CN116333035A - PET probe for targeting phosphorylated AKT protein, and synthetic method and application thereof - Google Patents

Abstract

The invention relates to a PET probe targeting phosphorylated AKT protein, and a synthesis method and application thereof. The invention develops a novel PET probe for targeted detection of phosphorylated AKT (p-AKT), which is a precursor DOTA-Gly-pAKTi of the PET probe for targeted detection of p-AKT for the first time in the world, and radionuclides ⁶⁸ Ga-marked molecular probe for PET detection ⁶⁸ Ga-DOTA-Gly-pAKTi. The PET probe has specificity of targeting combination of the AKT protein modified by phosphorylation, and is used for detecting the expression of the AKT protein modified by phosphorylation in tumor tissues. Aberrant activation of phosphorylated AKT is responsible for tumor developmentImportant mechanisms of growth and development, AKT is often activated by PIK3CA mutation or loss of the cancer suppressor gene PTEN is highly phosphorylated and activates signaling pathways such as downstream mTOR to promote tumor development and growth infiltration. Therefore, the PET probe can provide an important and reliable molecular imaging tool for detecting clinical tumor focus, detecting p-AKT molecular phenotype of tumor tissues, and predicting and early evaluating anti-tumor curative effects of various kinase inhibitors targeting PI3K/AKT and downstream signal paths.

Description

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

technical field

The invention belongs to the technical field of probe synthesis and the field of radiopharmaceutical labeling, and in particular relates to a PET probe targeting phosphorylated AKT protein and its synthesis method and application.

Background technique

The PI3K-AKT signaling pathway is one of the most important cell signal transduction pathways driving tumorigenesis and progression. Various receptors, such as receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), cytokine receptors, and integrins, can recruit phosphatidylinositol-4,5-bisphosphate 3 -kinase that catalyzes the phosphorylation of PIP2 by the PI3Kα subunit to trigger the PI3K/AKT pathway and promote 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 tightly 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 development. And PIK3CA mutation is one of the most common oncogenic mutations in breast cancer, ovarian cancer, glioma and other malignant tumors. Therefore, inhibitor drugs targeting PI3K, especially PI3Kα, have attracted great attention from the biomedical industry. The FDA approved the first PI3Kα inhibitor for the treatment of hormone receptor-positive advanced breast cancer in 2019, and there are currently 835 clinical trials for the clinical transformation of PI3K inhibitors. There will be broader indications.

However, multiple mechanisms can lead to resistance of tumor cells to PI3Kα inhibitors. Among them, the loss of the tumor suppressor gene PTEN is the most common cause of resistance to PI3Kα inhibitors. The loss of PTEN leads to the blockage of the conversion of PIP3 to PIP2, thereby continuously and abnormally high-level activation of AKT phosphorylation, making tumors 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 serve as a target for identifying tumor resistance to PI3Kα inhibitors.

Positron emission tomography (PET) is a highly sensitive and noninvasive examination technique capable of detecting molecular signatures in tissues throughout the body. Because it is non-invasive, this inspection 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 inhibitors in the early stage. Therefore, the simple preparation, good stability, and high specificity of the PET probe for p-AKT can help clinicians to early predict/monitor the sensitivity of tumor patients to PI3K inhibitors, select tumor treatment strategies and evaluate the efficacy very important.

Contents of the invention

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, its synthesis method and application.

The purpose of the present invention can be achieved through the following technical solutions:

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

In the second aspect, the present invention provides a preparation method of the compound DOTA-Gly-pAKTi.

A preparation method of compound DOTA-Gly-pAKTi, the compound DOTA-Gly-pAKTi is prepared from the compound shown in formula (3):

In one embodiment of the present invention, in the preparation method of the compound DOTA-Gly-pAKTi, the compound shown in formula (3) reacts in the first solvent under the condition that the first acid exists, and removes the first solvent under reduced pressure, After the first post-treatment, the compound DOTA-Gly-pAKTi was obtained.

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

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

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

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

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

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

In one embodiment of the present invention, the compound of formula (2) reacts with the compound DOTA-tris (t-Bu ester) in the second solvent under the condition that the second base and the second condensing agent exist, and the second compound is removed under reduced pressure. Second solvent, after the second post-treatment, the compound of formula (3) is obtained.

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

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

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

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.

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

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.

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.

In one embodiment of the present invention, the reaction time of the reaction between the compound of formula (2) and the compound DOTA-tris (t-Bu ester) may be 8-12 h, and the reaction conditions may be room temperature.

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

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 after a third post-treatment.

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

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

In one embodiment of the present invention, the third post-treatment may include pouring into diethyl ether, separating out solids, and filtering to obtain the product

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 as 80mg, and the number of moles is 0.13mmol, The third acid has a molar concentration of 4.0M and a volume of 8 mL.

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

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

In one embodiment of the present invention, the compound GDC-0068 reacts with the compound Boc-Glycine in the second solvent in the presence of the fourth base and the fourth condensing agent, the fourth solvent is removed under reduced pressure, and after the fourth Processing yields compound formula (1).

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

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

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

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.

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

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

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

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

In the third aspect, the present invention provides the use of the compound DOTA-Gly-pAKTi or a pharmaceutically acceptable salt thereof. Application of the compound DOTA-Gly-pAKTi or a pharmaceutically acceptable salt thereof in the preparation of products for detecting diseases related to p-AKT.

In one embodiment of the present invention, the p-AKT-related diseases may include tumors.

In one embodiment of the present invention, the tumor is a tumor type with abnormal activation of PI3K/AKT signaling pathway or loss of PTEN mutation, for example, it may include breast cancer, prostate cancer and the like.

In one embodiment of the invention, said product comprises a diagnostic tracer.

In the application of the compound DOTA-Gly-pAKTi or its pharmaceutically acceptable salt provided by the present invention in the preparation of products for detecting diseases related to p-AKT, it can specifically be used for clinical/preclinical diagnosis and/or efficacy evaluation The application of drugs can be used in the research of radiochemical labeling, preclinical animal imaging and other methods, as well as its application in the products of p-AKT related diseases.

The PET probe small molecule precursor targeting phosphorylated AKT (p-AKT) protein obtained in the present invention can be further modified by DOTA-Gly-pAKTi, and the modification method includes but is not limited to replacing radiolabeled nuclides, and Conventional modification of DOTA-Gly-pAKTi.

In a fourth aspect, the present invention provides a method for radiolabeling the precursor DOTA-Gly-pAKTi with a ⁶⁸ Ga-labeled tracer.

A ⁶⁸ Ga-labeled tracer radioactive labeling method, comprising the following steps:

1) 0.1M HCl rinses the germanium-gallium generator to obtain ⁶⁸ GaCl ₃ solution;

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

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

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

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

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

In the fifth aspect, the present invention provides a ⁶⁸ Ga-DOTA-Gly-pAKTi molecular probe that can be used for PET detection and is produced based on a ⁶⁸ Ga-labeled tracer radioactive labeling method. ⁶⁸ Ga-DOTA-Gly-pAKTi molecular probe is a ⁶⁸ Ga-labeled radiolabeled PET probe, which can be used for PET detection. The structure of ⁶⁸ Ga-DOTA-Gly-pAKTi molecular probe is as follows:

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

The application of the molecular probe in the preparation of drugs for clinical/preclinical diagnosis and/or efficacy evaluation;

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

The application of the molecular probe in the research of preclinical animal imaging methods;

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

The PET probe small molecule precursor DOTA-Gly-pAKTi or the molecular probe can be further modified, and the modification methods include but not limited to the replacement of radiolabeled nuclides and the conventional modification of DOTA-Gly-pAKTi.

The compound DOTA-Gly-pAKTi or its pharmaceutically acceptable salt provided by the present invention is simple to prepare, short in preparation time, high in radiochemical yield, good in specificity, high in ingestion, and has good performance in normal saline or serum The stability of p-AKT can detect the expression level of p-AKT in the tumor at the cellular level and in breast cancer tumor-bearing mice.

In addition, the ⁶⁸ Ga-labeled radiolabeled DOTA-Gly-pAKTi method provided by the present invention produces ⁶⁸ Ga-DOTA-Gly-pAKTi molecular probes that can be used for PET detection. The labeling method is simple to operate, short in preparation time, and produces High rate.

The PET probe provided by the invention has the specificity of targeting and binding phosphorylated modified AKT protein, and is used for detecting the expression of phosphorylated modified AKT protein in tumor tissue. Abnormal activation of phosphorylated AKT is an important mechanism driving tumorigenesis and development. AKT is often activated by PIK3CA mutation or loss of tumor suppressor gene PTEN, highly phosphorylated and activates downstream mTOR and other signaling pathways to promote tumor occurrence, growth and infiltration. Therefore, this PET probe can provide important information 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 antitumor efficacy of various kinase inhibitors targeting PI3K/AKT and downstream signaling pathways. and reliable molecular imaging tools.

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

(1) ^{The 68} Ga-DOTA-Gly-pAKTi provided by the present invention has higher specificity for p-AKT.

(2) ^{The 68} Ga-DOTA-Gly-pAKTi provided by the present invention has better stability.

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

(4) PET/CT imaging using ⁶⁸ Ga-DOTA-Gly-pAKTi probe can effectively reflect the drug resistance of tumors to PI3Kα inhibitors and the molecular characteristics of PTEN loss in tumor cells.

Description of drawings

FIG. 1 shows the mass spectrum of the compound DOTA-Gly-pAKTi prepared in Example 1.

FIG. 2 shows the analysis of DOTA-Gly-pAKTi and ⁶⁸ Ga-DOTA-Gly-pAKTi from Example 2 by HPLC.

Fig. 3 shows the stability of ⁶⁸ Ga-DOTA-Gly-pAKTi in mouse serum and physiological saline obtained from Example 3.

Fig. 4 shows the blood clearance rate of ⁶⁸ Ga-DOTA-Gly-pAKTi in mice in Example 4.

Figure 5 shows the results of uptake of ⁶⁸ Ga-DOTA-Gly-pAKTi by p-AKT high expression and low expression cells in Example 5; where the left figure is ⁶⁸ Ga-DOTA-Gly-pAKTi in HCC-1806, HCC-1937 , SUM-159, MDA-MB-231, MDA-MB-468, and BT-549 cells incubated for 120 minutes, of which HCC-1806, HCC-1937, and SUM-159 are triple negatives with low expression of p-AKT Breast cancer cell lines, MDA-MB-231, MDA-MB-468, and BT-549 are triple-negative breast cancer cell lines with high expression of p-AKT; the right picture shows incubation in ⁶⁸ Ga-DOTA-Gly-pAKTi for 120 minutes After MDA-MB-231, MDA-MB-468, BT-549 cells were replaced with non-radioactive medium and incubated for 90 minutes, the percentage of radioactive retention indicated that ⁶⁸ Ga-DOTA-Gly-pAKTi could be quickly taken up by tumor cells, and tumor The uptake of ^68Ga -DOTA-Gly-pAKTi by cells is positively correlated with the expression of p-AKT. After ^68Ga -DOTA-Gly-pAKTi is taken up by tumor cells, it will be metabolized by cells within a certain period of time and can be used as a PET imaging probe. Characteristics.

Fig. 6 shows that utilize p-AKT inhibitor GDC-0068 and ⁶⁸ Ga-DOTA-Gly-pAKTi to carry out competitive inhibition binding test in MDA-MB-231, MDA-MB-468, BT-549 cells in embodiment 6 . In MDA-MB-468, BT-549 and MDA-MB-231, the IC ₅₀ values of GDC-0068 are 0.6nM, 0.039nM and 0.038nM, respectively.

Fig. 7 shows the PET/CT imaging by the tumor-bearing animal model in embodiment 7; Wherein, figure A is ⁶⁸ Ga-DOTA-Gly-pAKTi in HCC-1806, HCC-1937, MDA-MB-231, MDA-MB PET imaging in -468 tumor models, in which HCC-1806 and HCC-1937 were tumors with low expression of p-AKT, MDA-MB-231 and MDA-MB-468 were tumors with high expression of p-AKT; Figure B is the tumor pair ⁶⁸ Uptake values of Ga-DOTA-Gly-pAKTi. This result demonstrates high ⁶⁸ Ga-DOTA-Gly-pAKTi radioactive uptake in p-AKT high tumors, but only low ⁶⁸ Ga-DOTA-Gly-pAKTi radioactive uptake in p-AKT low tumors. Therefore, ^{the 68} Ga-DOTA-Gly-pAKTi PET probe has good sensitivity and specificity in detecting pAKT in living tumors.

Fig. 8 shows the biodistribution diagram of ^68Ga -DOTA-Gly-pAKTi in Example 8 in HCC-1806, HCC-1937, MDA-MB-231, MDA-MB-468 tumor-bearing mouse models for 1.5h. ^{The 68} Ga-DOTA-Gly-pAKTi probe had a significantly higher biodistribution in PTEN-null tumors (high p-AKT) compared to most normal tissues and PTEN-normal tumors (low p-AKT). This result further suggested the specificity of ⁶⁸ Ga-DOTA-Gly-pAKTi PET probe in detecting tumor p-AKT levels in vivo, and suggested that liver and kidney tissues would have higher background uptake.

Figure 9 shows the PET/CT imaging of the control tumor-bearing model animals with PTEN deficiency and PTEN normal in Example 9; among them, Figure A is ^68Ga -DOTA-Gly-pAKTi in HCC-1806PTEN WT, HCC-1806PTEN KO, PET imaging in HCC-1937PTEN WT and HCC-1937PTEN KO tumor models; Panel B shows the expression of PTEN and p-AKT in tumor tissue detected by Western Blot. The results proved that after PTEN knockout, tumor cells lost PTEN, and the expression of p-AKT was not inhibited, but increased significantly. The uptake of ⁶⁸ Ga-DOTA-Gly-pAKTi was increased in tumors with high p-AKT expression.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

Embodiment 1: the preparation of compound DOTA-Gly-pAKTi

1) Preparation of formula (1) compound

Synthesis of the compound of formula (1): Add 10mL DMF to a 50mL eggplant-shaped bottle, and then add the compound Boc-Glycine (2,36.72mg, 0.21mmol, 1.2eq), HATU (73.06mg, 0.19mmol, 1.1eq) and DIPEA (58.70mg, 0.45mmol, 2.6eq), after stirring at room temperature for 30min, compound GDC-0068 (1,80mg, 0.17mmol, 1eq) was added, and the reaction system was stirred overnight at room temperature. After the reaction was completed, _NaHCO3 saturated solution (50mL) was added to the reaction solution and continued to stir for 15min, then ethyl acetate ( _50mL ) was added to extract 3 times, the organic phases were combined, dried over anhydrous _Na2SO4 , filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain product 3 (65.06 mg, 60.55%) as solid powder.

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).

2) Preparation of formula (2) compound

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

3) Preparation of formula (3) compound

Add 10mL DMF to a 50mL eggplant-shaped bottle, and add the compound tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (5,67.63mg, 0.12 mmol, 1.3eq), HATU (37.99mg, 0.010mmol, 1.1eq) and DIPEA (37.99mg, 0.24mmol, 2.6eq), after stirring at room temperature for 30min, compound 2 (50mg, 0.090mmol, 1eq) was added, and the reaction system was Stirring overnight. After the reaction was completed, _NaHCO3 saturated solution (50mL) was added to the reaction solution and continued to stir for 15min, then ethyl acetate ( _50mL ) was added to extract 3 times, the organic phases were combined, dried over anhydrous _Na2SO4 , filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain product 3 (35.18 mg, 36.21%) as solid powder.

4) Preparation of compound DOTA-Gly-pAKTi

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

The mass spectrum of the compound DOTA-Gly-pAKTi is shown in Figure 1.

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.

Embodiment 2: the preparation of tracer

The preparation of the tracer using the compound DOTA-Gly-pAKTi as a precursor comprises:

1) 0.1M HCl rinses the germanium-gallium generator to obtain ⁶⁸ GaCl ₃ solution;

2) Adjust the pH value of the ⁶⁸ GaCl ₃ solution to 2.3-3.5 with 0.5M NaAC 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 tracers.

The results of analyzing DOTA-Gly-pAKTi and ⁶⁸ Ga-DOTA-Gly-pAKTi by HPLC are shown in FIG. 2 .

Results: The tracer ⁶⁸ Ga-DOTA-Gly-pAKTi was successfully prepared within 20 minutes from the ⁶⁸ GaCl ₃ solution obtained by elution, with a radiochemical purity of over 95%, which was consistent with the retention time of its standard in HPLC.

Embodiment 3: Stability experiment

Blood was collected from Balb/c mice, and serum was prepared for use. Take several EP tubes and add 200 μL serum or 200 μL saline to them respectively. Add 20 μL of freshly prepared ⁶⁸ Ga-DOTA-Gly-pAKTi to serum and normal saline, respectively, and incubate at 37°C for different times (0h, 0.5h, 1h, 1.5h, 2h, 2.5h) , 3h); after the incubation, add 200 μL of methanol to the serum tube, centrifuge at room temperature (1000 rpm, 5min), take the supernatant to measure the radiochemical purity by iTLC; The radiochemical purity was determined directly by iTLC.

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

Results: More than 95% of the tracer ⁶⁸ Ga-DOTA-Gly-pAKTi remained in the prototype in vitro at 37°C for 3 hours in mouse serum; nearly 95% of the tracer remained in the prototype in vitro at room temperature in saline for 3 hours Not decomposed. The tracer ⁶⁸ Ga-DOTA-Gly-pAKTi has good stability in both serum and normal saline.

Embodiment 4: Blood clearance experiment

Prepare several Balb/c mice and inject 1.7MBq ⁶⁸ Ga-DOTA-Gly-pAKTi solution through the tail vein of each mouse; puncture the blood vessel at the end of the tail vein on the opposite side, and collect blood with capillary tubes. 2.0min, 4.0min, 10.0min, 20.0min, 40.0min, 60.0min, 90.0min, 120.0min, 180.0min, 240.0min blood, measure the radioactive count of the collected blood with a gamma counter, and parallel 5 mice at each time point . The biphasic attenuation fitting was performed on the processed gamma counter measurement results by GraphPad Prism software, and the fitting formula was

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

Figure 4 shows the blood clearance rate of ⁶⁸ Ga-DOTA-Gly-pAKTi in mice.

Results: The distribution phase half-life t1/2 of ⁶⁸ Ga-DOTA-Gly-pAKTi in mice was 2.0 min, and the blood clearance phase half-life t1/2 was 56.2 min by biphasic decay analysis.

Example 5: Cellular uptake and efflux experiments

After preliminary experiments, it was found that triple-negative breast cancer HCC-1806, HCC-1937, and SUM-159 cell lines had low expression of p-AKT, and triple-negative breast cancer MDA-MB-231, MDA-MB-468, and BT-549 cell lines High expression of p-AKT. Spread HCC-1806 cells, HCC-1937 cells, SUM-159 cells, MDA-MB-231 cells, MDA-MB-468 cells, BT-549 cells in 24-well plates (1*10 ⁵ /well), After attachment, tumor cells were co-incubated with ⁶⁸ Ga-DOTA-Gly-pAKTi (74kBq/well) at 37°C for 15, 30, 45, 60, 90 and 120 minutes.

After incubation, tumor cells were gently washed three times with refrigerated phosphate buffered saline and detached from the 24-well plate with 0.25% trypsin/0.02% EDTA. Observe under a light microscope to ensure that cells are completely detached and collected. The radioactivity of the collected cell suspension was detected with a gamma counter. The radioactivity in the cell suspension was corrected for attenuation and calculated as a percentage of the total radioactivity. Experiments were repeated three times.

MDA-MB-231 cells, MDA-MB-468 cells, and BT-549 cells with high expression of p-AKT were plated in 24-well plates (1*10 ⁵ /well), and the tumor cells were mixed with ⁶⁸ Ga -DOTA-Gly-pAKTi (74kBq/well) were co-incubated at 37°C for 120 minutes. Remove the radioactive medium, add non-radiative medium and incubate for 0, 15, 30, 45, 60 and 90 minutes, remove the culture medium, wash the cells twice with cold PBS solution, digest with trypsin, and detect with a gamma counter radioactivity.

The results of uptake of ⁶⁸ Ga-DOTA-Gly-pAKTi by cells with high and low p-AKT expression are ^shown in Figure 5; Uptake values of 159, MDA-MB-231, MDA-MB-468, and BT-549 cells incubated for 120 minutes, among which HCC-1806, HCC-1937, and SUM-159 are triple-negative breast cancer cells with low expression of p-AKT MDA-MB-231, MDA-MB-468, and BT-549 are triple-negative breast cancer cell lines with high expression of p-AKT; the right picture shows MDA incubated in ⁶⁸ Ga-DOTA-Gly-pAKTi for 120 minutes -MB-231, MDA-MB-468, BT-549 cells were replaced with the radioactive retention percentage within 90 minutes of incubation with non-radioactive culture medium, suggesting that ⁶⁸ Ga-DOTA-Gly-pAKTi can be quickly taken up by tumor cells, and tumor cells have a positive effect on ⁶⁸ The uptake of Ga-DOTA-Gly-pAKTi is positively correlated with the expression of p-AKT. ⁶⁸ After being taken up by tumor cells, Ga-DOTA-Gly-pAKTi is metabolized by cells within a certain period of time, and has the characteristics of being a PET imaging probe.

Results: The triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-468, and BT-549 with high expression of p-AKT had a higher uptake of ⁶⁸ Ga-DOTA-Gly-pAKTi, and the uptake values of 2h reached 1.15 %±0.56, 2.02%±0.26, 1.97%±0.35; 2h uptake value of ⁶⁸ Ga-DOTA-Gly-pAKTi by triple-negative breast cancer cell lines HCC-1806, HCC-1937 and SUM-159 with low p-AKT expression Respectively reach 0.37%±0.09, 0.47%±0.1, 0.34%±0.12.

For the expulsion experiment, ^68Ga -DOTA-Gly-pAKTi remained in MDA-MB-231, MDA-MB-468, BT-549 cells for more than 90min.

Embodiment 6: cell binding experiment

The 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. Cells were gently washed twice with cold PBS, and then cells were co-incubated with ⁶⁸ Ga-DOTA-Gly-pAKTi and different concentrations of GDC-0068 for 1 hour. After gently 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 gamma counter. Non-linear regression fitting was performed on the data by computer software GraphPad Prism, and the optimal 50% inhibitory concentration (IC ₅₀ ) was calculated. Experiments were repeated three times.

Figure 6 shows the results of the competitive inhibition binding assays using the p-AKT inhibitor GDC-0068 and ⁶⁸ Ga-DOTA-Gly-pAKTi in MDA-MB-231, MDA-MB-468, and BT-549 cells.

Results: In MDA-MB-468, BT-549 and MDA-MB-231 cells, the IC ₅₀ values of GDC-0068 for blocking the uptake of ^68Ga -DOTA-Gly-pAKTi were 0.6nM, 0.039nM and 0.038nM, respectively.

Example 7: PET imaging experiment of tumor-bearing model animals

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

The PET/CT images of the tumor-bearing model animals are shown in Figure 7; among them, Figure A shows the expression of ^68Ga -DOTA-Gly-pAKTi in HCC-1806, HCC-1937, MDA-MB-231, MDA-MB-468 tumors PET imaging in the model, in which HCC-1806 and HCC-1937 are tumors with low expression of p-AKT, MDA-MB-231 and MDA-MB-468 are tumors with high expression of p- ^AKT ; Uptake values of DOTA-Gly-pAKTi.

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

Example 8: In vivo biodistribution experiment

1.5 hours after injecting 1.7MBq ⁶⁸ Ga-DOTA-Gly-pAKTi solution into tumor-bearing nude mice via tail vein, the nude mice were sacrificed and dissected to obtain heart, liver, spleen, lung, kidney, muscle, bone, brain, stomach, and intestine , blood, tumors and other tissues and organs. After these tissues and organs were weighed, the radioactivity was measured with a gamma counter, and the radioactivity per gram of tissue was calculated. The uptake value in tissues and organs is expressed as the percentage of the radioactive dose taken up per gram of tissue to the injected dose (%ID/g).

The biodistribution diagram of ⁶⁸ Ga-DOTA-Gly-pAKTi in HCC-1806, HCC-1937, MDA-MB-231, MDA-MB-468 tumor mouse models at 1.5h is shown in Fig. 8 . ^{The 68} Ga-DOTA-Gly-pAKTi probe had a significantly higher biodistribution in PTEN-null tumors (high p-AKT) compared to most normal tissues and PTEN-normal tumors (low p-AKT).

Results: The uptake of ⁶⁸ Ga-DOTA-Gly-pAKTi was high in MDA-MB-231 and MDA-MB-468 tumor tissues with high p-AKT expression, and in HCC-1806 and HCC-1937 tumor tissues with low p-AKT expression low intake. Muscle, bone, brain and other organs have low uptake of ⁶⁸ Ga-DOTA-Gly-pAKTi. ⁶⁸ Ga-DOTA-Gly-pAKTi is mainly metabolized by the kidney and liver. This result further suggested the specificity of ^68Ga -DOTA-Gly-pAKTi PET probe in detecting tumor phosphorylated AKT levels in vivo, and reminded that liver and kidney tissues would have higher background uptake.

Example 9: PET imaging experiment of tumor-bearing model animals with normal PTEN and loss of PTEN

HCC-1806PTEN WT, HCC-1806PTEN KO, HCC-1937PTEN WT, HCC-1937PTEN KO tumor models were constructed, and imaging was performed when the tumor grew to 1 cm ³ . The labeled probe was injected into the tumor-bearing mice through the tail vein, and each tumor-bearing mouse was injected with 7.4MBq. PET/CT imaging was performed 1 hour after the probe was injected through the tail vein, and the uptake of the probe by each tumor tissue was analyzed with Inveon Research Workplace image analysis software.

The PET/CT images of PTEN-deficient and PTEN-normal control tumor burden model animals are shown in Figure 9, in which, Figure A shows the expression of ^68Ga -DOTA-Gly-pAKTi in HCC-1806PTEN WT, HCC-1806PTEN KO, and HCC-1937PTEN PET imaging in WT and HCC-1937PTEN KO tumor models; Panel B shows the expression of PTEN and p-AKT in tumor tissue detected by Western Blot.

Results: The tumor-bearing models constructed by PTEN-deficient tumor cells HCC-1806PTEN KO and HCC-1937PTEN KO had high uptake of ⁶⁸ Ga-DOTA-Gly-pAKTi, and the expression level of p-AKT in tumor tissue was significantly increased by Western Blot verification; PTEN The normal tumor cells HCC-1806PTEN WT and HCC-1937PTEN WT constructed tumor-bearing model tumors had low uptake of ⁶⁸ Ga-DOTA-Gly-pAKTi and low expression of p-AKT in tumor tissues. The results proved that after PTEN knockout, tumor cells lost PTEN, and the expression of p-AKT was not inhibited, but increased significantly. The uptake of ⁶⁸ Ga-DOTA-Gly-pAKTi was increased in tumors with high p-AKT expression.

Embodiment 3-embodiment 9 can draw the following conclusions:

(1) Stability experiments showed that ⁶⁸ Ga-DOTA-Gly-pAKTi had good stability in both serum and saline.

(2) The blood clearance experiment shows that ⁶⁸ Ga-DOTA-Gly-pAKTi can be rapidly distributed in the tissues and organs of the whole body after intravenous injection. At the same time, the half-life in the blood is short, so it is suitable as an imaging probe.

(3) Cell uptake experiments showed that the uptake of ⁶⁸ Ga-DOTA-Gly-pAKTi by cells was related to the p-AKT expressed by cells.

(4) Cell binding experiments showed that ⁶⁸ Ga-DOTA-Gly-pAKTi had good specificity to p-AKT.

(5) The uptake of the probe by tumor tissue was related to the expression level of p-AKT.

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

The above descriptions of the embodiments are for those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described here to other embodiments without creative efforts. Therefore, the present invention is not limited to the above-mentioned embodiments. Improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.

Claims (10)

1. A PET probe small molecule precursor targeting phosphorylated AKT protein, which is the compound DOTA-Gly-pAKTi or a pharmaceutically acceptable salt thereof

2. The preparation method of compound DOTA-Gly-pAKTi is characterized in that, compound DOTA-Gly-pAKTi is prepared by the compound shown in formula (3), wherein the compound shown in formula (3) is as follows:

3. according to the preparation method of the described compound DOTA-Gly-pAKTi of claim 2, it is characterized in that, the compound shown in formula (3) is under the condition that the first acid exists, reacts in the first solvent, removes the first under reduced pressure Solvent, after the first post-treatment, the compound DOTA-Gly-pAKTi is obtained; And/or the first acid includes hydrochloric acid; and/or the first solvent includes at least one of water or dioxane; and/or the first post-treatment includes: pouring into ether to precipitate a solid, Filter to get the product. 4. according to the preparation method of compound DOTA-Gly-pAKTi described in claim 2, it is characterized in that, described formula (3) compound is prepared by compound shown in formula (2) and DOTA-tris (t-Bu ester) into, wherein, the structures of compound shown in formula (2) and DOTA-tris (t-Bu ester) are as follows respectively:

The compound of formula (2) reacts with DOTA-tris (t-Buester) in the second solvent under the condition that the second base and the second condensing agent exist, and the second solvent is removed under reduced pressure, and after the second post-treatment, the formula (3) compound; And/or the second base includes diisopropylethylamine; and/or the second condensing agent includes 2-(7-azobenzotriazole)-N,N,N',N' -Tetramethylurea hexafluorophosphate; and/or the second solvent includes N,N-dimethylformamide; and/or the second post-treatment includes: extraction, drying, filtration, concentration under reduced pressure, The product was purified by column chromatography. 5. according to the preparation method of compound DOTA-Gly-pAKTi described in claim 4, it is characterized in that, described formula (2) compound is prepared from formula (1) compound, and wherein formula (1) compound structure is as follows:

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 through a third post-treatment; And/or the third acid includes hydrochloric acid; and/or the third solvent includes dioxane hydrochloride solution; and/or the third post-treatment includes: pouring into ether, separating out solids, and filtering to obtain the product. 6. according to the preparation method of compound DOTA-Gly-pAKTi described in claim 5, it is characterized in that, described formula (1) compound is prepared from compound GDC-0068 and compound Boc-Glycine, wherein, compound GDC-0068 and compound Boc-Glycine structures are as follows:

The compound GDC-0068 reacts with the compound Boc-Glycine in the fourth solvent in the presence of the fourth base and the fourth condensing agent, removes the fourth solvent under reduced pressure, and undergoes the fourth post-treatment to obtain the compound formula (1); And/or the fourth base includes diisopropylethylamine; and/or the fourth condensing agent includes 2-(7-azobenzotriazole)-N,N,N',N' -Tetramethylurea hexafluorophosphate; and/or the fourth solvent includes N,N-dimethylformamide; and/or the fourth post-treatment includes: extraction, drying, filtration, concentration under reduced pressure, The product was purified by column chromatography. 7. the application of the PET probe small molecule precursor of target phosphorylation AKT albumen described in claim 1, it is characterized in that, compound DOTA-Gly-pAKTi or its pharmaceutically acceptable salt are in preparation detection and p-AKT related disease applications; and/or, The diseases related to p-AKT include tumors, and the tumors are tumor types with abnormal activation of PI3K/AKT signaling pathway or loss of PTEN mutation; And/or, the product includes a diagnostic tracer. 8. A ⁶⁸ Ga-labeled tracer radioactive labeling method, characterized in that, comprising the following steps: 1) 0.1M HCl rinses the germanium-gallium generator to obtain ⁶⁸ GaCl ₃ solution; 2) Adjust the pH value of the ⁶⁸ GaCl ₃ solution to 2.3-3.5 with 0.5M NaAC to form a labeling system; 3) Add the precursor solution to the labeling system, and react at 100°C for 10 minutes to obtain a ⁶⁸ Ga-labeled tracer; The precursor is the compound DOTA-Gly-pAKTi or a pharmaceutically acceptable salt thereof. 9. ⁶⁸ Ga-DOTA-Gly-pAKTi molecular probes that can be used for PET detection based on the tracer radiolabeling method of ⁶⁸ Ga labels described in claim 8, ⁶⁸ Ga-DOTA-Gly-pAKTi molecular probes The structure is as follows:

10. The application of the ⁶⁸ Ga-DOTA-Gly-pAKTi molecular probe that can be used for PET detection according to claim 9 is characterized in that it is selected from one of the following applications: The application of the molecular probe in the preparation of drugs for clinical/preclinical diagnosis and/or efficacy evaluation; The application of the molecular probe in the preparation of radiochemically labeled products; The application of the molecular probe in the research of preclinical animal imaging methods; The application of the molecular probe in the preparation of products for diagnosing/treating diseases related to p-AKT.

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