Tumor probe of fluorine-18 labeled targeted PCNA protein as well as preparation method and application of tumor probe

Abstract

The invention provides a PET (Polyethylene Terephthalate) micromolecule probe [18F]-N-(2-((2-(3-(2-fluorooxy) phenyl) phenyl) amine)-2-oxoethyl)-1-naphthamide (abbreviated to [18F]-FPON) of a targeted proliferating cell nuclear antigen (PCNA), a synthesis scheme of the PET micromolecule probe, and application of the PET micromolecule probe as a radioactive tracer to diagnosis of tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical imaging technology, and particularly relates to a fluorine-18 labeled tumor PET molecular probe targeting proliferating cell nuclear antigen (PCNA) as well as a preparation method and application thereof. BACKGROUND

[0002] PCNA (Proliferating Cell Nuclear Antigen) is a highly conserved protein that plays a central role in DNA replication, repair, and cell proliferation, and is considered a key therapeutic target in various cancers, particularly in glioma and colorectal cancer (Wang T, et al. Targeting the "Undruggable": Small-Molecule Inhibitors of Proliferating Cell Nuclear Antigen (PCNA) in the Spotlight in Cancer Therapy. J Med Chem. 2025;68(3):2058-2088.). In glioma, the upregulation of PCNA expression is associated with cell proliferation signaling pathways, such as FKBP10 activating AKT-CREB-PCNA cascade by interacting with Hsp47, promoting tumor growth (Cai HQ, et al. FKBP10 promotes proliferation of glioma cells via activating AKT-CREB-PCNA axis. J Biomed Sci. 2021;28(1):13.). In addition, PCNA-related biomarkers such as PCBP2 and circNALCN are highly expressed in glioma and associated with poor prognosis. In colorectal cancer, PCNA, as a marker of cell proliferation, is significantly involved in cancer progression, and its expression is reduced in PCNA-positive cells under the action of the chemopreventive agent DMC, thereby inhibiting tumor formation (Karami Fath M, et al. Anti-cancer peptide-based therapeutic strategies in solid tumors. Cell Mol Biol Lett. 2022;27(1):33.). PCNA inhibitors such as ATX-101 and AOH1996 exhibit the ability to selectively kill cancer cells by interfering with transcription and repair processes, becoming a potential therapeutic strategy, and this target has the advantage of its broad application prospects across cancers (Gu L, et al. Small molecule targeting of transcription-replication conflict for selective chemotherapy. Cell Chem Biol. 2023;30(10):1235-1247.e6.).

[0003] PET technology dynamically monitors the biological characteristics of tumors through radioactive tracers, and its functional imaging advantage has unique value in early diagnosis, molecular typing and efficacy evaluation of tumors (Kim D, et al. Correction to: Recent Update on PET / CT Radiotracers for Imaging Cerebral Glioma. Nucl Med Mol Imaging. 2025; 59(1): 91.).

[0004] Although PCNA plays a key role in tumor development, there is still a lack of non-invasive molecular imaging probes targeting this target. Compared with traditional imaging methods (MRI, CT, X-ray, ultrasound), the development of PET molecular probes targeting PCNA has multiple advantages: first, the expression level of PCNA can be traced to realize real-time quantitative evaluation of tumor proliferation activity; second, it can overcome the limitations of existing tracers in grading identification; more importantly, the design of probes based on PCNA-specific subtypes is expected to realize specific imaging of malignant tumors, providing a new tool for precise diagnosis and treatment of tumors. With the progress of new radio-labeling technology and molecular probe design strategy, such probes show broad application prospects in the fields of tumor molecular typing, treatment target screening and efficacy dynamic monitoring. SUMMARY

[0005] AOH1996 is a small molecule inhibitor targeting PCNA, which has good affinity with PCNA and good therapeutic effect on most tumors in animal models (Gu L, et al. Small molecule targeting of transcription-replication conflict for selective chemotherapy. Cell Chem Biol. 2023; 30(10): 1235-1247.e6.).

[0006] Based on the structure of AOH1996, the present application optimizes and modifies it, and invents a similar probe precursor OTs-FPON and a non-radioactive probe standard FPON. With the designed precursor and 18 F ion as raw material, a 18 F-labeled small molecule PET probe is prepared, the reaction process is simple, the raw material is easy to synthesize, and in the orthotopic mouse model of brain glioblastoma and the subcutaneous tumor model of colon cancer, the tumor has good specific uptake of the probe.

[0007] The purpose of the present application is to provide the above 18F-labeled small molecule PET probe targeting PCNA for tumor, application in reagent for positron emission tomography. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 Differential analysis of PCNA expression in common tumors and normal tissues Figure 2 Structure of the screening molecule of the probe, parameter table, optimal structure and docking mode diagram of AOH1996 and PCNA (PDB: 1vym) Figure 3 Synthesis process of probe standard FPON (1a) Figure 4 Synthesis process of probe precursor OTs-FPON (1b) Figure 5 Results of using BLI technology to detect the affinity of FPON and PCNA (HIS-tag) Figure 6 PET molecular probe 18 Synthesis process of F-FPON and TLC and HPLC results for evaluating the synthesis quality of the probe Figure 7 Results of in vivo metabolic stability and biodistribution of the probe in SD rats and results of biodistribution of the probe in tumor-bearing mice Figure 8 MRI image of U87 glioma orthotopic model and PET imaging results and quantification of the probe Figure 9 Radioautography results of the probe in the brain tissue of U87 glioma orthotopic model mice, fusion with MRI and quantification Figure 10 Immunohistochemical staining results of PCNA in U87 glioma tissue Figure 11 PET imaging results and quantification of the probe in MC38 colon cancer subcutaneous tumor model

[0009] The specific technical solutions of the present application are as follows:

[0010] Differential analysis of PCNA expression in common tumors and normal tissues Figure 1 )

[0011] (1) Experimental design: Using the Internet database GEPIA2, PCNA gene expression in tumors such as glioma (GBM, LGG), colorectal adenocarcinoma (COAD), invasive breast cancer (BRCA), esophageal cancer (ESCA), hepatocellular carcinoma (LIHC), squamous cell carcinoma of the lung (LUSC), pancreatic cancer (PAAD), and gastric cancer (STAD) and normal tissues was evaluated by matching Cancer Genome Atlas (TCGA) and GTEx data. (2) Experimental results: We can see that there are significant differences in PCNA expression between most tumors and normal tissues.

[0012] Probe structure screening ( Figure 2 )

[0013] (1) Experimental design: Based on the structure of AOH1996, we designed a series of possible experiments. 18 The F-labeled product structure is named as probe 1-28 ( Figure 2 A) Using SWISSDOCK and Chemdraw, predicted parameters for each structure were obtained, and the labeled products were evaluated based on scores for binding energy (docking energy), lipophilicity (clogP), molecular weight, and molecular area (tPSA). (Scoring criteria: Docking energy absolute value: <7.0=0 points, 7.0-7.2=1 point, 7.2-7.4=2 points; MW<500=1 point; Clog: <3=0 points, 3-3.5=1 point, 3.5-4=2 points, 4.0-4.5=3 points, 4.5-5=4 points, >5=5 points; tPSA>70=0 points, 60-70=1 point, <60=2 points.) (2) Experimental results: We selected the structure with the highest comprehensive score as... 18 The probe structure 14 of the F-fluorooxyethyl substituted methyl ether group was finally named FPON. Figure 2 B). Docking results of AOH1996, FPON, and target PCNA molecules showed that the fluorooxyethyl substitution of FPON did not affect the binding with PCNA, and the naphthalene ring and fluorooxyethyl group of FPON formed new hydrophobic bonds with the amino acid residues I128, Y250, and P234 of PCNA, respectively, so the binding was more stable than that of AOH1996. Figure 2 C).

[0014] Synthesis of probe standard FPON(1a) Figure 3 )

[0015] (1) Glycine (3.94 g, 52.5 mmol) was dissolved in 1.0 M NaOH, and 1-naphthoyl chloride (10.00 g, 52.5 mmol) was dissolved in dioxane (20 mL) and added to the reaction system, which was stirred at room temperature for 2 h. The reaction system was washed with EtOAc, and the aqueous phase was adjusted to be acidic with 5.0 M HC1. The precipitate was obtained by suction filtration. The obtained residue was purified by silica gel column chromatography [V(EtOAc) / V(Petroleum ether) = 0 / 100-50 / 50] to obtain compound 4 as a white solid.

[0016] (2) 2-Fluoronitrobenzene (15.00 g, 106.3 mmol) was dissolved in DMF (150 mL), and K2CO3(58.80 g, 425.2 mmol) was added. Then, resorcinol (15.22 g, 138.2 mmol) and 18-crown-6 (281.0 mg, 1.1 mmol) were added in batches. The reaction was stirred at room temperature for 12 h. TLC showed that the reaction was complete. The reaction system was poured into water and stirred. The organic phase was extracted with CH2Cl2three times, and then washed with 1.0 M NaOH once. The aqueous phase was separated and adjusted to be acidic with 1.0 N HC1. The organic phase was extracted with CH2Cl2three times, washed with saturated NaCl once, dried over anhydrous MgSO4, and filtered to remove the drying agent. The filtrate was evaporated on a rotary evaporator to obtain a residue. The residue was purified by silica gel column chromatography [V(EtOAc) / V(Petroleum ether) = 0 / 100-15 / 85] to obtain compound 7 as a yellow oily liquid.

[0017] (3) Compound 7 (1.00 g, 4.33 mmol) was dissolved in DMF (10 mL), and 1-fluoro-2-iodoethane (0.90 g, 5.19 mmol) and potassium carbonate (1.79 g, 12.98 mmol) were added. The reaction system was stirred under nitrogen protection, and the temperature was raised to 70°C. The reaction was stirred for 3 h. TLC showed that the reaction was complete. The reaction mixture was poured into water and stirred. The organic phase was extracted with EtOAc three times, washed with saturated NaCl solution twice, dried over anhydrous MgSO4, and filtered to remove the drying agent. The filtrate was evaporated on a rotary evaporator to obtain a residue. The residue was purified by silica gel column chromatography [V(EtOAc) / V(Petroleum ether) = 0 / 100-7 / 93] to obtain compound 8 as a yellow oily liquid.

[0018] (4) Compound 8 (0.90 g, 3.25 mmol) was dissolved in MeOH (10 mL), 10% Pd / C (5% by w / w) was added, and hydrogenation was carried out at room temperature under room pressure (balloon) for 12 h according to the standard procedure. TLC showed that the reaction was complete. The 10% Pd / C was removed by filtration with the aid of celite, and the filtrate was evaporated on a rotary evaporator to remove the methanol. The obtained crude product 9 was a red oily liquid, 0.90 g, which was used directly in the next step without further purification and characterization.

[0019] (5) Compound 4 (0.81 g, 3.52 mmol) was dissolved in DMF (10 mL), DIPEA (0.91 g, 7.04 mmol), compound 9 (0.58 g, 2.35 mmol) and HATU (1.34 g, 3.52 mmol) [3] were added successively, and stirring was carried out at room temperature under nitrogen protection for 12 h. TLC detection showed that the reaction was complete. The reaction mixture was poured into water, stirred, extracted with EtOAc for 3 times, and the organic phases were combined. The organic phase was washed successively with saturated NaHCO3, 0.1 N HCl and saturated NaCl, dried over anhydrous MgSO4, and the drying agent was removed by suction filtration. The filtrate was evaporated on a rotary evaporator to remove the solvent. The obtained residue was purified by silica gel column chromatography [V(EtOAc) / V(Petroleum ether) = 40 / 60-50 / 50] and slurried from an ethyl acetate / petroleum ether mixture to obtain the product la, a white solid.

[0020] Synthesis of probe precursor OTs-FPON (lb) Figure 4 )

[0021] (1) Compound 7 (4.00 g, 17.30 mmol) was dissolved in DMF (40 mL), 2-bromoethanol (2.59 g, 20.76 mmol) and potassium carbonate (7.17 g, 51.90 mmol) were added, and stirring was carried out under nitrogen protection. The temperature of the reaction system was raised to 90°C, and the reaction was carried out for 3 h. TLC detection showed that the reaction was complete. The reaction mixture was poured into water, stirred, extracted with EtOAc for 3 times, and the organic phases were combined. The combined organic phase was washed with saturated NaCl solution for 2 times, dried over anhydrous MgSO4, and the drying agent was removed by suction filtration. The filtrate was evaporated on a rotary evaporator to remove the solvent. The obtained residue was purified by silica gel column chromatography [V(EtOAc) / V(Petroleum ether) = 0 / 100-20 / 80] to obtain compound 10, a light yellow solid.

[0022] (2) Compound 10 (3.40 g, 12.35 mmol) was dissolved in dry CH2Cl2 (30 mL), stirred under nitrogen protection, and the reaction system was cooled to 0 °C. Triethylamine (2.50 g, 18.53 mmol) was added dropwise to the system using a syringe. After the addition was complete, p-TsCl (3.53 g, 18.53 mmol) was added to the reaction system in portions. The reaction system was then stirred at room temperature until the reaction was completed by TLC (generally 4 h). The reaction system was poured into water and extracted three times with CH2Cl2. The organic phases were combined, washed with saturated NaCl, dried over anhydrous MgSO4, and the desiccant was removed by filtration. The solvent was evaporated from the filtrate using a rotary evaporator. The residue was purified by silica gel column chromatography [V(EtOAc) / V(Petroleum ether) = 0 / 100-30 / 70] to obtain compound 11, a yellow oily liquid.

[0023] (3) Compound 11 (4.84 g, 11.28 mmol) was dissolved in MeOH (50 mL), and 10% Pd / C (5% by w / w) was added. Hydrogenation was carried out at room temperature (balloon) for 12 h according to the standard procedure. TLC showed that the reaction was complete. The 10% Pd / C was removed by filtration with diatomaceous earth. Methanol was evaporated from the filtrate on a rotary evaporator. The residue was added to EtOAc (10 mL) and stirred at room temperature. The solid was collected by suction filtration and dried at room temperature to obtain compound 12, a white solid.

[0024] (4) Compound 4 (1.29 g, 5.63 mmol) was dissolved in DMF (15 mL), and DIPEA (1.46 g, 11.27 mmol), compound 12 (1.5 g, 3.76 mmol) and HATU (2.14 g, 5.63 mmol) were added sequentially. The mixture was stirred at room temperature for 12 h under nitrogen protection, and the reaction was detected by TLC to indicate completeness. The reaction mixture was poured into water and stirred. The mixture was extracted three times with EtOAc, and the organic phases were combined. The organic phases were washed sequentially with saturated NaHCO3, 0.1 N HCl and saturated NaCl, dried over anhydrous MgSO4, and the desiccant was removed by filtration. The solvent was evaporated from the filtrate using a rotary evaporator. The residue was purified by silica gel column chromatography [V(EtOAc) / V(Petroleum ether) = 40 / 60-50 / 50] to obtain compound 1b, a white solid.

[0025] FPON receptor targeting ( Figure 5 ) (1) Experimental method: Biomembrane interference (BLI) technique was used to detect the affinity between the probe and PCNA.

[0026] (2) Experimental results: The results show that the affinity of AOH1996 with PCNA is 945 ± 212 nM, and the affinity Kd of FPON is 900.8 ± 131.2 nM, indicating that PCNA has good binding affinity with FPON.

[0027] 18 Synthesis of F-FPON Figure 6 ) (1) Labeling method: 18F ions are washed from the QMA column into a vial with a mixed solution of 1 mL (acetonitrile: water = 7:3) of K2CO3 (1 mg) and K 222 (5 mg). Dry with nitrogen at 110°C (10 min), then add 1 mL of acetonitrile azeotropically (5 min) to make the system as dry as possible, add 500ul of dissolved precursor (1a) at 110°C, and press the lid for 20 min. Add 5 mL of acetonitrile to the vial and inject it into the alumina column (filter membrane) to purify the product. Add 1-2 ml of ultrapure water for dilution, and use HPLC for further purification. The collected product is diluted with 10 ml of ultrapure water, and the diluted liquid is passed through a C18 column, and the product is adsorbed on the C18 column. Then elute it with 1.5 ml of anhydrous ethanol, and dilute the eluted product with physiological saline so that the ethanol concentration is not higher than 10%. At this time, the drug can be used for intravenous injection of experimental animals.

[0028] (2) Labeling results: 18 F - TLC analysis of the crude product after labeling shows that the conversion rate of the reaction is about 90%. After mixing the purified product with the standard product and passing it through HPLC, the peak time is consistent, indicating 18 F-FPON is successfully synthesized. The radiochemical yield (RCY) of this reaction is 11.43 ± 4.03% (n = 6), and the specific activity (SA) is 2.057 ± 0.8569 GBq / μmol (n = 6).

[0029] 18 Performance detection of F-FPON in animal experiments: 1. Metabolic stability in normal SD rats Figure 7 A)

[0030] (1) Experimental design: Take 3 SD rats, and inject about 500 μCi of 18F-FPON, respectively in 5 min, 30 min, 60 min dissection, take 1 ml blood, 1 g liver, 1 g brain tissue homogenate centrifugation, take supernatant HPLC, collection flow through fluid, every minute to collect a tube, a total of 20 minutes. The collected liquid on the gamma counter CPM value, the readings of each group is plotted into a curve, fitting to each peak area.

[0031] (2) experimental results: from the image, the probe in blood and liver metabolism faster, and more stable in brain tissue. With origin software to curve fitting algorithm peak area, obtained in 60 min, brain tissue probe still about 87%, indicating that its metabolism in the brain more stable, again to illustrate its as brain tumor PET probe has a high potential.

[0032] 2. normal SD rat biodistribution experiment ( Figure 7 B) (1) experimental design: first take 10 tube drug, measure the corresponding activity, then the 10 tube drug with gamma counter detection corresponding CPM value, according to the activity and CPM value to calculate the conversion coefficient K. The injection dose of each rat into CPM value (total CPM).

[0033] (2) 16 SD rats were divided into 30 min, 60 min, 90 min, 120 min four groups, each group of four. Each injection of about 200 μCi of 18 F-FPON, then in the corresponding time to kill rats take organs, with gamma counter detection corresponding CPM value, after decay, the organs were weighed. Finally according to the formula [(organ CPM / total CPM) / organ weight]*100% to calculate the ID% / g value of each organ.

[0034] (3) experimental results: 18 F-FPON mainly in liver metabolism, indicating that its fat solubility is strong, in brain tissue probe also uptake, also further illustrate its as brain tumor disease PET probe potential and feasibility.

[0035] 3. 18 F-FPON in U87 glioma in situ model of biological distribution of mice ( Figure 7 B) (1) experimental design: divided into non blocking group and blocking group, each group of 3 tumor-bearing mice. Blocking group in 10 minutes before injection of 18 F-FPON injection AOH1996 blocking. Tumor-bearing mice each injection of about 200-300 μCi of 18F-FPON, then the tumor was taken out after 60 minutes, and the corresponding CPM value was detected by a gamma counter. After the decay, the tumor was weighed. Finally, the ID% / g value of the tumor was calculated according to the formula [(organ CPM / total CPM) / organ weight]*100%.

[0036] (2) Experimental results: the uptake of the probe in the non-blocked group of U87 glioma tissue was higher than that in the blocked group of tumor tissue, which showed that 18 F-FPON had specificity as a PET probe for intracranial tumors.

[0037] 4. 18 PET imaging of F-FPON in U87 glioma orthotopic model mice Figure 8 ) (1) Experimental design: divided into non-blocked group and blocked group, and the blocked group was injected with PCNA inhibitor AOH1996 10 minutes before injecting 18 F-FPON. Each tumor-bearing mouse was injected with 10 μCi / g of 18 F-FPON, and then immediately underwent 1-hour PET / CT dynamic scanning. The PET image was fused with the MRI image.

[0038] (2) Experimental results: the probe had good imaging effect in the U87 glioma orthotopic model, the tumor localization shown by MRI had high coincidence with the tumor localization shown by PET, and the SUVr showed that the tumor had good contrast difference with the control brain tissue, which showed that 18 F-FPON was feasible as a PET probe for intracranial tumors. The SUV value of the blocked group was significantly lower than that of the non-blocked group, which showed that 18 F-FPON had specificity.

[0039] 5. 18 Radioautography of F-FPON in brain tissue of U87 glioma orthotopic model mice Figure 9 ) (1) Experimental design: each tumor-bearing mouse was injected with 1 mCi 18 F-FPON, and 30 minutes later, the brain tissue was taken out for frozen section. After processing the section samples, radioautography was performed using an ultrafast real-time digital radioautography system, and the results were fused with the MRI image.

[0040] (2) Experimental results: the uptake of the probe in the U87 glioma tissue had good contrast difference with the control brain tissue, which showed that 18 F-FPON was feasible as a PET probe for intracranial tumors.

[0041] 6. Whole brain PCNA immunohistochemistry in U87 glioma orthotopic model mice Figure 10 ) (1) Experimental design: The whole brain tissue of glioma-bearing mice was taken for PCNA immunohistochemical staining.

[0042] (2) Experimental results: The expression of PCNA in U87 glioma was significantly higher than that in the contralateral glioma, which was consistent with the PET results.

[0043] 7. 18 F-FPON6. PET imaging in MC38 colon cancer subcutaneous tumor model mice Figure 11 ) (1) Experimental design: Each tumor-bearing mouse was injected with 10 μCi / g of F-FPON, followed by 1-hour dynamic scanning. 18

[0044] (2) Experimental results: The probe had good imaging effect in MC38 colon cancer subcutaneous tumor model mice, and SUVr showed that there was a good contrast difference between the tumor and the contralateral brain tissue, indicating the feasibility of F-FPON in diagnosing colon cancer tumor. 18 F-FPON6. PET imaging in MC38 colon cancer subcutaneous tumor model mice Figure 11 ) (1) Experimental design: Each tumor-bearing mouse was injected with 10 μCi / g of F-FPON, followed by 1-hour dynamic scanning. 18

[0044] (2) Experimental results: The probe had good imaging effect in MC38 colon cancer subcutaneous tumor model mice, and SUVr showed that there was a good contrast difference between the tumor and the contralateral brain tissue, indicating the feasibility of F-FPON in diagnosing colon cancer tumor. 18 F-FPON6. PET imaging in MC38 colon cancer subcutaneous tumor model mice Figure 11 ) (1) Experimental design: Each tumor-bearing mouse was injected with 10 μCi / g of F-FPON, followed by 1-hour dynamic scanning. 18

[0044] (2) Experimental results: The probe had good imaging effect in MC38 colon cancer subcutaneous tumor model mice, and SUVr showed that there was a good contrast difference between the tumor and the contralateral brain tissue, indicating the feasibility of F-FPON in diagnosing colon cancer tumor. 18 F-FPON6. PET imaging

Claims

1. A probe targeting proliferating cell nuclear antigen (PCNA) for tumor PET imaging [ 18 F]-N-(2-((2-(3-(2-fluoroethoxy)phenoxy)phenyl)amino)-2-oxoethyl)-1-naphthamide, abbreviation 18 F-FPON has the following structure:

2. Probes for targeting proliferating cell nuclear antigen (PCNA) for tumor PET imaging 18 F-FPON, whose synthetic precursor is 2-(3-(2-(2-(1-naphthamido)acetamido)phenoxy)phenoxy)ethyl-4-methylbenzenesulfonate, possesses a methylbenzenesulfonate leaving group with a specific labeling microstructure:

3. A kind 18 The method for preparing F-FPON probes is characterized by: Through Na 18 F-labeling of the probe precursor of claim 1 includes the following steps: Use 1 mL of (acetonitrile:water = 7:3) containing K2CO3 (1 mg) and K 222 A mixed solution of (5 mg) will 18 F ions were washed down from the QMA column into a vial. The solution was dried under nitrogen at 110 °C for 10 min, followed by azeotropic drying with 1 mL of acetonitrile for 5 min to make the system as anhydrous as possible. Then, 500 μL of the dissolved 4 mg / mL precursor was added, and the reaction was capped at 110 °C for 20 min.

4. The one described in claim 2 18 F-FPON-labeled PCNA targeting probe, characterized in that Specifically targets the PIP domain of PCNA.

5. The one claimed in claim 2 18 F - The application of labeled PCNA-targeting probes in the preparation of positron emission tomography (PET) for tumors, including gliomas.

6. The one claimed in claim 2 18 F - The application of labeled PCNA-targeting probes in the preparation of positron emission tomography for proliferative diseases.

7. The one claimed in claim 2 18 F - The application of labeled PCNA-targeting probes in the preparation of positron emission tomography for efficacy evaluation of PCNA-targeted therapies.

8. The one claimed in claim 2 18 F - The application of labeled PCNA-targeting probes in the preparation of positron emission tomography for evaluating the efficacy of radiotherapy and chemotherapy.

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