60°C; (e) Compound 5 reacts with 5,6-dimethoxyisoindoline in the presence of potassium carbonate and triethylamine in acetonitrile to obtain compound 6; (f) Compound 6 reacts with trifluoroacetic acid in dichloromethane at room temperature to obtain compound 7; (g) Compound 7 was reacted with 1-bromo-2-fluoroethane in the presence of potassium carbonate and tetrabutylammonium iodide in N,N-dimethylformamide to obtain compound 8; the reaction temperature was 65°C; (h) Compound 7 reacts with (2-bromoethoxy)-tert-butyldimethylsilane in the presence of potassium carbonate and tetrabutylammonium iodide in N,N-dimethylformamide to obtain compound 9; the reaction temperature is 60°C; (i) Compound 9 reacts with 4 mol / L methanolic hydrochloric acid in methanol at room temperature to obtain compound 10; (j) Compound 10 reacts with p-toluenesulfonyl chloride in the presence of triethylamine and 4-dimethylaminopyridine at room temperature in dichloromethane to obtain compound 11; p-toluenesulfonyl chloride is added at 0°C; (k) Under sealed conditions, compound 11 and 4,7,13,16,21,24-hexaoxo-1,10-diazabicyclo[8.8.8]hexacosane (K 2.2.2 ) reacts under the action of potassium carbonate to obtain 18 F-labeled imidazopyridone σ2 receptor / TMEM97 compounds [ 18 F]8.

3. A molecular probe for binding to σ2 receptor / TMEM97, characterized in that Containing the general formula (I) of claim 1 wherein R is 18 F is the corresponding compound.

4. An imaging agent targeting σ2 receptor / TMEM97, characterized in that Containing the general formula (I) of claim 1 wherein R is 18 F is the corresponding compound.

5. Use of the molecular probe for binding to σ2 receptor / TMEM97 according to claim 3 in the preparation of a positron emission tomography imaging agent for diseases associated with σ2 receptor / TMEM97.

6. Use of the molecular probe for binding to σ2 receptor / TMEM97 according to claim 3 in the preparation of products for diagnosis, staging or efficacy evaluation of cancer patients.

7. Use of the imaging agent targeting σ2 receptor / TMEM97 according to claim 4 in the preparation of a positron emission tomography imaging agent for diseases associated with σ2 receptor / TMEM97.

8. Use of the imaging agent targeting σ2 receptor / TMEM97 according to claim 4 in the preparation of a product for diagnosis, staging or efficacy evaluation of cancer patients.

9. Use of the imaging agent targeting σ2 receptor / TMEM97 according to claim 4 in the preparation of a product for diagnosis, staging or efficacy evaluation of patients with atherosclerosis.

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