FAPI tripolymer compound as well as preparation and application thereof

Abstract

The invention provides an FAPI trimer compound, a preparation method of the FAPI trimer compound, a PET developer based on the FAPI trimer compound and a preparation method of the developer. Amphipathic polyethylene glycol chains and trimeric structures in molecules of the FAPI trimer compound can improve in-vivo dynamic characteristics of the compound, and the in-vivo dynamic characteristics of the compound can be improved. The residence time in the tumor is prolonged; the PET imaging agent based on the FAPI trimer compound can improve the uptake and imaging effects of the PET imaging agent in tumors, and has good application prospects in PET imaging, preparation of drugs for diagnosing FAP-q expressed tumors and drugs for marking therapeutic nuclide (177Lu or 90Y) to treat the FAP-q expressed tumors in the future.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of radiopharmaceutical chemistry, and particularly relates to a FAPI trimer compound and preparation and application thereof. BACKGROUND

[0002] Early diagnosis, treatment and individualized comprehensive treatment of tumors are the most effective measures to reduce tumor mortality. Precise imaging diagnostic techniques, especially tumor molecular imaging, are one of the key links to achieve early diagnosis, early treatment and individualized treatment of tumors. Among various molecular imaging diagnostic techniques, single photon emission computed tomography (SPECT) and positron emission computed tomography (PET) in nuclear medicine molecular imaging technology have been widely used in clinical practice. Through radionuclide tracing technology, PET and SPECT can sensitively and clearly display the molecular imaging characteristics of deep tissues in patients, bringing hope for early diagnosis, early treatment and individualized comprehensive treatment of cancer patients. With the continuous exploration of the mechanism of tumor formation, the occurrence, development and metastasis of tumors have a close relationship with tumor microenvironment. Tumor is considered to be the product of continuous interaction between tumor cells and different cell types in the surrounding supporting tissue or tumor stroma. Tumor-associated fibroblasts (CAFs) are an important component of the tumor microenvironment, which almost exist in all fibroblasts in solid tumors, and the proportion in some tumor interstitial tissues is as high as 90%. Clinical studies have shown that CAFS are an important prognostic factor for breast cancer, gastric cancer and liver cancer and other tumors.

[0003] FAP protein refers to fibroblast activation protein, also known as FAPα. It is a cell surface protease mainly expressed on the surface of fibroblasts, tumor-associated fibroblasts and some cancer cells. FAP protein plays an important role in physiological and pathological processes. FAP protein is a serine protease with specific degradation activity of fibronectin and collagen. In the tumor microenvironment, the expression of FAP protein is usually associated with tumor progression, invasion and metastasis. In addition, FAP protein can also regulate the tumor immune microenvironment and affect the response of tumor to immunotherapy. Because FAP protein is overexpressed in many tumor types and plays an important role in tumor progression, it has become a potential target for tumor diagnosis, treatment and prognosis evaluation. Antibody drugs, immune cell therapy and other treatment strategies targeting FAP protein are being developed and evaluated in clinical trials to improve tumor treatment effect. In recent years, FAP imaging targeting 68 Ga-FAPI46 PET / CT has made rapid progress in clinical tumor diagnosis. Although 68Ga-FAPI-04 can quickly enter the tumor, but its retention time is relatively short, resulting in its uptake and imaging effect in the tumor with low FAP expression are not ideal. SUMMARY

[0004] The application provides a FAPI trimer compound and preparation and application thereof, and the PET imaging agent based on the FAPI trimer compound provided by the application has a longer retention time in a tumor and better imaging effect.

[0005] The application provides a FAPI trimer compound, which has a structure shown in formula I.

[0006]

[0007] The application discloses a preparation method of the FAPI trimer compound, and comprises the following steps:

[0008] (1) compound 1 and CTC resin are swelled in DCM, N,N-diisopropylethylamine is added, and the reaction is carried out on a shaker at room temperature; methanol is added to terminate the reaction, and compound 2 is obtained;

[0009] (2) 20% piperidine / DMF solution is added to compound 2, nitrogen is blown for reaction, and then the solid is washed with DMF for four times; Fmoc-Glu(OBzl)-OH, HBTU, DMF and N,N-diisopropylethylamine are mixed and blown with nitrogen twice for reaction, so that Fmoc is removed and condensation reaction is carried out to obtain compound 3;

[0010] (3) 20% piperidine / DMF solution is added to compound 3, nitrogen is blown for reaction, and then the solid is washed with DMF for four times; DOTA(OtBu)3, HBTU, DMF and N,N-diisopropylethylamine are mixed and blown with nitrogen twice for reaction, so that Fmoc of compound 3 is removed, and DOTA(OtBu)3 is coupled to obtain compound 4;

[0011] (4) compound 4 is dissolved in a 25% TFA / HFIP / DCM mixed solution, stirred at room temperature, filtered, and the filter cake is washed with DCM for three times, so that the deprotection base HOCTCResin is completed to obtain compound 5;

[0012] (5) compound 5 is dissolved in methanol, replaced with nitrogen for three times, mixed with 10% palladium-carbon, replaced with hydrogen for three times, stirred at room temperature under a hydrogen atmosphere for 4-5h, filtered, and the filter cake is washed with methanol for three times, so that compound 5 is deprotected and three carboxylic acids are leaked out to obtain compound 6;

[0013] (6) Compound 6, HATU, FAPI monomer and DIPEA are dissolved in DMF, and after reaction for 3h, the compound is analyzed by reverse column chromatography to obtain compound 7;

[0014] (7) The FAPI trimer compound is finally obtained by removing Boc from compound 7;

[0015]

[0016]

[0017] Further, the shaking table reaction time in step (1) is 3-4h.

[0018] Further, the nitrogen blowing reaction time in steps (2) and (3) is 25-40min.

[0019] Further, the secondary nitrogen blowing reaction time in steps (2) and (3) is 50-60min.

[0020] Further, in step (4), the stirring time at room temperature is 45-60min.

[0021] The application discloses a PET imaging agent based on a FAPI trimer compound, which comprises a FAPI trimer compound and a diagnostic nuclide; the FAPI trimer compound is the FAPI trimer compound or the FAPI trimer compound obtained by the preparation method; and the diagnostic nuclide comprises 68 Ga, 177 Lu, 131 I, 126 I, 191 Ir, and 193 Ir.

[0022] Further, the diagnostic nuclide is 68 Ga and 177 Lu.

[0023] The application discloses a preparation method of the PET imaging agent, and the steps are as follows:

[0024] The FAPI trimer compound and the solution containing the diagnostic nuclide are mixed, the pH value is adjusted to 3.3-3.6, and then radio labeling is carried out to obtain the PET imaging agent based on the FAPI trimer.

[0025] The application further discloses application of the FAPI trimer compound, the FAPI trimer compound obtained by the preparation method, the PET imaging agent or the PET imaging agent obtained by the preparation method in PET imaging or preparation of a diagnostic FAP-q expressing tumor drug.

[0026] Compared with the prior art, the at least one technical scheme adopted by the embodiments of the present specification can achieve the beneficial effects at least including:

[0027] (1) The amphiphilic polyethylene glycol chain and the trimerization structure in the FAPI trimer compound molecule provided by the present application can improve the in vivo kinetic characteristics of the compound and prolong its residence time in the tumor;

[0028] (2) The PET imaging agent formed after labeling the diagnostic nuclide on the FAPI trimer compound provided by the present application can improve the uptake and imaging effect of the PET imaging agent in the tumor, and has good application prospects in PET imaging, preparation of drugs for diagnosing FAP-q expressing tumors, and future labeling of therapeutic nuclides (177Lu or 90Y) for treating FAP-q expressing tumors;

[0029] (3) The preparation method of the FAPI trimer compound of the present application has a short reaction route, simple operation, cheap and readily available raw materials, low production cost, and is suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0030] 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 as follows. 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.

[0031] Figure 1 HPLC spectrum of the FAPI trimer compound prepared for Example 1;

[0032] Figure 2 LC-MS spectrum of the FAPI trimer compound prepared for Example 1;

[0033] Figure 3 For 68 HPLC analysis of radiochemical purity results of Ga-FAPI-FUSCC-Tri1 under different conditions, wherein (a) is PBS, (b) is FBS, (c) is urine, and (d) is cell uptake results;

[0034] Figure 4 For 177 HPLC analysis of radiochemical purity results of LU-FAPI-FUSCC-Tri1 under different conditions, wherein (a) is PBS, (b) is FBS, and (c) is urine;

[0035] Figure 5 For 68Ga-FAPI-FUSCC-Tri1 in the model mice with tumor cells as FAPI high expression cells HT-1080, PET / CT imaging (a), imaging effect diagram after FAPI46 blocking (b) and 68 Ga-FAPI-04 imaging effect diagram (c);

[0036] Figure 6 For 177 Lu-FAPI-FUSCC-Tri1 in the model mice with tumor cells as FAPI high expression cells HT-1080, PET / CT imaging effect diagram. DETAILED DESCRIPTION

[0037] The embodiments of the present application will be described in detail below with reference to the drawings.

[0038] The embodiments of the present application will be described in detail below with reference to the drawings.

[0039] Example 1

[0040] Preparation of FAPI trimer compound:

[0041] (1) Compound 1 (3 mmol, 1.38 g) and CTC resin (1 mmol, 1 g) were swelled in 20 mL DCM (dichloromethane), N,N-diisopropyl ethylamine (3 mmol, 417 mg) was added, and the reaction was carried out on a shaking bed at room temperature for 3 h. 3 mL of methanol was added to terminate the reaction, the CTC resin was filtered, the resin was washed with DMF (dimethylformamide) for 3 times, and was dried for standby use;

[0042] (2) 20 mL of 20% piperidine / DMF solution was added to compound 2 (1 mmol), and nitrogen was blown for 25 min. After being washed with DMF for 4 times, it was dried for standby. Ninhydrin color reaction was blue. Fmoc-Glu(OBzl)-OH (3 mmol, 1.38 g) and HBTU (3 mmol, 1.14 g) were added to the CTC resin, and then 10 mL of DMF and N,N-diisopropyl ethylamine (3 mmol, 417 mg) were added. Nitrogen was blown for 1 h. Ninhydrin color reaction was colorless. After being washed with DMF for 4 times, it was dried for standby.

[0043] (3) 20 mL of 20% piperidine / DMF solution was added to compound 3 (1 mmol), and nitrogen was blown for 25 min. After being washed with DMF for 4 times, it was dried for standby. Ninhydrin color reaction was blue. DOTA(OtBu)3 (3 mmol, 1.72 g) and HBTU (3 mmol, 1.14 g) were added to the CTC resin, and then 10 mL of DMF and N,N-diisopropyl ethylamine (3 mmol, 417 mg) were added. Nitrogen was blown for 1 h. Ninhydrin color reaction was colorless. After being washed with DMF for 4 times and methanol for 4 times, it was dried for standby.

[0044] (4) Compound 4 (18 mmol) was dissolved in 20 mL of 25% TFA / HFIP / DCM mixed solution, and stirred at room temperature for 1 h. After filtration, the filter cake was washed with DCM for 3 times. The filtrate was dried to obtain 910 mg of white powder compound 5 (yield, 90%);

[0045] (5) Compound 5 (910 mg, 0.9 mmol) was dissolved in 20 mL of methanol, and replaced with nitrogen for 3 times. 100 mg of 10% palladium-carbon was added, and replaced with hydrogen for 3 times. After stirring at room temperature for 4 h, the filter cake was washed with methanol for 3 times. The filtrate was dried to obtain 602 mg of white powder compound 6;

[0046] (6) Compound 6 (1 mmol), HATU (1.1 mmol), FAPI monomer (CAS: 2471983-20-5, 1 mmol) and DIPEA were dissolved in DMF. After reaction for 3 h, the compound was analyzed by reverse column chromatography to obtain compound 7;

[0047] (7) Compound 7 was dissolved in 95% TFA solution, and stirred at room temperature for 1 h to obtain the FAPI trimer compound.

[0048] The HPLC spectrum and LC-MS spectrum analysis of the FAPI trimer compound are shown in Figure 1 and Figure 2 , and the specific reaction route of the FAPI trimer compound is shown as follows:

[0049]

[0050] Example 2

[0051] 68 Labeling conditions and methods for Ga-FAPI-FUSCC-Tri1

[0052] FAPI trimer compound and 68 A mixture of GaCl3 radionuclide and aqueous solution was prepared, and the pH of the mixture was adjusted to 3.3-3.6 using sodium acetate solution (1M / L). The mixture was then heated at 100℃ for 10 min to obtain a FAPI trimer-based PET imaging agent for tumor diagnosis. 68 The specific reaction pathway of Ga-FAPI-FUSCC-Tri1 is shown below:

[0053]

[0054] Example 3

[0055] 177 Labeling conditions and methods for Lu-FAPI-FUSCC-Tri1

[0056] The compound DOTA-FAPI-FUSCC-Tri1 was dissolved in aqueous solution to prepare DOTA-FAPI-FUSCC-Tri1 solutions with a concentration of 1 μg / μL. 10 μL of each DOTA-FAPI-FUSCC-Tri1 solution was then mixed with 2.5 μL of 0.099 GBq / μL solution. 177 LuCl3 stock solution (in 0.04M HCl), 2 μL acetate-sodium acetate buffer, 10 μL gentianic acid, and 90 μL physiological saline were reacted at 90℃ for 30 min. After the reaction, the labeling rate was directly measured; the labeling rate reached 95% without further purification. 177 The specific reaction pathway of Lu-FAPI-FUSCC-Tri1 is shown below:

[0057]

[0058] Example 4

[0059] Stability test

[0060] Add 200 μL of normal saline, PBS, and serum to EP tubes (n=3), respectively. Add 4 μCi to each EP tube. 68 Ga-FAPI-FUSCC-Tri1 or 177Lu-FAPI-FUSCC-Tri1 was reacted at 37℃. iTLC analysis was performed at 30 min, 60 min, and 120 min to obtain the stability of each sample at different times.

[0061] Example 5

[0062] PET and SPECT Imaging Studies

[0063] To assess tumor-targeting capabilities and monitor the in vivo pharmacokinetics of these two radiotracers, experiments were conducted in mice bearing HT-1080-FAP tumors. 68 PET imaging of Ga-FAPI-FUSCC-Tri1. First, mice were injected with... 68 Ga-FAPI-FUS CC-Tri1 was used, followed by isoflurane anesthesia of the mice. After the mice were anesthetized, the OPEN button on the PET instrument was pressed, the mice were fixed onto the machine bed, the heating pad was turned on, and the bed was pushed into the PET instrument. Different PET / CT images were taken at different time points to obtain the desired images.

[0064] To assess tumor-targeting capabilities and monitor the in vivo pharmacokinetics of these two radiotracers, experiments were conducted in mice bearing HT-1080-FAP tumors. 177 PET imaging of Lu-FAPI-FUSCC-Tri1. Mice were first injected with... 177 Lu-FAPI-FU SCC-Tri1 was administered, followed by isoflurane anesthesia of the mice. After anesthesia, the SPECT instrument's OPEN button was pressed, the mice were secured to the machine bed, the heating pad was turned on, and the bed was pushed into the SPECT instrument. Different PET / CT images were captured at different time points to obtain the desired images.

[0065] 68 PET imaging results of Ga-FAPI-FUSCC-Tri1 are as follows Figure 5 As shown, relative to the blocking group Figure 5 b. The probe exhibited excellent tumor aggregation effects and maintained high uptake even after 240 minutes. Figure 5 a). Compared to 68 Ga-FAPI-04 ( Figure 5 c), 68 Ga-FAPI-FUSCC-Tri1 allows for faster uptake and more rapid accumulation of the probe at the tumor site. 68 Ga-FAPI-04 showed no significant uptake after 240 minutes, but 68 The Ga-FAPI-FUSCC-Tri1 uptake value reached its peak at 240 minutes, and in addition to tumor tissue, 68The in vivo metabolic tissue radioactivity of Ga-FAPI-FUSCC-Tri1 was significantly reduced.

[0066] 177 SPECT imaging results of Lu-FAPI-FUSCC-Tri1 are as follows Figure 6 As shown, 177 Lu-FAPI-FUSCC-Tri1 showed significant uptake at the tumor site, and the uptake was prolonged, remaining noticeable even after 120 hours, indicating that the probe has the potential to become a therapeutic probe.

[0067] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An FAPI trimer compound, characterized in that, It has the structure shown in Equation I:

2. The method for preparing the FAPI trimer compound as described in claim 1, characterized in that, Includes the following steps: (1) Compound 1 and CTC resin were swollen in DCM, N,N-diisopropylethylamine was added, and the reaction was carried out in a shaker at room temperature. The reaction was terminated by adding methanol to obtain compound 2. (2) Add 20% piperidine / DMF solution to compound 2, react under nitrogen, wash the solid with DMF 4 times, then add Fmoc-Glu(OBzl)-OH, HBTU, DMF and N,N-diisopropylethylamine, react under nitrogen for a second time, carry out de-Fmoc and condensation reaction to obtain compound 3; (3) Add 20% piperidine / DMF solution to compound 3, react under nitrogen, wash the solid with DMF 4 times, then add DOTA(OtBu)3, HBTU, DMF and N,N-diisopropylethylamine, react under nitrogen for a second time, remove Fmoc from compound 3, and then couple DOTA(OtBu)3 to obtain compound 4. (4) Compound 4 was dissolved in a 25% TFA / HFIP / DCM mixed solution, stirred at room temperature and filtered. The filter cake was washed three times with DCM to complete the deprotection of HOCTCResin and obtain compound 5. (5) Dissolve compound 5 in methanol, purge with nitrogen three times, add 10% palladium on carbon and mix, purge with hydrogen three times, stir at room temperature for 4-5 hours under hydrogen atmosphere and filter, wash the filter cake with methanol three times, deprotect compound 5 to release three carboxylic acids, and obtain compound 6. (6) Compound 6, HATU, FAPI monomer and DIPEA were dissolved in DMF and reacted for 3 h. The compounds were analyzed by reverse column chromatography to obtain compound 7. (7) Compound 7 was finally debocized to obtain the FAPI trimer compound; 3. The preparation method according to claim 2, characterized in that, The reaction time of the shaker in step (1) is 3-4 hours.

4. The preparation method according to claim 2, characterized in that, The nitrogen gas reaction time in steps (2) and (3) is 25-40 min.

5. The preparation method according to claim 2, characterized in that, The secondary nitrogen gas reaction time in steps (2) and (3) is 50-60 min.

6. The preparation method according to claim 2, characterized in that, In step (4), the stirring time at room temperature is 45-60 min.

7. A PET developer based on FAPI trimer compounds, characterized in that, The compound includes FAPI trimer compounds and diagnostic radionuclides; the FAPI trimer compound is the FAPI trimer compound of claim 1 or the FAPI trimer compound obtained by the preparation method of any one of claims 2-7; the diagnostic radionuclides include... 68 Ga, 177 Lu, 131 I, 126 I, 191 Ir, and 193 Ir.

8. The PET developer as described in claim 7, characterized in that, The diagnostic radionuclide is 68 Ga and 177 Lu.

9. The method for preparing the PET developer according to any one of claims 7-8, comprising the following steps: FAPI trimer compounds were mixed with a solution containing a diagnostic radionuclide, and the pH was adjusted to 3.3-3.6 before radiolabeling to obtain a PET imaging agent based on FAPI trimer.

10. The use of the FAPI trimer compound as described in claim 1, the FAPI trimer compound obtained by the preparation method according to any one of claims 2-6, the PET imaging agent according to any one of claims 7-8, or the PET imaging agent obtained by the preparation method according to claim 9 in PET imaging or in the preparation of tumor drugs for diagnosing FAP-q expression.

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