CN114315795B - 68 Ga-marked inhibitor radioactive probe for targeting fibroblast activation protein and preparation method thereof - Google Patents

Abstract

The present invention relates to a kind of ⁶⁸ Ga-labeled inhibitor radioactive probe of targeted fibroblast activation protein and a preparation method thereof, belonging to the technical field of radiopharmaceuticals chemistry; the structural formula of the radioactive probe is as follows:

the radioactive probe of the invention has the marking time of 5 minutes, the radiochemical yield of more than 99 percent, and the radiochemical purity of the product measured by radio-HPLC of more than 99 percent, and the product shows high affinity and high specificity to fibroblast activation protein, has high in-vitro cell uptake value, and is a more potential fibroblast activation protein PET imaging agent.

Description

68Ga-labeled inhibitor-like radioactive probe targeting fibroblast activation protein and preparation method thereof

technical field

The present invention relates to inhibitor radioactive probes ([ ⁶⁸ Ga]Ga-HBED-CC-04-DiF- ^Monomer , [ ⁶⁸ Ga]Ga- HBED-CC-04-DiF-Dimer) and a preparation method thereof belong to the technical field of radiopharmaceutical chemistry.

Background technique

Tumor mass is composed of tumor cells and tumor stroma. In highly desmoplastic tumors such as breast cancer, colon cancer and pancreatic cancer, tumor stroma accounts for more than 90% of the tumor mass. Cancer-associated fibroblasts (CAFs), also known as tumor-associated fibroblasts (TAFs) or activated fibroblasts, are an important part of the tumor stroma and participate in tumor growth, migration and development. CAFs express different markers such as fibroblast activation protein (fibroblast activation protein, FAP), α-smooth muscle actin (alpha-smooth muscle actin, α-SMA) and vimentin (vimentin).

Since α-SMA and vimentin are expressed in resting fibroblasts, pericytes and vascular smooth muscle cells, while FAP is highly specifically expressed only in activated fibroblasts and not expressed in benign tumors or normal adult tissues, Therefore, FAP is the most potential molecular marker of CAFs. In addition, FAP is also an essential factor in promoting tumor cell proliferation and metastasis, remodeling extracellular matrix, inducing angiogenesis, mediating immunosuppression, and participating in energy metabolism of tumor cells. Therefore, FAP has become a potential target for tumor imaging and treatment, and further study of FAP is of great significance for the diagnosis, treatment and prognosis of malignant tumors.

It is worth noting that activated fibroblasts are not only present in tumors but also in wound healing and matrix remodeling diseases such as chronic inflammation, myocardial infarction and fibrosis of the liver, lung or kidney, therefore, FAP is not only highly expressed between tumors In cytoplasmic CAFs, it is also highly expressed in many tissue remodeling processes. Therefore, FAP expression is not tumor specific. But at the same time, FAP-targeted radiopharmaceuticals can be applied not only to tumor imaging and therapy, but also to the imaging of many non-tumor diseases such as myocardial infarction, chronic inflammatory diseases, and lung, liver, or kidney fibrosis.

FAP is a 97 kDa type II transmembrane protein that is only active as a 170 kDa dimer (FAP-FAP). FAP-targeted inhibitor radiopharmaceuticals have been widely used in tumor imaging and treatment, and have become a research hotspot in recent years.

[ ⁶⁸ Ga]Ga-FAPI-02 was reported by Loktev et al. in 2018. [ ⁶⁸ Ga]Ga-FAPI-02 exhibited high FAP specificity and binding affinity both in vitro and in vivo, and could be rapidly taken up and internalized by cells with high FAP expression. In patients with breast cancer metastases, lung cancer metastases, and pancreatic cancer metastases, [ ⁶⁸ Ga]Ga-FAPI-02 is rapidly cleared, mainly excreted through the kidneys, and has low uptake in normal tissues. , a large accumulation of radioactivity was observed, and the images obtained were of high contrast. Comparing the imaging of [ ⁶⁸ Ga]Ga-FAPI-02 and [ ¹⁸ F]FDG in patients with locally advanced lung adenocarcinoma, it was found that [ ⁶⁸ Ga]Ga-FAPI-02 was significantly better than [ ¹⁸ F]FDG: [ ⁶⁸ Ga ]Ga-FAPI-02 has higher uptake in metastatic lesions, lower background, higher focus contrast, and better visibility; unlike [ ¹⁸ F]FDG's strong uptake in high-glucose metabolism tissues such as the brain, [ ⁶⁸ Ga]Ga-FAPI-02 selectively targets tissues with high FAP expression. Although [ ⁶⁸ Ga]Ga-FAPI-02 showed good initial tumor imaging properties, its rapid clearance rate may not reflect the situation of tumors such as head and neck cancer, ovarian cancer and liver cancer well. In addition, the short The tumor residence time is also unsuitable for treatment. Therefore, it is necessary to develop radiopharmaceuticals with longer tumor residence time for FAP-targeting inhibitors.

For this purpose, Lindner et al. reported FAPI-04 in the same year and applied it to the labeling of ⁶⁸ Ga and ¹⁷⁷ Lu. [ ⁶⁸ Ga]Ga-FAPI-04 exhibited similar rapid renal clearance, low background and high tumor uptake as [ ⁶⁸ Ga]Ga-FAPI-02. The researchers used ¹⁷⁷ Lu to label FAPI-04 and obtained [ ¹⁷⁷ Lu]Lu-FAPI-04. The results of biodistribution experiments in HT1080-FAP tumor-bearing mice showed that [ ¹⁷⁷ Lu]Lu-FAPI-04 was more active than [ ¹⁷⁷ Lu ]Lu-FAPI-02 had higher tumor uptake and longer tumor retention, and both backgrounds were very low. After 24 hours, the effective tumor uptake rate of [ ¹⁷⁷ Lu]Lu-FAPI-04 was higher than that of [ ¹⁷⁷ Lu]Lu-FAPI-02 was 100% higher, suggesting that FAPI-04 has therapeutic potential. Treatment with [ ⁹⁰ Y]Y-FAPI-04 in one patient showed a significant reduction in pain, metastatic lesions and low background were observed within 24 hours, and no side effects such as hematological toxicity were observed, but clinical data are limited , more clinical trials are still needed to verify its feasibility.

To further increase tumor uptake and tumor residence time, FAPI-21 and FAPI-46 were reported by Loktev et al. in 2019. The biodistribution results of HT1080-FAP tumor-bearing mice showed that the tumor uptake of [ ⁶⁸ Ga]Ga-FAPI-21 and [ ⁶⁸ Ga]Ga-FAPI-46 was higher than that of [ ⁶⁸ Ga]Ga-FAPI-04, but [ ⁶⁸ Ga] Ga-FAPI-21 has higher liver uptake and muscle uptake than [ ⁶⁸ Ga]Ga-FAPI-04; [ ⁶⁸ Ga]Ga-FAPI-46 has higher tumor/blood, tumor/muscle and tumor/liver ratios than [ ⁶⁸ Ga]Ga-FAPI-04 and [ ^68Ga ]Ga-FAPI-21. [ ¹⁷⁷ Lu]Lu-FAPI-21 and [ ¹⁷⁷ Lu]Lu-FAPI-46 had higher tumor accumulation than [ ¹⁷⁷ Lu]Lu-FAPI-04 at 1-4 hours after injection; at 24 hours after injection, the tumor retention rate of [ ¹⁷⁷ Lu]Lu-FAPI-21>[ ¹⁷⁷ Lu]Lu-FAPI-04>[ ¹⁷⁷ Lu]Lu-FAPI-46; the blood radioactivity ^levels of the three compounds were comparable, and the tumor/ Liver, tumor/kidney and tumor/brain ratios were improved. Intravenous injection of [ ⁶⁸ Ga]Ga-FAPI-21 and [ ⁶⁸ Ga]Ga-FAPI-46 in patients with mucoepidermoid, oropharyngeal, ovarian, and colorectal cancers, both in primary tumors and metastases After 1 hour of administration, the SUVmax were 11.9±3.33 and 12.76±0.90 respectively, and the radioactive uptake in normal tissues was low, and the radioactivity was rapidly cleared from the blood, mainly excreted through the kidneys; but it was observed that [ ⁶⁸ Ga]Ga-FAPI- The uptake of 21 in the oral mucosa, thyroid, parotid and submandibular glands was increased, suggesting that FAPI-21 may not be suitable as a therapeutic drug. Notably, tumor accumulation is highly dependent on tumor type. Lindner et al. found that within 3 hours after injection of [ ⁶⁸ Ga]Ga-FAPI-21 or [ ⁶⁸ Ga]Ga-FAPI-46, tumor radioactivity decreased in colorectal, ovarian, oropharyngeal, and pancreatic cancers. remained relatively stable, while in breast cancer, tumor radioactivity continued to decline; in addition, in a patient with a tumor of unknown origin, tumor uptake continued to increase within 1-3 hours after administration.

Human tumors form complex heterogeneous structures, and the number and distribution of FAP-expressing CAFs and the number of FAP molecules per cell may vary, resulting in different pharmacokinetic properties of radiopharmaceuticals in different tumor types. In clinical studies, differences in the uptake of [ ⁶⁸ Ga]Ga-FAPI-21 or [ ⁶⁸ Ga]Ga-FAPI-46 were observed in different types of tumors: in colorectal, ovarian, oropharyngeal and pancreatic cancers The uptake in breast cancer is relatively stable, while the uptake in breast cancer continues to decline. This may be due to a source of heterogeneity in CAFs. Due to differences in origin , these CAFs may exhibit distinct proteomes with strong variation or even lack of FAP expression. Therefore, radiopharmaceuticals with longer tumor residence times may better reflect the true status of the tumor than rapidly cleared radiopharmaceuticals. In addition, radiopharmaceuticals with longer tumor residence time and high tumor uptake are also clinical requirements for therapeutic drugs. At present, the most successful FAP-targeted inhibitor imaging drugs are FAPI-02, FAPI-04 and FAPI-46, and their ⁶⁸ Ga-labeled drugs are the most clinically used, but there is still room for improvement of these drugs to obtain Longer tumor retention time.

Therefore, providing a novel inhibitor-type radioactive probe that can exhibit high affinity and high specificity to fibroblast activating protein, has better tumor uptake and longer tumor retention time has become this technical field. Technical problems that need to be solved urgently.

Contents of the invention

One of the objects of the present invention is to provide ^68Ga -labeled targeting fibroblast activation protein (Fibroblast activation protein, FAP) inhibitor class radioactive probe, this type of radioactive probe shows high affinity and High specificity, better tumor uptake and longer tumor retention time, it is a potential FAP/PET imaging agent.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The structural formula of the ⁶⁸ Ga-labeled inhibitor-type radioactive probe targeting fibroblast activation protein is shown in FIG. 6 .

Another object of the present invention is to provide a method for preparing the aforementioned ⁶⁸ Ga-labeled inhibitor-type radioactive probe targeting fibroblast activation protein.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

⁶⁸ Ga-labelled inhibitor radioactive probe targeting fibroblast activation protein, the steps are as follows: in the presence of alkali and condensing agent, the bifunctional linker HBED-CC and fibroblast activation protein are inhibited Condensation reaction with reagent, remove the protective group by acid, dissolve the obtained product in dimethyl sulfoxide, add [ ⁶⁸ Ga]GaCl ₃ solution, mix evenly, and heat to obtain ^{the 68} Ga-labeled product shown in the above structural formula Inhibitor-like radioprobe targeting fibroblast activation protein.

The reaction formula is shown in Figure 7.

Preferably, the base is N,N-diisopropylethylamine, and the addition amount is 3-5 equivalents; the condensing agent is 1-hydroxybenzotriazole and 1-ethyl-(3-dimethyl Aminopropyl) carbodiimide, the addition is 1 equivalent; the fibroblast activation protein inhibitor is (S)-N-(2-(2-cyano-4,4-difluoropyrrolidine- 1-yl)-2-ethoxy)-6-(3-(piperazin-1-yl)propoxy)quinoline-4-formamide, the addition amount is 1-5 equivalents; the acid is three Fluoroacetic acid, the addition amount is 3 milliliters.

Preferably, the specific steps of the preparation method of the ⁶⁸ Ga-labeled inhibitor radioactive probe targeting fibroblast activation protein are as follows:

Step 1: Synthesis of labeled precursors of ⁶⁸ Ga-labeled inhibitor-like radioprobes targeting fibroblast activation protein

(S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-ethoxy)-6-(3-(piperazin-1-yl) Propoxy) quinoline-4-carboxamide was dissolved in anhydrous dimethylformamide, and 1-hydroxybenzotriazole, N,N-diisopropylethylamine, 1-ethyl- (3-Dimethylaminopropyl)carbodiimide and 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa- 6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxyl-3,1-phenyl))dipropionic acid, reacted at room temperature, after overnight, to Add ethyl acetate and saturated brine to the mixed solution, wash, and collect the organic phase filtrate; dry the organic phase filtrate with anhydrous sodium sulfate, filter, and remove solid impurities; use a rotary evaporator to depressurize, remove the solvent in the filtrate, and use two Chloromethane, methanol and 25% aqueous ammonia mixture (4/1/0.1, v/v/v) were separated through a silica gel column, the components were collected, and the rotary evaporator and oil pump decompressed to remove the solvent to obtain a brownish-yellow oil. The obtained brownish-yellow oil was dissolved in trifluoroacetic acid, stirred at room temperature, decompressed with a rotary evaporator and an oil pump, removed the solvent, and recrystallized with ether to obtain a brownish-yellow solid; the obtained brownish-yellow solid was dissolved in dimethyl In sulfoxide, purified by Semi-HPLC to obtain brown-yellow solid HBED-CC-04-DiF-Monomer;

Step 2: Labeling of ⁶⁸ Ga-labeled inhibitor-like radioprobe targeting fibroblast activation protein

Dissolve the HBED-CC-04-DiF-Monomer (labeled precursor) obtained in step 1 in dimethyl sulfoxide, then add sodium acetate solution to obtain the labeled precursor sodium acetate solution, and rinse germanium gallium with high-purity hydrochloric acid solution Generator (iThembalaboratories, 740MBq, 20mCi), add the obtained [ ⁶⁸ Ga]GaCl ₃ hydrochloric acid solution into the labeling precursor sodium acetate solution, mix well, react at 95°C, cool to room temperature, and use a radioactive detector (Flow-Count, Eckert&Ziegler) high-performance liquid chromatography (radio-HPLC, Agilent 1260Infinity II system) in the mobile phase of 0.1% trifluoroacetic acid aqueous solution (v/v) and 0.1% trifluoroacetic acid acetonitrile solution (v/v) to measure its labeling rate , to obtain [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer with a radiochemical yield greater than 99%.

Preferably, in step 2, the test condition of the high performance liquid chromatography with radioactive detector is: the first mobile phase is 0.1% trifluoroacetic acid aqueous solution (v/v), and the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution (v/v), the gradient elution condition is: 0-10 minutes, the first mobile phase of 100%-35%; 10-12 minutes, the first mobile phase of 35%-100%; 12-15 minutes, 100 % of the first mobile phase; the flow rate of the mobile phase was 1 ml/min.

Preferably, the specific steps of the preparation method of the ⁶⁸ Ga-labeled inhibitor radioactive probe targeting fibroblast activation protein are as follows:

Step 1: Synthesis of labeled precursors of ⁶⁸ Ga-labeled inhibitor-like radioprobes targeting fibroblast activation protein

(S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-ethoxy)-6-(3-(piperazin-1-yl) Propoxy) quinoline-4-carboxamide was dissolved in anhydrous dimethylformamide, and 1-hydroxybenzotriazole, N,N-diisopropylethylamine, 1-ethyl- (3-Dimethylaminopropyl)carbodiimide and 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa- 6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxyl-3,1-phenyl))dipropionic acid, reacted at room temperature, after overnight, to Add ethyl acetate and saturated brine to the mixed solution to wash, collect the organic phase filtrate; dry the organic phase filtrate with anhydrous sodium sulfate, filter to remove solid impurities; use a rotary evaporator to remove the solvent in the filtrate under reduced pressure, and dichloromethane , methanol and 25% ammonia water mixed solution (20/1/0.1, v/v/v) through silica gel column separation, collect components, rotary evaporator and oil pump decompression, remove solvent, obtain brownish red oily matter, will obtain Dissolve the brownish-red oil in trifluoroacetic acid, stir at room temperature, use a rotary evaporator and an oil pump to depressurize, remove the solvent, and recrystallize with ether to obtain a brownish-red solid; dissolve the obtained brownish-red solid in dimethyl sulfoxide , purified by Semi-HPLC to obtain a maroon solid HBED-CC-04-DiF-Dimer;

Step 2: Labeling of ⁶⁸ Ga-labeled inhibitor-like radioprobe targeting fibroblast activation protein

Dissolve HBED-CC-04-DiF-Dimer (labeling precursor) in dimethyl sulfoxide, and then add sodium acetate solution to obtain the labeling precursor sodium acetate solution, rinse the germanium gallium generator (iThemba Laboratories, 740MBq, 20mCi), the obtained [ ⁶⁸ Ga]GaCl ₃ hydrochloric acid solution was added to the labeling precursor sodium acetate solution, mixed uniformly, reacted at 95°C, cooled to room temperature, and used a radioactive detector (Flow-Count, Eckert&Ziegler) High performance liquid chromatography (radio-HPLC, Agilent 1260Infinity II system) measured its labeling rate in the mobile phase of 0.1% trifluoroacetic acid aqueous solution (v/v) and 0.1% trifluoroacetic acid acetonitrile solution (v/v), and obtained the radioactivity [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer in chemical yield greater than 99%.

Preferably, in step 2, the test condition of the high performance liquid chromatography with radioactive detector is: the first mobile phase is 0.1% trifluoroacetic acid aqueous solution (v/v), and the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution (v/v), the gradient elution condition is: 0-10 minutes, the first mobile phase of 100%-35%; 10-12 minutes, the first mobile phase of 35%-100%; 12-15 minutes, 100 % of the first mobile phase; the flow rate of the mobile phase was 1 ml/min.

Beneficial effect:

The ⁶⁸ Ga-labeled inhibitor radioactive probe targeting fibroblast activating protein of the present invention exhibits high affinity and high specificity for fibroblast activating protein, and has better tumor uptake and longer tumor retention Time is a more potential FAP/PET imaging agent.

The present invention will be further described below through the drawings and specific embodiments, but it does not mean to limit the protection scope of the present invention.

Description of drawings

Fig. 1 is a radioactive HPLC spectrum of the labeling reaction solution of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer prepared in Example 1 of the present invention.

Fig. 2 is the radioactive HPLC spectrum of the labeling reaction solution of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer prepared in Example 2 of the present invention.

Figure 3 shows the uptake of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer and [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer in vitro by HT1080-FAP cells in Application Example 1 of the present invention - Time graph.

Fig. 4 is the in vitro HT1080-FAP cell uptake experiment analysis of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer and [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer and Specific binding map of fibroblast activation protein.

Fig. 5 is a graph showing the binding affinity between HBED-CC-04-DiF-Monomer and HBED-CC-04-DiF-Dimer and fibroblast activating protein analyzed by the IC50 value determination experiment in Application Example 2 of the present invention.

Fig. 6 is the structural formula of the ⁶⁸ Ga-labeled inhibitor-type radioactive probe targeting fibroblast activation protein of the present invention.

Fig. 7 is the reaction formula of the ⁶⁸ Ga-labeled inhibitor-type radioactive probe targeting fibroblast activation protein of the present invention.

Fig. 8 is the synthesis reaction equation of HBED-CC-04-DiF-Monomer in step (1) in Application Example 1 of the present invention.

Fig. 9 is the labeling reaction equation of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer in step (2) of Application Example 1 of the present invention.

Fig. 10 is the synthetic reaction equation of HBED-CC-04-DiF-Dimer in the step (1) of the application example 2 of the present invention.

Fig. 11 is the labeling reaction equation of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer in step (2) of application example 2 of the present invention.

Detailed ways:

Unless otherwise specified, the reagents and raw materials used in the preparation methods and detection methods described in the following examples and comparative examples are all commercially available commodities, and the equipment used are all commonly used equipment.

Unless otherwise specified, the concentrations and ratios described in the following examples and comparative examples are all in weight units.

Example 1 ([ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer)

Step 1: Synthesis of labeled precursors of ⁶⁸ Ga-labeled inhibitor-like radioprobes targeting fibroblast activation protein

HBED-CC-04-DiF-Monomer is (S)-3-(5-methyl-((3-carboxymethyl)(2-((3-carboxymethyl)(5-(3-(4- (4-(4-(2-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)- 4-oxopropyl) piperazin-1-yl)-3-oxopropyl)-2-hydroxybenzyl) amino) ethyl) amino)-4-hydroxyphenyl) propionic acid synthetic reaction equation as shown in Figure 8 shown.

resolve resolution:

(S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-ethoxy)-6-(3-(piperazin-1-yl) Propoxy) quinoline-4-carboxamide (52 mg, 0.1 mmol) was dissolved in 2 ml of anhydrous dimethylformamide, and 1-hydroxybenzotriazole (17 mg, 0.1 mmol) was added to the mixed solution ), N,N-diisopropylethylamine (68 mg, 0.5 mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (25 mg, 0.1 mmol) and 3 ,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl ) two (methylene)) two (4-hydroxyl-3,1-phenyl)) dipropionic acid (57 mg, 0.1 mmol), react at room temperature, after overnight, add 30 milliliters of ethyl acetate to the mixed solution ester and 10 milliliters of saturated brine, washed twice, and the organic phase filtrate was collected; the organic phase filtrate was dried with anhydrous sodium sulfate, and filtered to remove solid impurities; the organic solvent in the filtrate was removed by a rotary evaporator, and dichloromethane/methanol was used to /25% ammonia water (4/1/0.1, v/v/v) is separated through a silica gel column (refined column chromatography silica gel, 200-300 mesh), the components are collected, and the components are removed by a rotary evaporator and an oil pump. organic solvent to obtain a brown-yellow oil, which was dissolved in 3 milliliters of trifluoroacetic acid, and stirred at room temperature for 7 hours, using a rotary evaporator and an oil pump to remove the solvent, and recrystallized with ether to obtain a brown-yellow solid ; The resulting brown-yellow solid was dissolved in dimethyl sulfoxide and purified by Semi-HPLC (0.1% trifluoroacetic acid water/acetonitrile=7/3, v/v) to obtain brown-yellow solid HBED-CC-04- DiF-Monomer 16 mg (yield: 20%);

Confirmation of Compound HBED-CC-04-DiF-Monomer:

¹ H NMR (600MHz, DMSO) δ8.91-8.85 (m, 1H), 8.05 (dd, J = 9.2, 2.0Hz, 1H), 7.89 (s, 1H), 7.66-7.59 (m, 1H), 7.55 -7.51(m,1H),7.07(t,J=7.7Hz,4H),6.80(d,J=8.2Hz,2H),5.11(d,J=7.4Hz,1H),4.45-4.35(m, 2H),4.34-4.16(m,8H),4.16-3.96(m,10H),3.33-3.28(m,2H),3.23-3.21(m,4H),2.95-2.80(m,2H),2.69( t,J＝7.3Hz,4H),2.60(d,J＝8.3Hz,2H),2.26-2.17(m,2H).

HRMS (ESI) theoretical molecular weight C ₅₀ H ₅₈ F ₂ N ₈ O ₁₂ [M+H] ⁺ 1001.4221, measured molecular weight 1001.4239 [M+H] ⁺ ;

Step 2: Labeling of ⁶⁸ Ga-labeled inhibitor-like radioprobe targeting fibroblast activation protein

The labeling reaction equation of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer is shown in Figure 9 .

Marking method:

Dissolve 8 micrograms of the labeling precursor HBED-CC-04-DiF-Monomer in 80 microliters of dimethyl sulfoxide, and add 135 microliters of sodium acetate buffer with a concentration of 3 mol/L to obtain the labeling precursor sodium acetate buffer solution; rinse the germanium gallium generator ((iThemba laboratories, 740MBq, 20mCi) with 6 ml of high-purity 0.6 mol/L hydrochloric acid solution to obtain [ ⁶⁸ Ga]GaCl ₃ hydrochloric acid solution, take 0.3 ml of it, and add the labeling precursor In sodium acetate buffer, mix well, react at 95°C for 5 minutes, cool to room temperature, and perform high-performance liquid chromatography (radio-HPLC, Agilent 1260 Infinity II system) with a radioactive detector (Flow-Count, Eckert&Ziegler) at 0.1% Tris The labeling rate was measured in the mobile phase of fluoroacetic acid aqueous solution (v/v) and 0.1% trifluoroacetic acid acetonitrile solution (v/v), and the radiochemical yield of [ ⁶⁸ Ga]Ga-HBED-CC-04 was 99.56%. -DiF-Monomer;

As shown in Figure 1, it is the radioactive HPLC spectrum of the labeled reaction solution of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer prepared in Example 1 of the present invention. The spectrum shows that [ ⁶⁸ Ga]Ga-HBED- The radiochemical purity of CC-04-DiF-Monomer is greater than 99%;

In the radio-HPLC measurement described in the above steps, the first mobile phase is 0.1% trifluoroacetic acid aqueous solution (v/v), the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution (v/v), gradient elution conditions For: 0-10 minutes, 100%-35% of the first mobile phase; 10-12 minutes, 35%-100% of the first mobile phase; 12-15 minutes, 100% of the first mobile phase; mobile phase The flow rate was 1 ml/min.

Example 2 ([ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer)

Step 1: Synthesis of labeled precursors of ⁶⁸ Ga-labeled inhibitor-like radioprobes targeting fibroblast activation protein

HBED-CC-04-DiF-Dimer is 2,2'-(ethane-1,2-diylbis(5-(3-(4-(4-(4-(2-(2-((S )-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-7-yl)-4-oxopropyl)piperazine-1- Base)-3-oxopropyl)-2-hydroxybenzyl)) azacyclodiacetic acid synthesis reaction equation is shown in Figure 10.

resolve resolution:

(S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-ethoxy)-6-(3-(piperazin-1-yl) Propoxy) quinoline-4-carboxamide (100 mg, 0.2 mmol) was dissolved in 2 ml of anhydrous dimethylformamide, and 1-hydroxybenzotriazole (23 mg, 0.2 mmol) was added to the mixed solution ), N,N-diisopropylethylamine (84 mg, 0.6 mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (32 mg, 0.2 mmol) and 3 ,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl ) two (methylene)) two (4-hydroxyl-3,1-phenyl)) dipropionic acid (23 mg, 0.04 mmol), after reacting overnight at room temperature, add 30 milliliters of ethyl acetate in the mixed solution and 10 milliliters of saturated brine, washed twice, and collected the organic phase filtrate; the organic phase filtrate was dried with anhydrous sodium sulfate, filtered to remove solid impurities; the solvent in the filtrate was removed under reduced pressure by a rotary evaporator, and dichloromethane/methanol /25% ammonia water (20/1/0.1, v/v/v) was separated through a silica gel column (refined column chromatography silica gel, 200-300 mesh), the components were collected, and the components were removed by a rotary evaporator and an oil pump. organic solvent to obtain a brown-red oil, which was dissolved in 3 milliliters of trifluoroacetic acid, and stirred at room temperature for 7 hours, using a rotary evaporator and an oil pump to remove the solvent, and recrystallized with ether to obtain a brown-red solid ; The obtained maroon solid was dissolved in dimethyl sulfoxide, purified by Semi-HPLC (0.1% trifluoroacetic acid water/acetonitrile=7/3, v/v) to obtain a maroon solid HBED-CC-04-DiF -Monomer 20 mg (yield: 40%);

Confirmation of compound HBED-CC-04-DiF-Dimer:

¹ H NMR (600MHz, DMSO) δ8.84(d, J=4.3Hz, 2H), 8.03(d, J=9.2Hz, 2H), 7.88(d, J=2.2Hz, 2H), 7.56(d, J＝4.2Hz, 2H), 7.48(dd, J＝9.2, 1.5Hz, 2H), 7.13-7.06(m, 4H), 6.80(d, J＝8.0Hz, 2H), 5.13(dd, J＝9.2 ,2.3Hz,2H),4.28-4.18(m,12H),4.02-3.99(m,4H),3.66-3.63(m,8H),3.34-3.31(m,10H),3.21-3.19(m,6H ), 2.98-2.76(m,6H),2.72-2.69(m,4H),2.62-2.59(m,4H),2.25-2.20(m,4H).

HRMS (ESI) theoretical molecular weight C ₇₄ H ₈₄ F ₄ N ₁₄ O ₁₄ [M+H] ⁺ 1469.6306, measured molecular weight 1469.6306 [M+H] ⁺ ;

Step 2: Labeling of ⁶⁸ Ga-labeled inhibitor-like radioprobe targeting fibroblast activation protein

The labeling reaction equation of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer is shown in Figure 11 .

Marking method:

Dissolve 12 μg of the labeled precursor HBED-CC-04-DiF-Dimer in 120 μl of dimethyl sulfoxide, add 135 μl of 3 mol/L sodium acetate buffer to obtain a precursor solution; use 6 ml of high-purity Rinse the germanium-gallium generator (iThemba laboratories, 740MBq, 20mCi) with 0.6 mol/L hydrochloric acid solution, add 0.3 ml of the obtained [ ⁶⁸ Ga]GaCl ₃ hydrochloric acid solution to the precursor solution, mix well, and react at 95°C for 5 minutes , cooled to room temperature, and analyzed by high-performance liquid chromatography (radio-HPLC, Agilent 1260 Infinity II system) with a radioactive detector (Flow-Count, Eckert&Ziegler) in 0.1% trifluoroacetic acid aqueous solution (v/v) and 0.1% trifluoroacetic acid The labeling rate was measured in the mobile phase of acetonitrile solution (v/v), and the radiochemical yield of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer was 99.89%;

As shown in Figure 2, it is the radioactive HPLC spectrum of the labeled reaction solution of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer prepared in Example 2 of the present invention. The spectrum shows that [ ⁶⁸ Ga]Ga-HBED- The radiochemical purity of CC-04-DiF-Dimer is greater than 99%;

In the radio-HPLC measurement described in the above steps, the first mobile phase is 0.1% trifluoroacetic acid aqueous solution (v/v), the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution (v/v), gradient elution conditions For: 0-10 minutes, 100%-35% of the first mobile phase; 10-12 minutes, 35%-100% of the first mobile phase; 12-15 minutes, 100% of the first mobile phase; mobile phase The flow rate was 1 ml/min.

Application Example 1

In Vitro Cellular Uptake of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer and [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer

(1) Seed HT1080-FAP (FAP positive) cells (~5×10 ⁵ /well) in a 6-well plate, culture in an incubator for 60 hours, and the cell coverage rate is 90-100%; after 60 hours, suck Remove the culture medium, wash the cells twice with phosphate buffered saline, add 12μCi of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer or [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF to the well plate -Dimer or [ ⁶⁸ Ga]Ga-FAPI-04, incubated at 37°C for 10, 30, 60, 90 and 120 minutes, using 10 μM UAMC-1110 (FAP inhibitor) to block cellular uptake at 60 minutes, after incubation , aspirate the solution, quickly wash the cells three times with phosphate buffered saline to stop the cell uptake; lyse all the cells with sodium hydroxide, collect the lysate for radioactivity counting;

(2) From the results of in vitro cell uptake experiments, it can be known that [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer and [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer are in vitro HT1080-FAP (FAP positive ) The uptake in cells was high and increased with time, the uptake at 10 minutes was higher than the highest uptake of [ ⁶⁸ Ga]Ga-FAPI-04, and the uptake at 120 minutes (>60%) was significantly higher than that of [ ⁶⁸ Ga]Ga-FAPI-04; the uptake of both was blocked after adding the fibroblast activation protein inhibitor UAMC-1110, indicating that [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer and [ ⁶⁸ Ga ]Ga-HBED-CC-04-DiF-Dimer specifically binds fibroblast activation protein.

As shown in Figure 3, it is the uptake of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer and [ 68 Ga]Ga-HBED-CC-04-DiF-Monomer and [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF- Dimer intake-time curve, n=3.

As shown in Figure 4, it is the analysis of [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer and [ ⁶⁸ Ga]Ga-HBED-CC-04- Specific binding of DiF-Dimer to fibroblast activation protein, n=3, the significant difference in cell uptake between the group without UAMC1110 and the group with UAMC1110 shows: [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer and [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer specifically binds to fibroblast activation protein.

Application Example 2

Determination of IC50 value of HBED-CC-04-DiF-Monomer and HBED-CC-04-DiF-Dimer

(1) Seed HT1080-FAP (FAP positive) cells (~5×10 ⁵ /well) in a 6-well plate, culture in an incubator for 60 hours, the cell coverage rate is 90-100%, after 60 hours, suck Remove the culture medium, wash the cells twice with phosphate buffered saline, add 12 μCi of [ ⁶⁸ Ga]Ga-FAPI-04 and HBED-CC-04-DiF-Monomer or HBED-CC-04-DiF-Dimer to the well plate or FAPI-04, so that the final concentrations of HBED-CC-04-DiF-Monomer or HBED-CC-04-DiF-Dimer or FAPI-04 are 10 ^-9.5 , 10 ^-9 , 10 ^-8.5 , 10 ^{-8 ,} respectively , 10 ^-7 and 10 ^-6 mol/L, after incubating at 37°C for 60 minutes, aspirate the solution, wash the cells three times quickly with phosphate buffered saline, stop the cell uptake, lyse all the cells with sodium hydroxide, collect the lysate for radioactivity count;

As shown in Figure 5, it is the binding affinity diagram of HBED-CC-04-DiF-Monomer and HBED-CC-04-DiF-Dimer and fibroblast activating protein in the IC50 value determination experiment analysis in Application Example 2 of the present invention, n =3, the IC50 value at nM level shows: HBED-CC-04-DiF-Monomer and HBED-CC-04-DiF-Dimer have high binding affinity to fibroblast activation protein;

(2) From the experimental results of IC50 value determination, it can be seen that HBED-CC-04-DiF-Monomer and HBED-CC-04-DiF-Dimer have high binding affinity to fibroblast activation protein, and the IC50 values are 5.06±1.09nM and 5.10±1.41nM, basically consistent with FAPI-04 (5.16±0.75nM).

The ⁶⁸ Ga-labeled inhibitor radioactive probe targeting fibroblast activation protein of the present invention has excellent ⁶⁸ Ga labeling properties, benefiting from N,N'-bis[2-hydroxyl-5-(carboxyethyl )-benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC), an excellent Ga ³⁺ bifunctional linker, has a high thermodynamic stability constant between HBED-CC and Ga ³⁺ (logK _ML : 38.5 ), the energy required for coordination is low, therefore, the labeling of [ ⁶⁸ Ga]Ga-HBED-CC is fast and efficient. Compared with [ ⁶⁸ Ga]Ga-FAPI-04, which needs to be heated at 100°C for 20 minutes to prepare a product with a radiochemical purity greater than 90%, ^{the 68} Ga-labeled inhibitor-like radioactive probe targeting fibroblast activation protein of the present invention It can be rapidly prepared by heating at 95° C. for 5 minutes, and the product has high radiochemical yield and radiochemical purity (both >99%) and high stability.

The ⁶⁸ Ga-labeled inhibitor radioactive probe targeting fibroblast activation protein of the present invention contains (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl) -2-ethoxy)-6-(3-(piperazin-1-yl)propoxy)quinoline-4-carboxamide group, which has a good affinity for fibroblast activation protein; Additionally, the introduction of HBED-CC may increase tumor uptake. We expected [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer and [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Dimer to have good affinity and tumor uptake values for tumors expressing fibroblast activation protein High and long tumor retention time. Therefore, the ⁶⁸ Ga-labeled inhibitor-type radioactive probe targeting fibroblast activation protein of the present invention can be used as a tumor positron molecular probe for tumor imaging.

The above is only a preferred embodiment of the present invention, so the scope of the present invention cannot be limited accordingly, that is, equivalent changes and modifications made according to the patent scope of the present invention and the content of the specification should still be covered by the present invention In the range.

Claims (6)

1. ⁶⁸ Ga-labeled inhibitor-like radioactive probes targeting fibroblast activation protein, respectively: [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer or [ ⁶⁸ Ga]Ga-HBED-CC- 04-DiF-Dimer, its structural formula is as follows:

2. The preparation method of the inhibitory radioactive probe targeting fibroblast activation protein labeled with ⁶⁸ Ga, the steps are as follows: in the presence of alkali and condensing agent, the bifunctional linker HBED-CC and fibroblasts are activated Condensation reaction of protein inhibitors, use acid to remove the protective group, dissolve the obtained product in dimethyl sulfoxide, add [ ⁶⁸ Ga]GaCl ₃ solution, mix well, and heat to obtain ⁶⁸ Ga-labeled targeting component Inhibitor-like radioprobe for fibroblast activation protein. 3. The preparation method of the inhibitor class radioactive probe targeting fibroblast activation protein according to claim 2, characterized in that: the base is N, N-diisopropylethylamine, and the addition amount is 3 -5 equivalents; the condensing agent is 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and the addition is 1 equivalent; the fibroblast activation The protein inhibitor is (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-ethoxy)-6-(3-(piperazine-1 -yl) propoxy) quinoline-4-carboxamide, the addition amount is 1-5 equivalents; the acid is trifluoroacetic acid, the addition amount is 3 milliliters. 4. the preparation method of the inhibitor class radioactive probe targeting fibroblast activation protein according to claim 2, is characterized in that: concrete steps are as follows: Step 1: Synthesis of Labeled Precursors of Inhibitor-Class Radioprobes Targeting Fibroblast Activation Protein (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-ethoxy)-6-(3-(piperazin-1-yl) Propoxy) quinoline-4-carboxamide was dissolved in anhydrous dimethylformamide, and 1-hydroxybenzotriazole, N,N-diisopropylethylamine, 1-ethyl- (3-Dimethylaminopropyl)carbodiimide and 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa- 6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxyl-3,1-phenyl))dipropionic acid, reacted at room temperature, after overnight, to Add ethyl acetate and saturated brine to the mixed solution to wash, collect the organic phase filtrate; dry the organic phase filtrate with anhydrous sodium sulfate, filter to remove solid impurities; use a rotary evaporator to remove the solvent in the filtrate under reduced pressure, and dichloromethane , methanol and 25% ammonia water mixture (4:1:0.1, v/v/v) was separated through a silica gel column, the components were collected, and the organic solvent in the components was removed by a rotary evaporator and an oil pump to obtain a brownish-yellow oil, Dissolve the obtained brown-yellow oil in trifluoroacetic acid, stir at room temperature, use a rotary evaporator and an oil pump to remove the solvent, and recrystallize with ether to obtain a brown-yellow solid; dissolve the obtained brown-yellow solid in dimethyl sulfoxide , purified by Semi-HPLC to obtain brown-yellow solid HBED-CC-04-DiF-Monomer; Step 2: Labeling of inhibitor-like radioprobes targeting fibroblast activation protein Dissolve the HBED-CC-04-DiF-Monomer obtained in step 1 in dimethyl sulfoxide, and then add sodium acetate solution to obtain the labeling precursor sodium acetate solution, rinse the germanium gallium generator with high-purity hydrochloric acid solution, and the obtained [ ⁶⁸ Ga]GaCl ₃ hydrochloric acid solution was added to the labeling precursor sodium acetate solution, mixed evenly, reacted at 95°C, cooled to room temperature, and the labeling rate was measured by high-performance liquid chromatography (radio-HPLC) with a radioactive detector to obtain radioactivity [ ⁶⁸ Ga]Ga-HBED-CC-04-DiF-Monomer with a chemical yield greater than 99%. 5. the preparation method of the inhibitor class radioactive probe targeting fibroblast activation protein according to claim 2, is characterized in that: concrete steps are as follows: Step 1: Synthesis of Labeled Precursors of Inhibitor-Class Radioprobes Targeting Fibroblast Activation Protein (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-ethoxy)-6-(3-(piperazin-1-yl) Propoxy) quinoline-4-carboxamide was dissolved in anhydrous dimethylformamide, and 1-hydroxybenzotriazole, N,N-diisopropylethylamine, 1-ethyl- (3-Dimethylaminopropyl)carbodiimide and 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa- 6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxyl-3,1-phenyl))dipropionic acid, reacted at room temperature, after overnight, to Add ethyl acetate and saturated brine to the mixed solution to wash, collect the organic phase filtrate; dry the organic phase filtrate with anhydrous sodium sulfate, filter to remove solid impurities; use a rotary evaporator to remove the solvent in the filtrate under reduced pressure, and dichloromethane , methanol and 25% ammonia water mixture (20:1:0.1, v/v/v) was separated through a silica gel column, the volume ratio of the mixture was 20:1:0.1, the components were collected, and the components were removed using a rotary evaporator and an oil pump. The organic solvent in the mixture was obtained to obtain a brown-red oil, which was dissolved in trifluoroacetic acid, stirred at room temperature, and the solvent was removed by using a rotary evaporator and an oil pump, and recrystallized with ether to obtain a brown-red solid; The obtained maroon solid was dissolved in dimethyl sulfoxide, and purified by Semi-HPLC to obtain a maroon solid HBED-CC-04-DiF-Dimer; Step 2: Labeling of inhibitor-like radioprobes targeting fibroblast activation protein Dissolve the HBED-CC-04-DiF-Dimer obtained in step 2 in dimethyl sulfoxide, then add sodium acetate solution to obtain the labeling precursor sodium acetate solution, rinse the germanium gallium generator with high-purity hydrochloric acid solution, and the obtained [ ⁶⁸ Ga]GaCl ₃ hydrochloric acid solution was added to the labeling precursor sodium acetate solution, mixed evenly, reacted at 95°C, cooled to room temperature, and the labeling rate was measured by high-performance liquid chromatography with a radioactive detector, and the radiochemical yield was greater than 99 % of [ ^68Ga ]Ga-HBED-CC-04-DiF-Dimer. 6. according to claim 4 or the preparation method of the inhibitor class radioprobe of targeting fibroblast activation protein described in 5, it is characterized in that: in step 2, the test condition of the high performance liquid chromatography with radioactive detector is : the first mobile phase is 0.1% trifluoroacetic acid aqueous solution, the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution, the gradient elution condition is: 0-10 minutes, 100%-35% of the first mobile phase; 10- 12 minutes, 35%-100% of the first mobile phase; 12-15 minutes, 100% of the first mobile phase; the flow rate of the mobile phase is 1 ml/min.

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