Small molecule compound targeting pAKT, PET molecular probe precursor, PET molecular probe and preparation method and application thereof

Abstract

The invention provides a pAKT-targeted small molecule compound, a PET molecular probe precursor, a PET molecular probe and a preparation method and application of the PET molecular probe. The PET molecular probe disclosed by the invention is a radioactive probe which is modified on the basis of a chemical structure of a medicine Capivastib (AZD5363) and is marked by 68Ga and is targeted to pAKT. According to the probe, structural modification is carried out on Capivasertib, the area, combined with PKA, of Capivasertib is changed, precursor molecules combined with pAKT in a high-specificity mode are obtained, and the precursor molecules are further researched, developed and modified into the radioactive probe targeting pAKT capable of being used for PET imaging. The probe has relatively low abdominal nonspecific uptake, can effectively distinguish tumors with high expression and low expression of pAKT in vivo, and can predict and early evaluate the response condition of the tumors to a PI3K / AKT pathway related inhibitor. Therefore, the probe has a very strong clinical application prospect, on one hand, the probe can be used for analyzing the expression quantity of pAKT in tumor tissues; on the other hand, the probe can also be used for predicting and early evaluating the effect of the PI3K / AKT pathway related inhibitor in anti-tumor treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nuclear medicine, and particularly relates to a small-molecule compound targeting pAKT, a PET molecular probe precursor, a PET molecular probe and a preparation method and application thereof. BACKGROUND

[0002] AKT is a serine / threonine kinase. The activation of AKT needs to be mediated by the PI3K (phosphatidylinositol 3-kinase) signaling pathway. PI3K catalyzes the generation of PIP3, which promotes the transfer of AKT from the cytoplasm to the cell membrane. On the cell membrane, PIP3 provides a binding site for AKT and promotes the activation of the upstream kinase of AKT. The main upstream kinase is PDK1, which partially activates AKT through the Thr308 site of pAKT. In addition, the upstream kinase TORC2 can further promote the complete activation of AKT through the Ser473 site of pAKT. AKT, as the core molecule in the PI3K / AKT signaling pathway, plays an important role in regulating various physiological processes such as cell proliferation, survival, and metabolism. Abnormal activation of AKT plays a key role in various cancers.

[0003] Capivasertib is a highly efficient pan-AKT phosphorylation small molecule inhibitor, which blocks multiple important signaling pathways downstream of AKT by inhibiting the phosphorylation of AKT. Capivasertib has been approved by FDA on November 16, 2023 for use in combination with fulvestrant for the treatment of adult patients with hormone receptor (HR) positive, human epidermal growth factor receptor 2 (HER2) negative locally advanced or metastatic breast cancer. Most of these patients have one or more PIK3CA / AKT1 / PTEN genetic alterations, and the disease progresses after receiving at least one endocrine therapy in the metastatic stage, or relapses within 12 months after completing adjuvant therapy. The study of phase III clinical trial CAPItello-291 showed that Capivasertib combined with fulvestrant significantly prolonged the progression-free survival in the above patient population, and reduced the risk of disease progression or death by 50%.

[0004] Although Capivasertib shows good efficacy in clinical trials, its clinical accurate use is still challenging. First, the pAKT status of tumors is highly heterogeneous, and the pAKT inhibition of Capivasertib in whole body tumors needs to be systematically evaluated. Second, normal cells also need pAKT to maintain cell survival, and the toxic side effects of Capivasertib on normal cells need to reduce unnecessary drug exposure. Third, the therapeutic effect of Capivasertib may be affected by the tumor microenvironment, cancer cell mutations, and their cross with other signaling pathways. Therefore, how to non-invasively visualize pAKT and systematically predict and evaluate the therapeutic effect of Capivasertib is an important research direction that needs to be carried out urgently.

[0005] Various important oncogene mutations (such as PIK3CA, HER2, EGFR) activate pAKT through the PI3K-AKT signaling pathway, of which the most direct is that PIK3CA mutation causes persistent activation of pAKT. On the other hand, the loss of the common tumor suppressor PTEN will cause the loss of the channel that "turns off" pAKT, leading to persistent activation of pAKT. Various small molecule inhibitors or ADCs targeting PIK3CA, HER2, EGFR, etc. target drugs play an important role in clinical anti-tumor therapy, and PI3Kα inhibitors have been officially approved by FDA for breast cancer treatment. It is particularly noted that PI3Kα inhibitors have good therapeutic effect on tumors with pAKT abnormally activated by PIK3CA mutation. PTEN loss tumors are resistant to PI3Kα inhibitors, and PTEN loss is also significantly associated with poor prognosis of patients with HER2 and EGFR targeted drugs. Positron emission computed tomography (PET), a highly sensitive and non-invasive molecular imaging technology, is widely used in the treatment monitoring of tumors and other diseases. Therefore, the development of PET radioactive molecular probes targeting pAKT based on the structure of Capivasertib can not only non-invasively detect the pAKT level of tumors, but also predict and early monitor the therapeutic effect of Capivasertib and a series of PI3K-AKT pathway related inhibitors / ADCs and other targeted drugs in anti-tumor therapy, providing an important basis for clinical treatment strategy selection and efficacy evaluation. SUMMARY

[0006] Based on the defects in the prior art described above, the purpose of the present application is to provide a small molecule compound targeting pAKT, a PET molecular probe precursor, a PET molecular probe and a preparation method and application thereof. The tracer is obtained by chemically modifying Capivasertib (AZD5363) as the mother skeleton to obtain compound I, and then synthesizing precursor compound DOTA-oncopAKTi from compound I through a series of reactions, and finally labeling with a positron emitting nuclide (such as 68The application discloses a PET tracer precursor compound for targeting pAKT, and a preparation method thereof. The tracer precursor compound is DOTA-oncopAKTi, and has a structure as shown in any one of formula II, formula III or formula IV. The tracer precursor compound is labeled by Ga to obtain a molecular probe capable of specifically combining with a pAKT target and used for PET imaging. Capivasertib is a pAKT and PKA double-target inhibitor approved by FDA for breast cancer treatment, and has good affinity to phosphorylated Akt1, Akt2, Akt3 and PKA. At present, no probe based on Capivasertib is reported. The PET molecular probe for targeting pAKT provided by the application is mainly used for pAKT molecular detection, accurate diagnosis, treatment decision and efficacy evaluation of tumors, and is especially suitable for pAKT and PTEN-PI3K signal pathway axis targeting imaging which is highly expressed in various solid tumors or hematological tumors. The probe has the following advantages: (1) The preparation process is simplified, and the radiochemical purity is high; (2) The molecular structure has excellent stability in vitro and in vivo, can maintain high targeting pAKT affinity, and blocks the combination of the combination region with PKA; (3) High selectivity and high uptake can be realized at the tumor site, so that a clear and high-contrast image can be obtained in PET imaging; (4) The chemical structure of the probe is highly related to the pAKT inhibitor which has been approved or is in clinical trials, and is beneficial to subsequent clinical conversion application.

[0007] (5) The tracer precursor compound for targeting pAKT provided by the application has the following advantages: 68 The Ga-labeled pAKT targeting PET tracer can specifically combine with pAKT, and can be used for early detection of overexpression or activation of pAKT in tumors in clinic, and can assist in diagnosis and provide a basis for treatment decision.

[0008] (6) The probe can provide accurate imaging information in aspects of clinical staging, efficacy monitoring and recurrence and metastasis evaluation.

[0009] (7) The tracer is derived from Capivasertib which has been verified in safety and effectiveness in clinic or clinical trials, and is beneficial to subsequent expansion of a new precise diagnosis and treatment mode combining imaging and therapy.

[0010] The technical scheme of the application comprises the following steps: The technical scheme 1 of the application provides a small-molecule compound for targeting pAKT, which has a structure as shown in formula I. Formula I; The technical scheme 2 of the application provides a PET tracer precursor compound for targeting pAKT, and the PET tracer precursor compound is DOTA-oncopAKTi, and has a structure as shown in any one of formula II, formula III or formula IV. Formula II; Formula III; Formula IV; or a pharmaceutically acceptable salt thereof, wherein, in Formulas II-IV: n is an integer selected from 1 to 10.

[0011] Further, in some embodiments of the present application, when n = 2, the structure of the PET tracer precursor compound as shown in Formula II is specifically as shown in Formula V: Formula V; or a pharmaceutically acceptable salt thereof.

[0012] The technical solution 3 of the present application provides a preparation method of the above-mentioned PET tracer labeled precursor targeting pAKT, taking compound V as an example, the preparation method is according to the route of compound I→compound A→compound B→compound C→compound V, comprising the following steps: Step S1: mixing compound I, FmocNH-PEG2-COOH, HATU, and DIPEA to obtain compound A; the equivalent relationship of the compound I, FmocNH-PEG2-COOH, HATU, and DIPEA is preferably 1.0 eq: 1.3 eq: 1.3 eq: 2.6 eq; The raw material FmocNH-PEG2-COOH can be replaced by FmocNH-PEGn-COOH, and the subscript n is equal to n in the structure of the PET tracer labeled precursor compound DOTA-oncopAKTi as shown in Formula II, that is, by selecting raw materials FmocNH-PEGn-COOH with different n numbers, compounds A, B, C, and the PET tracer labeled precursor compound DOTA-oncopAKTi as shown in Formula II with different structures can be obtained. The raw material FmocNH-PEGn-COOH can be further replaced by FmocNH-(CH2)n-COOH or FmocNH-(Gly)n-COOH; the FmocNH-(CH2)n-COOH corresponds to the PET tracer labeled precursor compound as shown in Formula III, and the FmocNH-(Gly)n-COOH corresponds to the PET tracer labeled precursor compound as shown in Formula IV.

[0013] Step S2: Compound A is dissolved in dichloromethane, and a diethylamine solution is added to obtain target compound B; Step S3: Compound B, DOTA-TRIS(ACID), HATU and DIPEA are mixed to obtain compound C; the equivalent relationship of the compound B, DOTA-TRIS(ACID), HATU and DIPEA solvent is 1.0 eq:1.3 eq:1.1 eq:2.6 eq; Step S4: Compound C is dissolved in dichloromethane, and a trifluoroacetic acid (TFA) solution is added to obtain a PET tracer labeling precursor compound DOTA-oncopAKTi as shown in formula V.

[0014] Further, the compounds A, B, C are intermediate products in the above steps, and are different according to different raw materials FmocNH-PEGn-COOH or FmocNH-(CH2)n-COOH or FmocNH-(Gly)n-COOH.

[0015] Further, in some embodiments of the present application, the compound A has the following structural formula: Formula A; Further, in some embodiments of the present application, the compound B has the following structural formula: Formula B; Further, in some embodiments of the present application, the compound C has the following structural formula: Formula C.

[0016] The technical solution 4 of the present application provides a PET tracer targeting pAKT, which is obtained by radiolabeling the above-mentioned PET tracer labeling precursor. The radiolabeled element is located in the DOTA chelating group. The radiolabeled element is combined with the compound DOTA-oncopAKTi through a coordination bond.

[0017] Further, the radiolabeled element is preferably 68 Ga, and can also be selected from 67 Ga, 111 In, 64 Cu, 89 Zr, 90 Y, 177 Lu, 153 Sm, or 166 Ho, etc.

[0018] Further, the radiolabeled element is preferably68 Ga and the compound as shown in formula V are taken as examples, the structural formula of the above-mentioned PET tracer targeting pAKT is provided: Formula VI; Further, the application discloses a preparation method of the above-mentioned PET tracer targeting pAKT, and the steps are as follows: (1) preparing an eluent containing radioactive ions; (2) adjusting the pH value of the eluent system to 4.0-4.2; (3) adding a PET tracer labeling precursor compound DOTA-oncopAKTi solution, after reaction at 90-100 DEG C for 10-20 min, cooling to room temperature; the reaction solution after reaction is separated and purified, and the radioactive labeled PET tracer targeting pAKT is obtained.

[0019] Further, the application further discloses the PET tracer when the above-mentioned radioactive element is 68 Ga, the preparation steps of the PET tracer (when other radioactive elements are labeled, the radioactive element ions are replaced), and the specific preparation steps are as follows: (1) injecting 5 mL of 0.1 M hydrochloric acid solution into a germanium gallium generator to obtain an eluent containing 68 GaCl3 ions; (2) adding a sodium acetate solution to 4 mL of the above-mentioned eluent, and adjusting the pH value of the system to 4.0-4.2; (3) adding a PET tracer labeling precursor compound DOTA-oncopAKTi solution, after reaction at 90-100 DEG C for 10 min, cooling to room temperature; diluting with water to obtain a diluted reaction solution, and separating and purifying the product through a C 18 column (SEP-PAK), so that the product is adsorbed to the C 18 column; injecting 70% ethanol aqueous solution to elute the product from the C 18 column, so that the PET tracer labeled with 68 Ga is obtained.

[0020] The technical scheme 5 of the application provides application of the above-mentioned PET tracer labeling precursor compound or the above-mentioned PET tracer labeled with 68 Ga in preparation of a drug for pAKT protein quantitative detection in a biological sample.

[0021] Further, the biological sample is derived from an ex vivo tumor or inflammation tissue sample; the biological sample has a disease or a condition related to pAKT protein, including but not limited to a tumor or inflammation.

[0022] Further, the tumor includes, but is not limited to, any one or several of breast cancer, prostate cancer, endometrial cancer, ovarian cancer, colorectal cancer, lung cancer.

[0023] Further, the drug for quantitative detection is used for detecting the pAKT expression level of a single PET lesion, and is also used for detecting the pAKT expression level before and after PI3K / AKT pathway related drug treatment. The detection result can be further used for guiding drug use, pathway research, mechanism research, drug development, etc. For example, by comparing the pAKT expression level before and after PI3K / AKT pathway related drug treatment, it can be distinguished whether the pAKT level is increased due to the activation of the PI3K / AKT pathway (such as PIK3CA mutation, HER2, EGFR mutation), or the pAKT level is increased due to the loss of PTEN, and then the efficacy of the PI3K / AKT pathway related drug is predicted to guide precise drug use.

[0024] The sixth technical solution of the present application provides a pharmaceutical composition comprising the small molecule compound targeting pAKT, or the PET tracer precursor compound, or the PET tracer targeting pAKT; and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0025] The seventh technical solution of the present application provides a kit comprising the small molecule compound targeting pAKT, or the PET tracer precursor compound, or the PET tracer targeting pAKT; and other detection related reagents.

[0026] Further, the kit has high affinity and specific recognition ability for pAKT; in various tumor models, the kit can clearly distinguish between tumors with high and low pAKT expression; after short-term PI3Kα inhibitor treatment, the kit can sensitively capture the decrease in pAKT level in PIK3CA mutant tumors and detect the treatment response.

[0027] Compared with the prior art, the above technical solutions adopted by the embodiments of the present application can achieve at least the following beneficial effects: (1) Safety and specificity are more guaranteed: based on the analog compound I of the small molecule drug Capivasertib which has been clinically applied or tested, the clinical transformation threshold is lower.

[0028] (2) High radiochemical purity and yield: the preparation process is simple and controllable, the ligand chelation and labeling efficiency are high, and it is easy to meet the clinical batch preparation demand.

[0029] (3) Excellent in vivo and in vitro stability: while ensuring high affinity to pAKT, the molecule has good stability in blood, tissue and tumor microenvironment, which can reduce non-specific binding and off-target effects.

[0030] (4) Good imaging contrast: Compared with GDC0068-based precursor developed for 68 Ga-pAKTi probe (Patent No: CN116333035A), the Ga-oncopAKTi developed based on a completely different molecular structure in the present application has a high uptake at tumor sites and a low uptake in background tissues, especially in tissues such as liver and gastrointestinal tract, and can provide clearer and more accurate image information. The present application meets the need for new specific compounds for tumor tissues and their use as tumor tracers and therapeutic drugs for pAKT high expression in nuclear medicine. 68 Ga-oncopAKTi has a high uptake at tumor sites and a low uptake in background tissues, especially in tissues such as liver and gastrointestinal tract, and can provide clearer and more accurate image information. The present application meets the need for new specific compounds for tumor tissues and their use as tumor tracers and therapeutic drugs for pAKT high expression in nuclear medicine.

[0031] (5) Wide application: Abnormal activation of the PI3K-AKT signaling pathway and PTEN deletion leading to persistent abnormal activation of pAKT are common in various solid tumors and hematological tumors, which can provide precise diagnosis and evaluation means for patients with various types of tumors. It can not only realize qualitative diagnosis of pAKT in various tumors, and then realize non-invasive precise quantification of pAKT in tumors, and provide reliable basis for clinical staging, treatment decision-making, and efficacy monitoring; it can also detect the changes of pAKT before and after short-term treatment of PI3K-AKT drugs through PET imaging comparison, to predict the long-term efficacy of the drugs, and then guide the precise use of PI3K-AKT drugs, and ultimately realize precise diagnosis and treatment. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0033] Figure 1 The preparation schematic diagram of the PET tracer labeling precursor DOTA-oncopAKTi for targeting pAKT, wherein formula A, B and C represent compounds A, B and C respectively, and the starting compound is compound I; Figure 2 The radio-HPLC spectrum of the precursor DOTA-oncopAKTi (A) and the radioactive probe 68 Ga-DOTA-oncopAKTi (B), and 68 The radioactive probe obtained after Ga labeling 68 Ga-DOTA-oncopAKTi (C); Figure 3 The radioactive probe 68In vitro stability experiment of Ga-DOTA-oncopAKTi; Label: (A) in PBS buffer, (B) in mouse serum; Figure 4 For radioactive probe 68 Blood clearance experiment of Ga-DOTA-oncopAKTi in mice; Figure 5 For blocking agent Capivasertib and radioactive probe 68 Competitive inhibition binding experiment of Ga-DOTA-oncopAKTi in MDA-MB-468 cells; Figure 6 For radioactive probe 68 Biodistribution study of Ga-DOTA-oncopAKTi; Figure 7 For radioactive probe 68 Cellular uptake study of Ga-DOTA-oncopAKTi; Label: (A) Western Blot result figure of AKT pathway related protein expression level in 6 breast cancer cells; (B) radioactive probe 68 Uptake ability of Ga-DOTA-oncopAKTi over time; Figure 8 For radioactive probe 68 Micro-PET / CT imaging of Ga-DOTA-oncopAKTi in multiple mouse tumor models; Label: (A) whole body and tumor site PET / CT fusion images of 4 groups of tumor-bearing mice (HCC-1806, MDA-MB-157, MDA-MB-231, MDA-MB-468); (B) quantitative statistical box plot of probe uptake amount of tumor tissue of 4 groups of tumor models; Figure 9 For radioactive probe 68 Micro-PET / CT imaging of Ga-DOTA-oncopAKTi in PTEN WT and PTEN KO tumor models; Label: (A) protein expression level of PTEN, p-AKT (including Thr308 and Ser473), AKT, ACTIN (internal reference) in HCC-1806 cells under PTEN WT (wild type) and PTEN KO (knockout) states; (B) is a small animal micro-PET / CT whole body and local imaging figure; (C) is a quantitative statistical box plot; Figure 10 For radioactive probe 68Ga-DOTA-oncopAKTi micro-PET / CT imaging in PIK3CA Vector and PIK3CA H1047R tumor models; Label: (A) Protein expression levels of pAKT (including Ser473 and Thr308 sites), AKT, ACTIN (internal reference) in HCC-1806 cells, PIK3CA Vector (normal PIK3CA gene status) and PIK3CA H1047R (PIK3CA gene mutation status); (B) is the whole body and local micro-PET / CT imaging figure; (C) is the quantitative statistical box plot; Figure 11 For short-term PI3Kα inhibitor treatment before and after, radioactive probe 68 Ga-DOTA-oncopAKTi micro-PET / CT imaging in PTEN deletion and PIK3CA mutation tumor models; Label: (A) is the micro-PET / CT imaging figure of PTEN related tumor models (PTEN WT, PTEN KO) before and after PI3Kα inhibitor Alpelisib treatment; (B) is the micro-PET / CT imaging figure of PIK3CA related tumor models (PIK3CA Vector, PIK3CA H1047R) before and after Alpelisib treatment; (C) (D) are quantitative statistical scatter plots; Figure 12 For based on 68 Ga-oncopAKTi micro-PET / CT imaging evaluation of long-term PI3Kα inhibitor treatment on tumor-bearing mice; Label: (A) tumor real object comparison; (B) tumor growth curve; (C) tumor weight statistics; (D)-(F) PTEN related model supplementary verification; Figure 13 For mechanism summary schematic diagram. DETAILED DESCRIPTION

[0034] The embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0035] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] Example 1: Preparation of DOTA-oncopAKTi, a PET tracer-labeled precursor targeting pAKT like Figure 1 As shown, this embodiment provides a stepwise synthesis process for the PET tracer-labeled precursor DOTA-oncopAKTi targeting pAKT: Compound I → Compound A → Compound B → Compound C → PET tracer-labeled precursor DOTA-oncopAKTi. Through a series of chemical reactions and purification operations, the target product DOTA-oncopAKTi was successfully prepared. The specific steps are as follows: (1) Synthesis of compound A In a 50 mL flask, compound 9-fluorenyl methyl ester-diethylene glycol-carboxylic acid (FmocNH-PEG2-COOH, 1.3 eq, i.e., FmocNH-PEG in this example) was added sequentially. n The n in -COOH was selected as 2), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 1.3 eq), N,N-diisopropylethylamine (DIPEA, 2.6 eq), and compound I (a small molecule compound targeting pAKT after micro-modification of the Capivasertib structure, 1.0 eq) and dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF). The reaction system was stirred overnight at room temperature. After the reaction was monitored by TLC (a routine procedure well known to those skilled in the art, and therefore detailed procedures are not provided here), saturated sodium bicarbonate (NaHCO3) solution (2 × 30 mL) was added to the reaction system, followed by extraction with ethyl acetate (EA, 3 × 50 mL), and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate (Na2SO4), filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain compound A as a solid powder.

[0037] (2) Synthesis of compound B In a 50 mL round-bottom flask, compound A (1 eq) was added and dissolved in 5 mL of 20% diethylamine solution. The reaction system was stirred at room temperature for 3 h, and the reaction progress was monitored by TLC until it was complete. The reaction system was concentrated under reduced pressure, washed repeatedly with dichloromethane (DCM), and the remaining hydrochloric acid was removed by vacuum filtration to obtain compound B.

[0038] (3) Synthesis of compound C In a 50 mL round-bottom flask, compounds 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-triacid derivatives (DOTA-TRIS(ACID), 1.3 eq), HATU (1.1 eq), DIPEA (2.6 eq), and compound B (1 eq) were sequentially dissolved in 5 mL of anhydrous DMF. The reaction mixture was stirred overnight at room temperature. After the reaction was completed by TLC monitoring, saturated NaHCO3 solution (2 × 30 mL) was added to the reaction mixture, followed by extraction with EA (3 × 50 mL). The organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain compound C as a solid powder.

[0039] (4) Synthesis of DOTA-oncopAKTi, a precursor compound for PET tracer labeling In a 50 mL round-bottom flask, compound C (1 eq) was added and dissolved in 5 mL of 90% trifluoroacetic acid (TFA) solution (TFA / DCM = 9:1, v / v). The reaction system was stirred at room temperature for 3 h, and the reaction progress was monitored by TLC until completion. The reaction system was concentrated under reduced pressure, washed with diethyl ether, and the remaining hydrochloric acid was removed by vacuum filtration to obtain the PET tracer-labeled precursor compound DOTA-oncopAKTi.

[0040] The structural formula of compound AC mentioned in the above steps is as follows: Figure 1 The structure shown in Chinese AC is modified in other embodiments according to different raw materials.

[0041] Example 2 Compound 68 Preparation and Radiochemical Purity Verification of Ga-DOTA-oncopAKTi from 68 Ge- 68 4 mL was obtained by rinsing in the Ga generator. 68GaCl3 eluent was added sequentially with 40 μL of the DOTA-oncopAKTi (1 mg / mL) solution, a PET tracer-labeled precursor compound prepared in Example 1, and 0.408 mL of NaOAc (1.5 M) solution. The reaction system was incubated at 90-100 °C for 10 min, then cooled to room temperature. After dilution with 12 mL of sterile water, the labeling system was passed through a C1... 18 Column (SEP-PAK), product adsorption onto C 18 Column, inject 1.5 mL of ethanol-water solution (V) 乙醇 :V 水 =70% / 30%) will transfer the product from C 18 In-column elution. The products were evaluated by radio-iTLC (developing solvent NH4OAc (1M) / MeOH = 1 / 1) and radio-HPLC, respectively.

[0042] See results Figure 2 As shown in AB, after purification 68 The radiochemical purity of Ga-DOTA-oncopAKTi is still greater than 99%, meeting the standards for subsequent characterization and biological experiments. The labeled radioactive probe obtained in this embodiment... 68 Ga-DOTA-oncopAKTi structure as follows Figure 2 As shown in C.

[0043] Example 3 Radioactive probe 68 In vitro stability experiment of Ga-DOTA-oncopAKTi Blood was collected from BALB / c mice, and serum was separated using standard methods. Several EP tubes were taken, and 200 μL of PBS buffer (control group) or 200 μL of mouse serum (experimental group) was added to each tube. Then, 20 μL of freshly prepared PBS was added to each tube. 68 Ga-DOTA-oncopAKTi solution was incubated in a 37°C water bath, and observations were conducted at different time points (0, 1, 2, 3 h).

[0044] After incubation, an equal volume (200 μL) of acetonitrile was added to the serum samples, and the samples were centrifuged at 12000 g for 10 min at room temperature. The supernatant was collected, filtered through a 0.22 μm filter, and used for analysis. The PBS samples were analyzed directly without pretreatment. All samples were determined using high-performance liquid chromatography (radio-HPLC). 68 The radiochemical purity of Ga-DOTA-oncopAKTi was determined to assess its in vitro stability in different systems.

[0045] likeFigure 3 As shown, 68 Ga-DOTA-oncopAKTi incubated in PBS buffer and mouse serum for 0-3 h, the radiochemical purity is higher than 99%, no obvious degradation is observed, indicating 68 Ga-DOTA-oncopAKTi has good stability in vitro.

[0046] Example 4 Radioactive probe 68 Blood clearance experiment of Ga-DOTA-oncopAKTi in mice Select 5 BALB / c nude mice, each through the tail vein injection 68 Ga-DOTA-oncopAKTi solution (injection dose is 3.4 MBq per mouse). At the preset time points (3.0, 5.0, 10.0, 15.0, 30.0, 60.0, 90.0, 120.0 and 180.0 min), the blood samples were collected by piercing the end of the opposite tail vein vessel using a capillary. Blood samples from 5 mice at each time point were collected for parallel detection.

[0047] The collected blood samples were measured for radioactivity using a gamma counter and corrected for radioactive decay. The count data at each time point were analyzed using GraphPad Prism software, and the blood concentration-time curve was plotted as shown in Figure 4 The results show that, 68 Ga-DOTA-oncopAKTi has a relatively fast blood clearance characteristic in mice, and the clearance phase half-life (t 1 / 2 ) is 20.12 minutes.

[0048] Example 5 Drug Capivasertib and radioactive probe 68 Competitive inhibition binding experiment of Ga-DOTA-oncopAKTi in MDA-MB-468 cells Triple-negative breast cancer cells MDA-MB-468 were inoculated in a 24-well plate (1 × 10 5 cells per well) and incubated at 37°C overnight until the cells adhered. The next day, the cells were washed twice with pre-cooled PBS, and then 68 Ga-DOTA-oncopAKTi (0.37 MBq per well) and different concentrations (10 -5 , 10 -4 , 10 -3 , 10 -2 , 10 -1 , 10 1 , 10 2 , 10 3 , 104 10 5 Cells were co-incubated with the inhibitor Capivasertib (nM) for 1 hour to perform a competitive binding assay. After incubation, cells were gently washed three times with pre-cooled PBS, followed by trypsin to detach the cells. The cell suspension was collected, and the radioactivity of the cells was detected using a gamma counter. Nonlinear regression fitting was performed on the experimental data using GraphPad Prism software. The results are as follows: Figure 5 As shown, the calculated inhibition of Capivasertib is... 68 Ga-DOTA-oncopAKTi at a cell-binding activity concentration of 50% (IC50) 50 The value is 0.83 nM.

[0049] Example 6 Radioactive Probe 68 Biological distribution study of Ga-DOTA-oncopAKTi 3.4 MBq was injected via the tail vein into tumor-bearing female BALB / c nude mice. 68 Mice were euthanized and dissected at 30, 60, and 90 min after administration of Ga-DOTA-oncopAKTi solution. Organs including heart, liver, spleen, lungs, kidneys, brain, intestines, stomach, bladder, bones, muscles, blood, and tumors were collected. The radioactivity of these organs was measured using a gamma counter, and the radioactivity per gram of tissue was calculated. The uptake value within the organs was expressed as the percentage of the injected dose taken up per gram of tissue (%ID / g).

[0050] like Figure 6 As shown, 68 In the MDA-MB-468 tumor model, Ga-DOTA-oncopAKTi exhibited good tumor enrichment at 30, 60, and 90 minutes post-injection, demonstrating its highly specific binding to pAKT. Simultaneously, the probe showed extremely low uptake in the heart, gastrointestinal tract, bone, muscle, and brain tissues, indicating excellent tissue contrast and imaging background signal-to-noise ratio. The probe was primarily cleared via the kidneys, with high background radioactive uptake observed in kidney tissue.

[0051] Example 7 Radioactive Probe 68 Cellular uptake study of Ga-DOTA-oncopAKTi HCC-1806, MDA-MB-157, SUM-159PT, MDA-MB-231, MDA-MB-468, and BT-549 are all breast cancer cell lines. Figure 7 A represents the expression levels of PTEN and p-AKT proteins in cells. Figure 7 B represents cell pairs.68 Ga-DOTA-oncopAKTi uptake was assessed. The above cell lines were seeded into 6-well plates (1 × 10⁻⁶). 6 (1 cell / well) were cultured at 37°C and 5% CO2 until adherent.

[0052] After the cells adhered, add to each well 68 Ga-DOTA-oncopAKTi (0.37 MBq / well) was incubated at 37°C for 5, 10, 15, 30, and 60 min, respectively. After incubation, cells were gently washed three times with pre-chilled PBS buffer to remove unbound radioactive probes, followed by the addition of 0.25% trypsin / 0.02% EDTA solution to detach the cells from the culture plate. After confirming complete cell separation using an inverted microscope, the cell suspension was collected. The radioactivity of the collected cell suspension was measured using a gamma counter, and all data were attenuated. Figure 7 As shown in B, HCC-1806, MDA-MB-157, and SUM-159PT... 68 Ga-DOTA-oncopAKTi uptake is low, MDA-MB-231 has low uptake. 68 Ga-DOTA-oncopAKTi uptake is slightly higher, MDA-MB-468 and BT-549 uptake is lower. 68 Ga-DOTA-oncopAKTi had the highest uptake.

[0053] In conclusion, 68 Ga-DOTA-oncopAKTi exhibits good specificity for cells with different pAKT expression levels.

[0054] Example 8 Radioactive Probe 68 micro-PET / CT imaging of Ga-DOTA-oncopAKTi in various mouse tumor models Tumor models were established by subcutaneously inoculating female BALB / c nude mice with HCC-1806, MDA-MB-157, MDA-MB-231, and MDA-MB-468 cell lines. Tumors were cultured until they reached approximately 500-600 mm². 3 In vivo molecular imaging experiments were then conducted. Each tumor-bearing mouse was injected via the tail vein. 68 Ga-DOTA-oncopAKTi probe, injection dose is 7.4 MBq / insect.

[0055] One hour after injection, whole-body imaging was performed using small animal micro-PET / CT. After image acquisition, regions of interest (ROIs) were delineated and quantitatively analyzed using Inveon Research Workplace software to compare the probe uptake capabilities of different tumor models.

[0056] like Figure 8 As shown, Figure 8 The top row (A) shows whole-body 3D images, with the color bar "%ID / g" representing the "percentage injection dose per gram of tissue". It can be seen that the MDA-MB-468 tumor site (highlighted area in the image) is the reddest and has the highest uptake value, followed by the MDA-MB-231 tumor, indicating that the probe accumulates more within the tumor. The HCC-1806 and MDA-MB-157 tumors are darker and have lower uptake values. The bottom row shows tomographic images of the tumor site, providing a clearer comparison of the probe distribution across different models. Figure 8 B represents the ratio of radioactive probe uptake in tumor tissue (T) to that in non-tumor tissue (NT), more accurately reflecting "tumor-specific uptake." A higher ratio indicates more significant probe enrichment within the tumor compared to normal tissue, signifying stronger targeting. Therefore, the MDA-MB-231 and MDA-MB-468 tumor models with high pAKT expression are effective in... 68 Ga-DOTA-oncopAKTi showed higher uptake, while the HCC-1806 and MDA-MB-157 tumor models with low pAKT expression exhibited... 68 Ga-DOTA-oncopAKTi exhibited lower intake levels.

[0057] In conclusion, 68 Ga-DOTA-oncopAKTi exhibits good specificity for mouse tumor models with different pAKT expression levels.

[0058] Example 9 Radioactive Probe 68 Ga-DOTA-oncopAKTi in micro-PET / CT imaging of PTEN WT and PTEN KO tumor models Two mouse tumor models were constructed: HCC-1806 PTEN WT (low pAKT expression) and HCC-1806 PTEN KO (PTEN loss, high pAKT expression, KO indicating gene knockout). Tumors were tested when the tumor volume reached approximately 500–600 mm². 3 Imaging experiments were then performed. Each mouse was injected via the tail vein. 68 Ga-DOTA-oncopAKTi probe, injection dose is 7.4 MBq / insect.

[0059] One hour after injection, whole body imaging was performed using small animal micro-PET / CT. After image acquisition, the region of interest (ROI) was outlined and quantitatively analyzed using Inveon Research Workplace software to assess the uptake of the probe by each tumor model.

[0060] As shown in FIG. 1, in the in vivo experiment in mice, the protein level of pAKT in the PTEN KO group was significantly higher than that in the PTEN WT group due to the loss of PTEN protein expression caused by gene deletion. Figure 9 Figure 9 A) confirmed that the p-AKT (Thr308, Ser473 site) phosphorylation level in the PTEN KO group was significantly higher than that in the PTEN WT group due to the loss of PTEN protein expression caused by gene deletion. Figure 9 B-9C) visually and quantitatively showed that the uptake of the probe by the tumor in the PTEN WT group with low pAKT expression was lower, while the uptake of the probe by the tumor in the PTEN KO group with high pAKT expression was higher. This fully demonstrates that 68 Ga-DOTA-oncopAKTi can accurately reflect the abnormal activation of pAKT caused by the loss of PTEN.

[0061] In summary, 68 Ga-DOTA-oncopAKTi can accurately reflect the abnormal activation of pAKT caused by the loss of PTEN.

[0062] Example 10 Radioactive probe 68 Micro-PET / CT imaging of Ga-DOTA-oncopAKTi in PIK3CA Vector and PIK3CA H1047R tumor models Two mouse tumor models, HCC-1806 PIK3CA Vector (PIK3CA gene is in a normal state, the PI3Kα protein encoded by it functions normally, and the expression level of pAKT is low) and HCC-1806 PIK3CA H1047R (high expression of pAKT), were constructed. When the tumor volume reached about 500-600 mm 3 , the imaging experiment was performed. Each mouse was injected with 68 Ga-DOTA-oncopAKTi probe through the tail vein, and the injection dose was 7.4 MBq per mouse.

[0063] One hour after injection, whole body imaging was performed using small animal micro-PET / CT. After image acquisition, the region of interest (ROI) was outlined and quantitatively analyzed using Inveon Research Workplace software to assess the uptake of the probe by each tumor model. ​

[0064] As shown in Figure 10 Figure 1, in the mouse in vivo experimental system, protein level detection Figure 10 A) clearly showed that the p-AKT (Ser473, Thr308 sites) phosphorylation degree of the PIK3CA H1047R group was greatly improved compared with the PIK3CA Vector group, and there was no significant difference in AKT total protein expression; micro-PET / CT imaging Figure 10 B-10C) all showed that the tumor of the PIK3CA Vector group with low expression of pAKT had lower uptake of the probe. The tumor of the PIK3CA H1047R group with high expression of pAKT had higher uptake of the probe.

[0065] In summary, 68 Ga-DOTA-oncopAKTi can accurately reflect the abnormal activation of pAKT caused by PIK3CA mutation.

[0066] Example 11 Radioactive probe 68 Ga-DOTA-oncopAKTi micro-PET / CT before and after contrast imaging can distinguish high pAKT tumors caused by two mechanisms of PTEN deletion and PIK3CA mutation.

[0067] Four mouse tumor models of HCC-1806 PTEN WT, HCC-1806 PTEN KO, HCC-1806 PIK3CA Vector, and HCC-1806 PIK3CA H1047R were constructed. When the tumor volume reached about 500-600 mm 3 , the first imaging experiment was performed. After 3 days of treatment with PI3Kα inhibitor Alpelisib (30 mg / kg / d), the second imaging was performed. Each mouse was injected with 68 Ga-DOTA-oncopAKTi probe through the tail vein, and the injection dose was 7.4 MBq per mouse.

[0068] One hour after injection, whole body imaging was performed using a small animal micro-PET / CT, and after image acquisition, the Inveon Research Workplace software was used to draw and quantitatively analyze the region of interest (ROI) to evaluate the uptake of the probe by each tumor model.

[0069] As shown in Figure 11 In the mouse tumor model experiment, four models were constructed, and micro-PET / CT imaging Figure 11A-11B) Demonstration: In short-term (3 days) PI3K a inhibitor Alpelisib treatment, pAKT signal in PTEN loss tumor is persistently activated, while pAKT signal in PIK3CA mutant tumor is effectively inhibited. Quantitative scatter plot (left) and representative images (right) of pAKT micro-PET / CT imaging. Figure 11 C-11D) Further quantification of the change of probe uptake before and after treatment, PIK3CA H1047R model shows a significant decrease in uptake after treatment, while PTEN KO model shows no significant change.

[0070] In summary, 68 Ga-oncopAKTi can accurately distinguish the abnormal activation of pAKT caused by two different mechanisms of PTEN loss and PIK3CA mutation.

[0071] Example 12 Based on 68 Ga-oncopAKTi micro-PET / CT imaging, the efficacy of long-term PI3K a inhibitor treatment on tumor-bearing mice was evaluated.

[0072] Example 11 has demonstrated that short-term (3 days) PI3K a inhibitor Alpelisib (30mg / kg / d) treatment on tumor-bearing mice, by 68 Ga-oncopAKTi micro-PET / CT imaging, it can be seen that the pAKT signal in PTEN loss tumor is persistently activated after treatment, while the pAKT signal in PIK3CA mutant tumor is effectively inhibited.

[0073] On this basis, further long-term (30 days) Alpelisib (30mg / kg / d) treatment was performed on the two models to evaluate their efficacy. As shown in Figure 12 macro-therapeutic effect, PIK3CA mutant (H1047R) tumor after long-term treatment, the real volume significantly reduced, the growth curve slope dropped sharply, the endpoint weight statistics also verified the volume reduction ( Figure 12 A-12C); while PTEN loss type, the volume and weight of the treatment group and the control group have no difference ( Figure 12 D-12F). It can be seen that PIK3CA mutant tumor is significantly inhibited after long-term treatment, while PTEN loss tumor has no obvious response to treatment.

[0074] In summary, as shown in Figure 13 short-term PI3K a inhibitor treatment combined with 68 Ga-oncopAKTi micro-PET / CT imaging, can accurately evaluate the efficacy of long-term PI3K a inhibitor, and reflect the different mechanisms of PI3K-AKT activation and PTEN loss leading to elevated pAKT levels in vivo, and guide the precise use of PI3K-AKT pathway drugs.

[0075] The above description of the embodiments is intended to explain the technical concepts and features of the present application, and its purpose is to enable those skilled in the art to understand and use the application, and cannot be used to limit the protection scope of the present application. Those skilled in the art can make various modifications to the above embodiments, and apply the general principles of this description to other embodiments. Therefore, any equivalent improvements and modifications made without departing from the scope of the present application shall be covered by the protection scope of the present application.

Claims

1. A small molecule compound targeting pAKT, characterized in that, The small molecule compound targeting pAKT has a structure as shown in Formula I: Formula I.

2. A PET tracer precursor compound targeting pAKT, characterized in that, The PET tracer precursor compound is DOTA-oncopAKTi, which has a structure as shown in any one of Formula II, Formula III or Formula IV: Formula II; Formula III; Formula IV; or a pharmaceutically acceptable salt thereof, wherein, in Formula II~IV: n is an integer selected from 1 to 10.

3. The PET tracer precursor compound according to claim 2, characterized in that, The compound has a structure as shown in Formula V: Formula V; or a pharmaceutically acceptable salt thereof.

4. A process for the preparation of a PET tracer precursor compound according to claim 2 or 3, characterized in that, The preparation method comprises the following steps: Step S1: mixing compound I, FmocNH-PEGn-COOH or FmocNH-(CH2)n-COOH or FmocNH-(Gly)n-COOH, HATU and DIPEA to obtain compound A; the equivalent relationship of the compound I, FmocNH-PEGn-COOH, HATU and DIPEA is 1.0 eq:1.3 eq:1.3 eq:2.6 eq; The FmocNH-PEGn-COOH corresponds to the PET tracer labeling precursor compound as shown in Formula II in claim 2, the FmocNH-(CH2)n-COOH corresponds to the PET tracer labeling precursor compound as shown in Formula III in claim 2, and the FmocNH-(Gly)n-COOH corresponds to the PET tracer labeling precursor compound as shown in Formula IV in claim 2; n in the FmocNH-PEGn-COOH or FmocNH-(CH2)n-COOH or FmocNH-(Gly)n-COOH is equal to n in the structure of the PET tracer labeling precursor compound DOTA-oncopAKTi as shown in Formula II, III or IV in claim 2, respectively; Step S2: dissolving compound A in dichloromethane, and adding diethylamine solution to obtain target compound B; Step S3: mixing compound B, DOTA-TRIS(ACID), HATU and DIPEA to obtain compound C; the equivalent relationship of the compound B, DOTA-TRIS(ACID), HATU and DIPEA solvent is 1.0 eq:1.3 eq:1.1 eq:2.6 eq; Step S4: dissolving compound C in dichloromethane, and adding trifluoroacetic acid solution to obtain the PET tracer labeling precursor compound DOTA-oncopAKTi; The compounds A, B and C are intermediate products of the steps, and are different according to different FmocNH-PEGn-COOH or FmocNH-(CH2)n-COOH or FmocNH-(Gly)n-COOH.

5. A PET tracer targeting pAKT, characterized in that, The PET tracer is radiolabeled by the PET tracer precursor compound as claimed in claim 2 or 3; the radiolabeled element is located in the DOTA chelating group; and the radiological element is combined with the compound DOTA-oncopAKTi through coordination bond; said radiolabel is 68 Ga, 67 Ga, 111 In, 64 Cu, 89 Zr, 90 Y, 177 Lu, 153 Sm or 166 Ho; PET tracer precursor compounds having the structure of Formula V are prepared by 68 Ga-labeled have the structure of Formula VI: Formula VI.

6. A method of preparing a PET tracer as claimed in claim 5, characterized in that, The preparation method comprises the following steps: (1) preparing an eluent containing radioactive ions; (2) adding a sodium acetate solution to the eluent to adjust the pH value of the system to 4.0-4.2; (3) adding a PET tracer labeled precursor compound DOTA-oncopAKTi solution as claimed in claim 2 or 3, and after reaction at 90-100℃ for 10-20 min, cooling to room temperature; separating and purifying the reaction solution after reaction to obtain a PET tracer targeting pAKT labeled with radioactivity.

7. Use of the small-molecule compound targeting pAKT as claimed in claim 1, or the PET tracer precursor compound as claimed in claim 2 or 3, or the PET tracer targeting pAKT as claimed in claim 5 in the preparation of a medicament for the quantitative detection of pAKT protein in a biological sample.

8. Use according to claim 7, characterized in that, The biological sample is derived from an ex vivo tumor or inflammatory tissue sample; the tumor is any one or several of breast cancer, prostate cancer, endometrial cancer, ovarian cancer, colorectal cancer, lung cancer.

9. A pharmaceutical composition, characterized by, Any one of the small-molecule compound targeting pAKT as claimed in claim 1, or the PET tracer precursor compound as claimed in claim 2 or 3, or the PET tracer targeting pAKT as claimed in claim 5, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

10. A kit characterized in that, The kit comprises one or more of the small-molecule compound targeting pAKT as claimed in claim 1, or the PET tracer precursor compound as claimed in claim 2 or 3, or the PET tracer targeting pAKT as claimed in claim 5.

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