PET probe targeting phosphorylated AKT protein and its synthesis method and application

Abstract

The present invention relates to a PET probe targeting phosphorylated AKT protein, its synthesis method and application. The present invention has developed a novel PET probe for targeting and detecting phosphorylated AKT (p-AKT), the world's first PET probe precursor DOTA-Gly-pAKTi for targeting and detecting p-AKT, and a molecular probe <supgt;68< / supgt;Ga-DOTA-Gly-pAKTi labeled with the radionuclide <supgt;68< / supgt;Ga and applicable to PET detection. This PET probe has the specificity to target and bind to the phosphorylated AKT protein, and is used to detect the expression of the phosphorylated AKT protein in tumor tissues. The abnormal activation of phosphorylated AKT is an important mechanism driving tumorigenesis and development. AKT is often activated by PIK3CA mutations or highly phosphorylated and activated by the loss of the tumor suppressor gene PTEN, which activates downstream signaling pathways such as mTOR to promote tumorigenesis, growth and invasion. Therefore, this PET probe can provide an important and reliable molecular imaging tool for the detection of clinical tumor lesions, the detection of the p-AKT molecular phenotype in tumor tissues, and the prediction and early evaluation of the anti-tumor efficacy of various kinase inhibitors targeting the PI3K / AKT and downstream signaling pathways.

Description

Technical Field

[0001] The invention belongs to the field of probe synthesis technology and radioactive drug labeling, and in particular relates to a PET probe targeting phosphorylated AKT protein and a synthesis method and application thereof. Background Art

[0002] The PI3K-AKT signaling pathway is one of the most important cell signal transduction pathways that drive tumor occurrence and development. Various receptors, such as receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), cytokine receptors, and integrins, can trigger the PI3K / AKT pathway by recruiting phosphatidylinositol-4,5-bisphosphate 3-kinase and catalyzing the phosphorylation of PIP2 by the PI3Kα subunit, thereby promoting the generation of PIP3 from PIP2. PIP3 then activates the phosphorylation of AKT and initiates multiple signal transduction pathways such as downstream mTOR. In normal cells, the level of PIP3 is strictly regulated by PTEN, which converts PIP3 back to PIP2, thereby inhibiting the phosphorylation of AKT. However, in tumor cells, mutations in the PIK3CA gene encoding PI3Kα lead to abnormal and continuous phosphorylation of AKT, which in turn drives tumor occurrence and development. Moreover, PIK3CA mutations are one of the most common oncogenic mutations in a variety of malignant tumors, such as breast cancer, ovarian cancer, and brain glioma. Therefore, inhibitor drugs targeting PI3K, especially PI3Kα, have received great attention from the biopharmaceutical industry. In 2019, the FDA approved the first PI3Kα inhibitor for the treatment of hormone receptor-positive advanced breast cancer, and there are currently 835 clinical trials conducting clinical transformation of PI3K inhibitors. Inhibitor drugs targeting the PI3K pathway will have broader indications in tumor treatment.

[0003] However, multiple mechanisms can lead to tumor cell resistance to PI3Kα inhibitors. Among them, the loss of the tumor suppressor gene PTEN is the most common cause of resistance to PI3Kα inhibitors. PTEN loss leads to the obstruction of the conversion of PIP3 to PIP2, thereby continuously activating the phosphorylation of AKT at abnormally high levels, making the tumor resistant to PI3Kα inhibitors, and PTEN loss can promote tumor progression and metastasis. Therefore, abnormally high phosphorylated AKT in the early stage of treatment can be used as a target to identify tumor resistance to PI3Kα inhibitors.

[0004] Positron emission tomography (PET) is a highly sensitive and non-invasive examination technology that can detect molecular features in human tissues throughout the body. Because it is non-invasive, this examination technology can be used for real-time monitoring during the treatment of diseases such as tumors. PET molecular imaging targeting phosphorylated AKT can not only non-invasively detect systemic tumor lesions and detect the level of phosphorylated AKT in systemic tumors, but also effectively predict / monitor tumor resistance to PI3Kα inhibitor drugs at an early stage. Therefore, the preparation of a simple, stable, and highly specific PET probe for p-AKT can help clinicians predict / monitor the sensitivity of tumor patients to PI3K inhibitor drugs at an early stage, which is crucial for the selection of treatment strategies and efficacy evaluation of tumors. Summary of the invention

[0005] In order to further improve the specificity of the PET probe for p-AKT, the present invention provides a PET probe targeting phosphorylated AKT protein and a synthesis method and application thereof.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] In a first aspect, the present invention provides a small molecule precursor of a PET probe targeting phosphorylated AKT protein (abbreviated as p-AKT), which is a compound DOTA-Gly-pAKTi or a pharmaceutically acceptable salt thereof.

[0008]

[0009] In a second aspect, the present invention provides a method for preparing the compound DOTA-Gly-pAKTi.

[0010] A method for preparing a compound DOTA-Gly-pAKTi, wherein the compound DOTA-Gly-pAKTi is prepared from a compound represented by formula (3):

[0011]

[0012] In one embodiment of the present invention, in the preparation method of the compound DOTA-Gly-pAKTi, the compound represented by formula (3) is reacted in a first solvent in the presence of a first acid, the first solvent is removed under reduced pressure, and the compound DOTA-Gly-pAKTi is obtained by a first post-treatment.

[0013] In one embodiment of the present invention, the first acid may include hydrochloric acid.

[0014] In one embodiment of the present invention, the first solvent may include at least one of water or dioxane.

[0015] In one embodiment of the present invention, the first post-treatment may include pouring into diethyl ether, precipitating solids, and filtering to obtain the product.

[0016] In one embodiment of the present invention, the dosage of the compound of formula (3) and the first acid is in the following relationship: 0.018 mmol: 0.048 mmol. For example, the mass of the compound of formula (3) can be 20 mg, the molar number can be 0.018 mmol, the molar concentration of the first acid can be 6.0 M, and the volume can be 8 mL.

[0017] In one embodiment of the present invention, the reaction time of the reaction of the compound of formula (3) with the first acid may be 3 hours, and the reaction conditions may be room temperature.

[0018]

[0019] In one embodiment of the present invention, the compound of formula (3) is prepared from the compound of formula (2) and the compound DOTA-tris (t-Bu ester):

[0020] In one embodiment of the present invention, the compound of formula (2) is reacted with the compound DOTA-tris (t-Bu ester) in a second solvent in the presence of a second base and a second condensing agent, the second solvent is removed under reduced pressure, and a second post-treatment is performed to obtain a compound of formula (3).

[0021] In one embodiment of the present invention, the second base may include diisopropylethylamine.

[0022] In one embodiment of the present invention, the second condensing agent may include 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0023] In one embodiment of the present invention, the second solvent may include N,N-dimethylformamide.

[0024] In one embodiment of the present invention, the second post-treatment may include extraction, drying, filtration, concentration under reduced pressure, and purification by column chromatography to obtain the product.

[0025] In one embodiment of the present invention, the molar ratio of the compound of formula (2) to the compound DOTA-tris (t-Bu ester) can be 1.3:1.

[0026] In one embodiment of the present invention, the molar ratio of the second base to the compound of formula (2) may be 2.6:1.

[0027] In one embodiment of the present invention, the molar ratio of the second condensing agent to the compound of formula (2) may be 1.1:1.

[0028] In one embodiment of the present invention, the reaction time of the compound of formula (2) and the compound DOTA-tris (t-Bu ester) can be 8-12 hours, and the reaction conditions can be room temperature.

[0029] In one embodiment of the present invention, the compound of formula (2) is prepared from a compound of formula (1):

[0030]

[0031] In one embodiment of the present invention, the compound of formula (1) is reacted in a third solvent in the presence of a third acid, the third solvent is removed under reduced pressure, and the compound of formula (2) is obtained by a third post-treatment.

[0032] In one embodiment of the present invention, the third acid may include hydrochloric acid.

[0033] In one embodiment of the present invention, the third solvent may include a dioxane hydrochloride solution.

[0034] In one embodiment of the present invention, the third post-treatment may include pouring into ether, precipitating solids, filtering to obtain the product

[0035] In one embodiment of the present invention, the molar ratio of the compound of formula (1) to the third acid is 0.13mmol:0.032mmol. For example, the mass of the compound of formula (1) can be selected to be 80mg, the molar number can be 0.13mmol, the molar concentration of the third acid can be 4.0M, and the volume can be 8mL.

[0036] In one embodiment of the present invention, the reaction time of the compound of formula (1) and the third acid may be 2.5 hours, and the reaction conditions may be room temperature.

[0037] In one embodiment of the present invention, the compound of formula (1) is prepared from the compound GDC-0068 and the compound Boc-Glycine:

[0038]

[0039] In one embodiment of the present invention, compound GDC-0068 reacts with compound Boc-Glycine in the presence of a fourth base and a fourth condensing agent in a second solvent, the fourth solvent is removed under reduced pressure, and a fourth post-treatment is performed to obtain compound (1).

[0040] In one embodiment of the present invention, the fourth base may include diisopropylethylamine.

[0041] In one embodiment of the present invention, the fourth condensing agent may include 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0042] In one embodiment of the present invention, the fourth solvent may include N,N-dimethylformamide.

[0043] In one embodiment of the present invention, the fourth post-treatment may include extraction, drying, filtration, concentration under reduced pressure, and purification by column chromatography to obtain the product.

[0044] In one embodiment of the present invention, the molar ratio of the compound GDC-0068 to the compound Boc-Glycine is 1.2:1.

[0045] In one embodiment of the present invention, the molar ratio of the compound GDC-0068 to the fourth base may be 2.6:1.

[0046] In one embodiment of the present invention, the molar ratio of the compound GDC-0068 to the fourth condensing agent may be 1.1:1.

[0047] In one embodiment of the present invention, the reaction time of the compound GDC-0068 and the compound Boc-Glycine can be 8-12 hours.

[0048] In a third aspect, the present invention provides the use of the compound DOTA-Gly-pAKTi or a pharmaceutically acceptable salt thereof in preparing a product for detecting a disease associated with p-AKT.

[0049] In one embodiment of the present invention, the disease associated with p-AKT may include tumors.

[0050] In one embodiment of the present invention, the tumor is a tumor type in which the PI3K / AKT signaling pathway is abnormally activated or PTEN is mutated and lost, for example, it may include breast cancer, prostate cancer, etc.

[0051] In one embodiment of the invention, the product comprises a diagnostic tracer.

[0052] The compound DOTA-Gly-pAKTi or a pharmaceutically acceptable salt thereof provided by the present invention can be used in the preparation of products for detecting diseases associated with p-AKT, specifically, can be used as a drug for clinical / preclinical diagnosis and / or efficacy evaluation, can be used for the study of methods such as radiochemical labeling and preclinical animal imaging, and can be used in products for diseases associated with p-AKT.

[0053] The PET probe small molecule precursor targeting phosphorylated AKT (p-AKT) protein obtained by the present invention, DOTA-Gly-pAKTi, can be further modified, and the modification method includes but is not limited to replacing the radioactive labeled nuclide and conventional modification of DOTA-Gly-pAKTi.

[0054] In a fourth aspect, the present invention provides a 68 The precursor DOTA-Gly-pAKTi was radiolabeled with a Ga-labeled tracer.

[0055] A sort of 68 The Ga-labeled tracer radioactive labeling method comprises the following steps:

[0056] 1) 0.1M HCl was used to elute the germanium-gallium generator to obtain 68 GaCl 3 Solution;

[0057] 2) Adjust with 0.5M NaAC 68 GaCl 3 The pH value of the solution is 2.3-3.5 to form a labeling system;

[0058] 3) Add the precursor solution to the labeling system and react at 100°C for 10 minutes to obtain 68 Ga-labeled tracer;

[0059] The precursor is the compound DOTA-Gly-pAKTi or a pharmaceutically acceptable salt thereof, 68 The Ga-labeled tracer is a compound 68 Ga-DOTA-Gly-pAKTi molecular probe.

[0060] In one embodiment of the present invention, the concentration of the precursor solution is 1 mg / mL.

[0061] In one embodiment of the present invention, the solvent in the precursor solution may be pure water.

[0062] In a fifth aspect, the present invention provides a 68 Ga-labeled tracer radiolabeling method for PET detection 68Ga-DOTA-Gly-pAKTi molecular probe. 68 The Ga-DOTA-Gly-pAKTi molecular probe is a 68 Ga-labeled radioactive PET probe that can be used for PET detection. 68 The structure of the Ga-DOTA-Gly-pAKTi molecular probe is as follows:

[0063]

[0064] The application of the above-mentioned Ga-DOTA-Gly-pAKTi molecular probe that can be used for PET detection 68 is selected from one of the following applications:

[0065] The application of the molecular probe in the preparation of drugs for clinical / clinical pre-diagnosis and / or efficacy evaluation;

[0066] The application of the molecular probe in the preparation of radiochemically labeled products;

[0067] The application of the molecular probe in the study of pre-clinical animal imaging methods;

[0068] The application of the molecular probe in the preparation of products for diagnosing / treating p-AKT-related diseases.

[0069] The small molecule precursor DOTA-Gly-pAKTi of the PET probe or the molecular probe can be further modified. The modification methods include but are not limited to replacing the radioactive labeling nuclide and the conventional modification of DOTA-Gly-pAKTi.

[0070] The compound DOTA-Gly-pAKTi provided by the present invention or its pharmaceutically acceptable salt is simple to prepare, has a short preparation time, a high radiochemical yield, good specificity, a high uptake amount, and has good stability both in physiological saline and serum, and can detect the expression level of p-AKT in tumors at the cellular level and in breast cancer-bearing mice.

[0071] In addition, the 68 Ga-labeling method of the radioactive labeled DOTA-Gly-pAKTi provided by the present invention produces a Ga-DOTA-Gly-pAKTi molecular probe that can be used for PET detection. 68 The labeling method is simple to operate, has a short preparation time, and a high yield.

[0072] The PET probe provided by the present invention has specificity for specifically binding to phosphorylated AKT protein, and is used to detect the expression of phosphorylated AKT protein in tumor tissues. The abnormal activation of phosphorylated AKT is an important mechanism driving tumorigenesis and development. AKT is often activated by PIK3CA mutations or highly phosphorylated and activated by the loss of tumor suppressor gene PTEN, and activates downstream signaling pathways such as mTOR to promote tumorigenesis, growth and invasion. Therefore, this PET probe can provide an important and reliable molecular imaging tool for the detection of clinical tumor lesions, the detection of p-AKT molecular phenotypes in tumor tissues, and the prediction and early evaluation of the anti-tumor efficacy of various kinase inhibitors targeting the PI3K / AKT and downstream signaling pathways.

[0073] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0074] (1) 68 Ga-DOTA-Gly-pAKTi provided by the present invention has high specificity for p-AKT.

[0075] (2) 68 Ga-DOTA-Gly-pAKTi provided by the present invention has good stability.

[0076] (3) Tumors resistant to PI3Kα inhibitors 68 have high uptake of Ga-DOTA-Gly-pAKTi.

[0077] (4) 68 PET / CT imaging using the Ga-DOTA-Gly-pAKTi probe can effectively reflect the drug resistance of tumors to PI3Kα inhibitors and the molecular characteristics of PTEN deficiency in tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Shows the mass spectrum of the compound DOTA-Gly-pAKTi prepared in Example 1.

[0079] Figure 2 Shows the HPLC analysis of DOTA-Gly-pAKTi and 68 Ga-DOTA-Gly-pAKTi in Example 2.

[0080] Figure 3 Shows the 68 stability of Ga-DOTA-Gly-pAKTi in mouse serum and physiological saline in Example 3.

[0081] Figure 4 Shows the 68 blood clearance rate of Ga-DOTA-Gly-pAKTi in mice in Example 4.

[0082] Figure 5 The results show that the p-AKT high expression and low expression cells in Example 5 68 The uptake results of Ga-DOTA-Gly-pAKTi; the left picture is 68 The uptake values ​​of Ga-DOTA-Gly-pAKTi in HCC-1806, HCC-1937, SUM-159, MDA-MB-231, MDA-MB-468, and BT-549 cells after incubation for 120 minutes. HCC-1806, HCC-1937, and SUM-159 are triple-negative breast cancer cell lines with low p-AKT expression, and MDA-MB-231, MDA-MB-468, and BT-549 are triple-negative breast cancer cell lines with high p-AKT expression; the right figure shows the uptake values ​​of Ga-DOTA-Gly-pAKTi in HCC-1806, HCC-1937, and SUM-159 are triple-negative breast cancer cell lines with low p-AKT expression, and MDA-MB-231, MDA-MB-468, and BT-549 are triple-negative breast cancer cell lines with high p-AKT expression; 68 The radioactivity retention percentage of MDA-MB-231, MDA-MB-468, and BT-549 cells after incubation in Ga-DOTA-Gly-pAKTi for 120 minutes and then incubation in non-radioactive medium for 90 minutes indicated that 68 Ga-DOTA-Gly-pAKTi can be quickly taken up by tumor cells. 68 The uptake of Ga-DOTA-Gly-pAKTi was positively correlated with its expressed p-AKT. 68 After being taken up by tumor cells, Ga-DOTA-Gly-pAKTi is metabolized by the cells within a certain period of time and has the characteristics of being a PET imaging probe.

[0083] Figure 6 The p-AKT inhibitor GDC-0068 and 68 Competitive inhibition binding assays were performed on Ga-DOTA-Gly-pAKTi in MDA-MB-231, MDA-MB-468, and BT-549 cells. The IC of GDC-0068 was 50 The values ​​are 0.6nM, 0.039nM and 0.038nM respectively.

[0084] Figure 7 Figure 7 shows the PET / CT imaging of the tumor-bearing animal model in Example 7; wherein Figure A is 68 PET imaging of Ga-DOTA-Gly-pAKTi in HCC-1806, HCC-1937, MDA-MB-231, and MDA-MB-468 tumor models, of which HCC-1806 and HCC-1937 are tumors with low p-AKT expression, and MDA-MB-231 and MDA-MB-468 are tumors with high p-AKT expression; Figure B shows the tumor68 The uptake value of Ga-DOTA-Gly-pAKTi was higher in tumors with high p-AKT. 68 Ga-DOTA-Gly-pAKTi radioactivity uptake was only very low in tumors with low p-AKT. 68 Ga-DOTA-Gly-pAKTi radioactive uptake. 68 Ga-DOTA-Gly-pAKTi PET probe has good sensitivity and specificity in detecting pAKT in living tumors.

[0085] Figure 8 As shown in Example 8 68 Biodistribution of Ga-DOTA-Gly-pAKTi in HCC-1806, HCC-1937, MDA-MB-231, and MDA-MB-468 tumor-bearing mouse models for 1.5 h. Compared with most normal tissues and PTEN-normal tumors (low p-AKT), 68 The Ga-DOTA-Gly-pAKTi probe has a significantly higher biodistribution in PTEN-deficient tumors (high p-AKT). This result further suggests 68 The Ga-DOTA-Gly-pAKTi PET probe was specific for detecting tumor p-AKT levels in vivo, and suggested that there would be higher background uptake in liver and kidney tissues.

[0086] Fig. 9 Figure 9 shows the PET / CT imaging of the PTEN-deficient and PTEN-normal control tumor-bearing model animals; wherein Figure A is 68 PET imaging of Ga-DOTA-Gly-pAKTi in HCC-1806PTEN WT, HCC-1806PTEN KO, HCC-1937PTEN WT, and HCC-1937PTEN KO tumor models; Figure B is a Western Blot experiment to detect the expression of PTEN and p-AKT in tumor tissues. The results show that after PTEN knockout, tumor cells lack PTEN, and p-AKT expression is not inhibited and significantly increased. Tumors with high p-AKT expression 68 The uptake of Ga-DOTA-Gly-pAKTi was increased. DETAILED DESCRIPTION

[0087] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0088] Example 1: Preparation of compound DOTA-Gly-pAKTi

[0089]

[0090] 1) Preparation of the compound of formula (1)

[0091] Synthesis of the compound of formula (1): 10 mL of DMF was added to a 50 mL eggplant-shaped bottle, and the compounds Boc-Glycine (2, 36.72 mg, 0.21 mmol, 1.2 eq), HATU (73.06 mg, 0.19 mmol, 1.1 eq) and DIPEA (58.70 mg, 0.45 mmol, 2.6 eq) were added in sequence. After stirring at room temperature for 30 min, the compound GDC-0068 (1, 80 mg, 0.17 mmol, 1 eq) was added, and the reaction system was stirred at room temperature overnight. After the reaction was completed, NaHCO was added to the reaction solution. 3 The saturated solution (50 mL) was stirred for 15 min, then ethyl acetate (50 mL) was added and extracted three times, the organic phases were combined, and anhydrous Na 2 SO 4 The residue was dried, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain solid powder product 3 (65.06 mg, 60.55%).

[0092] 1H NMR(400MHz,Chloroform-d)δ:8.43(s,1H),7.27(d,J=8.4Hz,2H),7.15(d,J=8.0Hz,2H),5.55(d,J=4 .7Hz,1H),5.09(t,J=7.0Hz,1H),4.68(dd,J=8.9,5.2Hz,1H),3.93(d,J=4.8Hz,2H),3.77(td,J=16.4 ,15.5,6.0Hz,3H),3.70–3.52(m,3H),3.43(dt,J=16.2,8.4Hz,4H),3.35(d,J=7.2Hz,1H),3.23(td,J =8.4,3.7Hz,1H),2.22–2.04(m,2H),1.44(s,9H),1.08(dd,J=13.7,6.7Hz,6H),0.43(d,J=6.5Hz,3H).

[0093] 2) Preparation of the compound of formula (2)

[0094] Compound 1 (80 mg, 0.13 mmol) was added to a 50 mL eggplant-shaped bottle and dissolved in 8 mL of hydrogen chloride dioxane solution (4.0 M). The reaction was stirred at room temperature for 2.5 h. After the reaction was completed, the reaction system was concentrated under reduced pressure, ether was added for washing, and the remaining HCl was removed by ultrasonic filtration to obtain a solid powder product 4 (62.26 mg, 86.81%).

[0095] 3) Preparation of the compound of formula (3)

[0096] In a 50 mL eggplant-shaped bottle, 10 mL of DMF was added, followed by the addition of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (5, 67.63 mg, 0.12 mmol, 1.3 eq), HATU (37.99 mg, 0.010 mmol, 1.1 eq) and DIPEA (37.99 mg, 0.24 mmol, 2.6 eq), and the mixture was stirred at room temperature for 30 min before adding compound 2 (50 mg, 0.090 mmol, 1 eq). The reaction system was stirred at room temperature overnight. After the reaction was completed, NaHCO 3 The saturated solution (50 mL) was stirred for 15 min, then ethyl acetate (50 mL) was added and extracted three times, the organic phases were combined, and anhydrous Na 2 SO 4 The residue was dried, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain solid powder product 3 (35.18 mg, 36.21%).

[0097] 4) Preparation of compound DOTA-Gly-pAKTi

[0098] Compound 3 (20 mg, 0.018 mmol) was added to a 50 mL eggplant-shaped bottle and dissolved in 8 mL of 6.0 M hydrochloric acid solution. The reaction was stirred at room temperature for 3 h. After the reaction was completed, the reaction system was concentrated under reduced pressure, ether was added for washing, and the remaining HCl was removed by ultrasonic filtration to obtain a solid powder product DOTA-Gly-pAKTi (14.25 mg, 84.57%).

[0099] The mass spectrum of the compound DOTA-Gly-pAKTi is as follows Figure 1 shown.

[0100] MS-ESI(+)calcd for C 42 H 61 C l N 10 O 10 :900.4261,[M+Na-H]+found:922.8,[M+HCl+Na]+found:959.7.

[0101] Example 2: Preparation of tracer

[0102] The preparation of the tracer using the compound DOTA-Gly-pAKTi as a precursor comprises the following steps:

[0103] 1) 0.1M HCl was used to elute the germanium-gallium generator to obtain 68 GaCl 3 Solution;

[0104] 2) Adjust with 0.5M NaAC 68 GaCl 3 The pH value of the solution is 2.3-3.5 to form a labeling system;

[0105] 3) Add the precursor solution to the labeling system and react at 100°C for 10 minutes to obtain 68 Ga-labeled tracer.

[0106] DOTA-Gly-pAKTi and 68 The results of Ga-DOTA-Gly-pAKTi are as follows Figure 2 shown.

[0107] Results: From the elution 68 GaCl 3 The solution was started and the tracer was successfully prepared within 20 minutes. 68 The radiochemical purity of Ga-DOTA-Gly-pAKTi reaches more than 95%, and the retention time of the Ga-DOTA-Gly-pAKTi in HPLC is consistent with that of the standard product.

[0108] Example 3: Stability test

[0109] Blood was collected from Balb / c mice and serum was prepared for later use. Several EP tubes were taken and 200 μL of serum or 200 μL of saline was added to each tube. 20 μL of freshly prepared 68 Ga-DOTA-Gly-pAKTi was incubated for different time periods (0 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, and 3 h) in a 37 °C water bath and at room temperature. After the incubation period, 200 μL of methanol was added to the serum tube, and the supernatant was centrifuged at room temperature (1000 rpm, 5 min) and the radiochemical purity was determined by iTLC. The mixed solution in the saline tube was directly determined by iTLC for radiochemical purity after the incubation period.

[0110] 68 The stability of Ga-DOTA-Gly-pAKTi in mouse serum and saline Figure 3 shown.

[0111] Results: Tracer 68 Ga-DOTA-Gly-pAKTi remained undecomposed in the prototype after 3 hours in vitro at 37°C in mouse serum; and nearly 95% remained undecomposed in the prototype after 3 hours in vitro at room temperature in physiological saline. 68 Ga-DOTA-Gly-pAKTi has good stability in both serum and saline.

[0112] Example 4: Blood clearance experiment

[0113] Prepare several Balb / c mice and inject 1.7 MBq into each mouse through the tail vein. 68 Ga-DOTA-Gly-pAKTi solution; puncture the contralateral tail vein and collect blood using a capillary tube. Collect blood at 1.0min, 2.0min, 4.0min, 10.0min, 20.0min, 40.0min, 60.0min, 90.0min, 120.0min, 180.0min, and 240.0min after injection. Use a γ counter to measure the radioactivity count of the collected blood. Five parallels are used at each time point. The processed γ counter measurement results are fitted with a two-phase decay using GraphPad Prism software. The fitting formula is:

[0114] Y=Plateau+SpanFast*exp(-KFast*X)+SpanSlow*exp(-KSlow*X)

[0115] 68 Blood clearance rate of Ga-DOTA-Gly-pAKTi in mice Figure 4 shown.

[0116] Results: The biphasic decay analysis revealed 68 The half-life t1 / 2 of the distribution phase of Ga-DOTA-Gly-pAKTi in mice is 2.0min, and the half-life t1 / 2 of the blood elimination phase is 56.2min.

[0117] Example 5: Cellular uptake and efflux experiments

[0118] Preliminary experiments have shown that triple-negative breast cancer HCC-1806, HCC-1937, and SUM-159 cell lines have low p-AKT expression, while triple-negative breast cancer MDA-MB-231, MDA-MB-468, and BT-549 cell lines have high p-AKT expression. HCC-1806 cells, HCC-1937 cells, SUM-159 cells, MDA-MB-231 cells, MDA-MB-468 cells, and BT-549 cells were plated in 24-well plates (1*10 5 / hole), after adherence, the tumor cells were 68 Ga-DOTA-Gly-pAKTi (74 kBq / well) was co-incubated at 37°C for 15, 30, 45, 60, 90 and 120 minutes.

[0119] After incubation, the tumor cells were gently washed three times with refrigerated phosphate buffer and detached from the 24-well plate with 0.25% trypsin / 0.02% EDTA. Observe under an optical microscope to ensure that the cells are completely separated and collected. The radioactivity of the collected cell suspension was detected using a γ counter. The radioactivity in the cell suspension was corrected for attenuation and calculated as a percentage of the total radioactivity. The experiment was repeated three times.

[0120] MDA-MB-231 cells, MDA-MB-468 cells, and BT-549 cells with high p-AKT expression were plated in 24-well plates (1*10 5 / hole), after adherence, the tumor cells were 68 Ga-DOTA-Gly-pAKTi (74 kBq / well) was incubated at 37°C for 120 minutes. The radioactive medium was removed, and non-radioactive medium was added for incubation for 0, 15, 30, 45, 60 and 90 minutes. The culture medium was removed, and the cells were washed twice with cold PBS solution, digested with trypsin, and the radioactivity was detected with a γ counter.

[0121] p-AKT high expression and low expression of cells 68 The uptake results of Ga-DOTA-Gly-pAKTi are as follows Figure 5 As shown; the left picture is 68 The uptake values ​​of Ga-DOTA-Gly-pAKTi in HCC-1806, HCC-1937, SUM-159, MDA-MB-231, MDA-MB-468, and BT-549 cells after incubation for 120 minutes. HCC-1806, HCC-1937, and SUM-159 are triple-negative breast cancer cell lines with low p-AKT expression, and MDA-MB-231, MDA-MB-468, and BT-549 are triple-negative breast cancer cell lines with high p-AKT expression; the right figure shows the uptake values ​​of Ga-DOTA-Gly-pAKTi in HCC-1806, HCC-1937, and SUM-159 are triple-negative breast cancer cell lines with low p-AKT expression, and MDA-MB-231, MDA-MB-468, and BT-549 are triple-negative breast cancer cell lines with high p-AKT expression; 68 The radioactivity retention percentage of MDA-MB-231, MDA-MB-468, and BT-549 cells after incubation in Ga-DOTA-Gly-pAKTi for 120 minutes and then incubation in non-radioactive medium for 90 minutes indicated that 68 Ga-DOTA-Gly-pAKTi can be quickly taken up by tumor cells. 68 The uptake of Ga-DOTA-Gly-pAKTi was positively correlated with its expressed p-AKT. 68 After being taken up by tumor cells, Ga-DOTA-Gly-pAKTi is metabolized by the cells within a certain period of time and has the characteristics of being a PET imaging probe.

[0122] Results: The triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-468, and BT-549 with high p-AKT expression 68 The uptake of Ga-DOTA-Gly-pAKTi was high, and the uptake values ​​at 2h were 1.15%±0.56, 2.02%±0.26, and 1.97%±0.35, respectively; the triple-negative breast cancer cell lines HCC-1806, HCC-1937, and SUM-159 with low p-AKT expression had a significant effect on 68 The 2h uptake values ​​of Ga-DOTA-Gly-pAKTi reached 0.37%±0.09, 0.47%±0.1, and 0.34%±0.12, respectively.

[0123] For the discharge experiment, 68 Ga-DOTA-Gly-pAKTi remained in MDA-MB-231, MDA-MB-468, and BT-549 cells for more than 90 minutes.

[0124] Example 6: Cell binding assay

[0125] Triple negative breast cancer cells MDA-MB-231, MDA-MB-468, and BT-549 were seeded in 24-well plates (1*10 5 / well) overnight. The cells were gently washed twice with cold PBS and then 68 Ga-DOTA-Gly-pAKTi and different concentrations of GDC-0068 were co-incubated for 1 hour. After washing the cells three times with cold PBS, the cells were detached with trypsin, the cell suspension was collected, and the radioactivity of the cells was detected using a γ counter. The data were fitted with nonlinear regression using the computer software GraphPad Prism to calculate the optimal 50% inhibition concentration (IC 50 ). The experiment was repeated three times.

[0126] Using p-AKT inhibitor GDC-0068 and 68 The results of competitive inhibition binding assay of Ga-DOTA-Gly-pAKTi in MDA-MB-231, MDA-MB-468, and BT-549 cells are shown in Figure 6 shown.

[0127] Results: In MDA-MB-468, BT-549, and MDA-MB-231 cells, GDC-0068 blocked 68 IC of Ga-DOTA-Gly-pAKTi uptake 50 The values ​​are 0.6nM, 0.039nM and 0.038nM respectively.

[0128] Example 7: PET imaging experiment in tumor-bearing animal model

[0129] Construct HCC-1806, HCC-1937, MDA-MB-231, and MDA-MB-468 tumor models and wait until the tumor grows to 1 cm 3 The labeled probe was injected into the tumor-bearing mice via the tail vein, with each mouse injected with 7.4 MBq. PET / CT imaging was performed 1 hour after the probe was injected via the tail vein, and the uptake of the probe by each tumor tissue was analyzed using Inveon Research Workplace image analysis software.

[0130] PET / CT imaging of tumor-bearing animals Figure 7 As shown; Figure A is 68 PET imaging of Ga-DOTA-Gly-pAKTi in HCC-1806, HCC-1937, MDA-MB-231, and MDA-MB-468 tumor models, of which HCC-1806 and HCC-1937 are tumors with low p-AKT expression, and MDA-MB-231 and MDA-MB-468 are tumors with high p-AKT expression; Figure B shows the tumor 68 Uptake values ​​of Ga-DOTA-Gly-pAKTi.

[0131] Results: The tumor-bearing models constructed by MDA-MB-231 and MDA-MB-468 cell lines with high expression of p-AKT 68 Ga-DOTA-Gly-pAKTi high uptake, 68 After intravenous injection, Ga-DOTA-Gly-pAKTi specifically aggregated in tumor tissues, clearly showing tumor lesions; the tumor-bearing models constructed by HCC-1806 and HCC-1937 cell lines with low p-AKT expression 68 Ga-DOTA-Gly-pAKTi had low uptake.

[0132] Example 8: In vivo biodistribution experiment

[0133] 1.7 MBq was injected into tumor-bearing nude mice via the tail vein 68 1.5 hours after the injection of Ga-DOTA-Gly-pAKTi solution, the nude mice were killed and dissected to obtain the heart, liver, spleen, lungs, kidneys, muscles, bones, brain, stomach, intestines, blood, tumors and other tissues and organs. These tissues and organs were weighed and the radioactivity was measured with a γ counter to calculate the radioactivity per gram of tissue. The uptake value in the tissues and organs was expressed as the percentage of the radioactive dose taken by each gram of tissue to the injected dose (%ID / g).

[0134] 68The biodistribution diagram of Ga-DOTA-Gly-pAKTi in HCC-1806, HCC-1937, MDA-MB-231, and MDA-MB-468 tumor mouse models for 1.5 h is shown in the figure. Figure 8 Compared with most normal tissues and tumors with normal PTEN (low p-AKT), 68 The Ga-DOTA-Gly-pAKTi probe had significantly higher biodistribution in PTEN-deficient tumors (high p-AKT).

[0135] result: 68 Ga-DOTA-Gly-pAKTi was highly taken up in MDA-MB-231 and MDA-MB-468 tumor tissues with high p-AKT expression, and was lowly taken up in HCC-1806 and HCC-1937 tumor tissues with low p-AKT expression. 68 Ga-DOTA-Gly-pAKTi had low uptake. 68 Ga-DOTA-Gly-pAKTi is mainly metabolized by the kidneys and liver. This result further suggests 68 The Ga-DOTA-Gly-pAKTi PET probe is specific for detecting tumor phosphorylated AKT levels in vivo, and indicates high background uptake in liver and kidney tissues.

[0136] Example 9: PET imaging experiment in tumor-bearing animal models with normal PTEN and PTEN loss

[0137] Construct HCC-1806PTEN WT, HCC-1806PTEN KO, HCC-1937PTEN WT, and HCC-1937PTEN KO tumor models and wait until the tumor grows to 1 cm 3 The labeled probe was injected into the tumor-bearing mice via the tail vein, with each mouse injected with 7.4 MBq. PET / CT imaging was performed 1 hour after the probe was injected via the tail vein, and the uptake of the probe by each tumor tissue was analyzed using Inveon Research Workplace image analysis software.

[0138] PET / CT imaging of PTEN-deficient and PTEN-normal control tumor burden model animals Fig. 9 As shown, Figure A is 68 PET imaging of Ga-DOTA-Gly-pAKTi in HCC-1806PTEN WT, HCC-1806PTEN KO, HCC-1937PTEN WT, and HCC-1937PTEN KO tumor models; Figure B is a Western Blot experiment to detect the expression of PTEN and p-AKT in tumor tissues.

[0139] Results: The tumor-bearing models constructed by PTEN-deficient tumor cells HCC-1806PTEN KO and HCC-1937PTEN KO showed no significant difference in tumor growth and development. 68 Ga-DOTA-Gly-pAKTi was highly absorbed, and the expression level of p-AKT in tumor tissues was significantly increased as verified by Western Blot. The tumor-bearing models constructed by HCC-1806PTEN WT and HCC-1937PTEN WT tumor cells with normal PTEN showed no significant difference in expression level of p-AKT. 68 Ga-DOTA-Gly-pAKTi has low uptake and low p-AKT expression in tumor tissues. This result proves that after PTEN knockout, tumor cells lack PTEN and p-AKT expression is not inhibited but significantly increased. 68 The uptake of Ga-DOTA-Gly-pAKTi was increased.

[0140] The following conclusions can be drawn from Examples 3 to 9:

[0141] (1) Stability test shows 68 Ga-DOTA-Gly-pAKTi has good stability in both serum and saline.

[0142] (2) Blood clearance experiments showed 68 After intravenous injection, Ga-DOTA-Gly-pAKTi can be rapidly distributed throughout the body's tissues and organs. At the same time, it has a short half-life in the blood, making it suitable as an imaging probe.

[0143] (3) Cell uptake experiments showed that cells 68 The uptake of Ga-DOTA-Gly-pAKTi was correlated with the p-AKT expressed by the cells.

[0144] (4) Cell binding experiments showed 68 Ga-DOTA-Gly-pAKTi has good specificity for p-AKT.

[0145] (5) The uptake of the probe by tumor tissue is correlated with the level of p-AKT expressed by it.

[0146] (6) In summary, 68 Ga-DOTA-Gly-pAKTi can be used as an imaging probe to image p-AKT in tumors.

[0147] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A small molecule precursor of a PET probe targeting phosphorylated AKT protein, which is the compound DOTA-Gly-pAKTi or a pharmaceutically acceptable salt thereof 2. Preparation method of compound DOTA-Gly-pAKTi, It is characterized in that The compound represented by formula (3) is reacted in a first solvent in the presence of a first acid, the first solvent is removed under reduced pressure, and a first post-treatment is performed to obtain a compound DOTA-Gly-pAKTi; The first acid includes hydrochloric acid, the first solvent includes at least one of water or dioxane, and the first post-treatment includes: pouring into ether, precipitating solids, and filtering to obtain a product; The compound represented by formula (3) is as follows: The structure of the compound DOTA-Gly-pAKTi is as follows:

3. A method for preparing the compound DOTA-Gly-pAKTi according to claim 2, It is characterized in that The compound of formula (3) is prepared from the compound of formula (2) and DOTA-tris (t-Bu ester), wherein the structures of the compound of formula (2) and DOTA-tris (t-Bu ester) are as follows: The compound of formula (2) is reacted with DOTA-tris (t-Buester) in a second solvent in the presence of a second base and a second condensing agent, the second solvent is removed under reduced pressure, and a second post-treatment is performed to obtain a compound of formula (3); The second base includes diisopropylethylamine; the second condensing agent includes 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; the second solvent includes N,N-dimethylformamide; the second post-treatment includes: extraction, drying, filtration, concentration under reduced pressure, and purification by column chromatography to obtain the product.

4. A method for preparing the compound DOTA-Gly-pAKTi according to claim 3, It is characterized in that The compound of formula (2) is prepared from the compound of formula (1), wherein the compound of formula (1) has the following structure: The compound of formula (1) reacts in a third solvent in the presence of a third acid, removes the third solvent under reduced pressure, and undergoes a third post-treatment to obtain a compound of formula (2); The third acid includes hydrochloric acid; the third solvent includes a hydrochloric acid dioxane solution; The third post-treatment comprises: pouring into ether, precipitating solid, and filtering to obtain the product.

5. A method for preparing the compound DOTA-Gly-pAKTi according to claim 4, It is characterized in that The compound of formula (1) is prepared from compound GDC-0068 and compound Boc-Glycine, wherein the structures of compound GDC-0068 and compound Boc-Glycine are as follows: The compound GDC-0068 reacts with the compound Boc-Glycine in a fourth solvent in the presence of a fourth base and a fourth condensing agent, and the fourth solvent is removed under reduced pressure, and a fourth post-treatment is performed to obtain a compound of formula (1); The fourth base includes diisopropylethylamine; the fourth condensing agent includes 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; the fourth solvent includes N,N-dimethylformamide; the fourth post-treatment includes: extraction, drying, filtration, concentration under reduced pressure, and purification by column chromatography to obtain the product.

6. A 68 Ga-labeled tracer radiolabeling method, It is characterized in that The following steps are involved: 1) 0.1M HCl was used to elute the germanium-gallium generator to obtain 68 GaCl 3 Solution; 2) Adjust with 0.5M NaAC 68 GaCl 3 The pH value of the solution is 2.3-3.5 to form a labeling system; 3) Add the precursor solution to the labeling system and react at 100°C for 10 minutes to obtain 68 Ga-labeled tracer; The precursor is the compound DOTA-Gly-pAKTi or a pharmaceutically acceptable salt thereof as claimed in claim 1.

7. Based on claim 6 68 Ga-labeled tracer radiolabeling method for PET detection 68 Ga-DOTA-Gly-pAKTi molecular probe, 68 The structure of Ga-DOTA-Gly-pAKTi molecular probe is as follows:

8. The method for PET detection according to claim 7. 68 Application of Ga-DOTA-Gly-pAKTi molecular probe, It is characterized in that Select one of the following applications: Use of the molecular probe in the preparation of drugs for clinical / preclinical diagnosis and / or efficacy evaluation of diseases associated with p-AKT; Use of the molecular probe in preparing radiochemically labeled products; The molecular probe is used in preparing products for diagnosing diseases associated with p-AKT.

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