FAPI tripolymer compound based on GalNAc link chain as well as preparation method and application of FAPI tripolymer compound

Abstract

The invention provides a novel FAPI trimer compound based on a GalNAc (N-acetylgalactosamine) link chain, a preparation method of the FAPI trimer compound, a developing agent based on the FAPI trimer compound and a preparation method of a developing agent. The trimerization structure in the FAPI trimer compound molecule provided by the invention can improve the in-vivo dynamic characteristics of the compound and prolong the residence time of the compound in tumors; the imaging agent based on the FAPI trimer compound can improve the uptake and imaging effects in tumors, and has good application prospects in PET / SPECT imaging, preparation of drugs for diagnosing tumors expressed by FAP-q and drugs for marking therapeutic nuclide (177Lu or 90Y) to treat tumors expressed by FAP-q in the future.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of radiopharmaceutical chemistry, and particularly relates to a FAPI trimer compound based on a GalNAc (N-acetylgalactosamine) linking chain and a preparation method 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 have been evaluated in clinical trials to improve tumor treatment effect. In recent years, FAP-targeted imaging 68The research progress of Ga-FAPI46 PET / CT in clinical tumor diagnosis is rapid. 68 Ga-FAPI-04 can quickly enter tumors, but its retention time is relatively short, resulting in its uptake and imaging effect in tumors with low FAP expression. SUMMARY

[0004] The application provides a FAPI trimer compound based on a GalNAc linking chain and a preparation method and application thereof, and the FAPI trimer compound based on the GalNAc (N-acetylgalactosamine) linking chain provided by the application has the advantages of longer retention time in tumors and better imaging effect.

[0005] The application provides a FAPI trimer compound based on a GalNAc linking chain or a pharmaceutically acceptable salt thereof, and the FAPI trimer compound has the 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 Boc propanolamine are dissolved in DMF, HATU and triethylamine are added and reacted for 5 h, water is added to quench the reaction, ethyl acetate is added for extraction three times, and the DMF is removed by washing with salt water three times; after the solvent is spun dry, a DCM / TFA mixed solvent is added, stirring is performed at room temperature for 1 h, the solvent is spun dry, and compound 2 is obtained;

[0009] (2) Compound 2 and UAMC-1110 are dissolved in DMF, HATU and triethylamine are added and reacted for 5 h, water is added to quench the reaction, ethyl acetate is added for extraction three times, and the DMF is removed by washing with salt water three times; after the solvent is spun dry, MeOH and palladium carbon are added to the mixture, hydrogen is introduced to remove the protective gas, and compound 3 is obtained;

[0010] (3) Compound 3 and a DOTA derivative are dissolved in DMF, HATU and triethylamine are added and reacted for 5 h, water is added to quench the reaction, ethyl acetate is added for extraction three times, and the DMF is removed by washing with salt water three times; after the solvent is spun dry, a DCM / TFA mixed solvent is added, stirring is performed at room temperature for 1 h, the solvent is spun dry, and the FAPI trimer compound is obtained;

[0011]

[0012] Further, the volume ratio of DCM to TFA in the DCM / TFA mixed solvent of steps (1) and (3) is 8:1.

[0013] Further, the mass fraction of palladium in the palladium-carbon of step (2) is 10%.

[0014] The application further discloses a PET imaging agent obtained by labeling a FAPI trimer compound or a pharmaceutically acceptable salt thereof with a diagnostic nuclide; the FAPI trimer compound is 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.

[0015] Further, the diagnostic nuclide is 68 Ga and 177 Lu.

[0016] The application further discloses a preparation method of the imaging agent, and steps are as follows:

[0017] The FAPI trimer compound or the pharmaceutically acceptable salt thereof and a solution containing the diagnostic nuclide are mixed, and after the pH value is adjusted to 3.3-3.6, radioactive labeling is performed to obtain the imaging agent.

[0018] The application further discloses application of the FAPI trimer compound or the pharmaceutically acceptable salt thereof, the FAPI trimer compound obtained by the preparation method, the imaging agent or the imaging agent obtained by the preparation method in PET / SPECT imaging or preparation of a drug for diagnosing a tumor expressing FAP-q.

[0019] Compared with the prior art, the at least one technical solution adopted by the embodiments of the application can achieve the beneficial effects at least including:

[0020] (1) The FAPI trimer compound provided by the application is based on a GalNAc (N-acetylgalactosamine) linking chain and a trimer structure, which can improve the in-vivo kinetic characteristics of the compound and prolong the residence time of the compound in the tumor;

[0021] (2) The imaging agent provided by the application can improve the uptake and imaging effect in the tumor, and has a good application prospect in PET / SPECT imaging, preparation of a drug for diagnosing a tumor expressing FAP-q and future labeling of a therapeutic nuclide (177Lu or 90Y) to treat a tumor expressing FAP-q;

[0022] (3) The preparation method of the FAPI trimer compound has a short reaction route, simple operation, cheap and easily available raw materials, low production cost and suitability for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used 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 effort on the basis of the drawings.

[0024] Figure 1 HPLC spectrum of the FAPI trimer compound prepared for Example 1;

[0025] Figure 2 LC-MS spectrum of the FAPI trimer compound prepared for Example 1;

[0026] Figure 3 For 68 HPLC analysis radiochemical purity results of Ga-FAPI-FUSCC-Tri2 in different cases, wherein (a) is PBS, (b) is FBS, (c) is urine; (d) is cell uptake result figure;

[0027] Figure 4 For 177 HPLC analysis radiochemical purity results of LU-FAPI-FUSCC-Tri2 in different cases, wherein (a) is PBS, (b) is FBS, (c) is urine;

[0028] Figure 5 For 68 Tissue distribution figure of Ga-FAPI-FUSCC-Tri2;

[0029] Figure 6 For 68 PET / CT imaging results of Ga-FAPI-FUSCC-Tri2 in model mice with FAPI highly expressed cell HT-1080 as tumor cells (a), imaging results after FAPI46 blocking (b) and 68 Imaging results of Ga-FAPI-04 (c);

[0030] Figure 7 For 177 SPECT / CT imaging results of Lu-FAPI-FUSCC-Tri2 in model mice with FAPI highly expressed cell HT-1080 as tumor cells. DETAILED DESCRIPTION

[0031] The embodiments of the present application will be described in detail below with reference to the drawings.

[0032] Following, the embodiments of the present application are described through specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific embodiments, and various modifications or changes can be made to the details in the specification based on different views and applications without departing from the spirit of the present application. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0033] Embodiment 1

[0034] A FAPI trimer compound based on GalNAc linkage chain, the preparation steps are as follows:

[0035] (1) Compound 1 (1 g, 1 eq) and Boc propanolamine (1.44 g, 1.2 eq) were dissolved in 20 mL of DMF, HATU (0.88 g, 1.1 eq) and triethylamine (042 g, 2 eq) were added and reacted for 5 h, 15 mL of water was added to quench the reaction, and then 10 mL of ethyl acetate was added for extraction 3 times (10 mL*3), 15 mL of brine was added for washing 3 times (15 mL*3) to remove DMF, and then DCM / TFA mixed solvent 15 mL (volume ratio of DCM and TFA is 8:1) was added, stirred at room temperature for 1 h, and then the solvent was dried to obtain compound 2;

[0036] (2) Compound 2 (200 mg, 1 eq) and UAMC-1110 (291 mg, 1.1 eq) were dissolved in 20 mL of DMF, HATU (165.7 mg, 1.1 eq) and triethylamine (200 mg, 2 eq) were added and reacted for 5 h, 15 mL of water was added to quench the reaction, and then 10 mL of ethyl acetate was added for extraction 3 times (10 mL*3), 15 mL of brine was added for washing 3 times (15 mL*3) to remove DMF, and then 10 mL of MeOH and 10 eq of palladium on carbon (palladium content in palladium on carbon is 10%) were added to the mixture, and then hydrogen was introduced to remove the protective gas to obtain compound 3;

[0037] (3) Compound 3 (100 mg, 1 eq) was dissolved in 20 mL DMF, and reacted with a DOTA derivative (30 mg, 1.2 eq), HATU (23 mg, 1.1 eq) and triethylamine (30 mg, 2 eq) for 5 h, 15 mL water was added to quench the reaction, and then extracted with 10 mL ethyl acetate for 3 times (10 mL*3), washed with 3 times of brine to remove DMF, and then dried and concentrated to obtain the FAPI trimer compound based on the GalNAc linking chain by adding 3 mL of DCM / TFA mixed solvent (the volume ratio of DCM and TFA is 8:1), stirring at room temperature for 1 h, and drying the solvent.

[0038] The specific reaction route is as follows:

[0039]

[0040] Example 2

[0041] Developing agent 68 Labeling conditions and methods of Ga-FAPI-FUSCC-Tri2

[0042] The FAPI trimer compound DOTA-FAPI-FUSCC-Tri2 prepared in Example 1 (50 μg) and 10 mCi of GaCl3 radionuclide aqueous solution were mixed, the pH value of the mixture was adjusted to 3.3 by sodium acetate solution (1 M / L), and then heated at 100°C for 10 min to obtain the tumor diagnosis PET imaging agent based on the FAPI trimer compound 68 GaCl3 radionuclide aqueous solution, and then the pH value of the mixture was adjusted to 3.3 by sodium acetate solution (1 M / L), and then heated at 100°C for 10 min to obtain the tumor diagnosis PET imaging agent based on the FAPI trimer compound 68 Ga-FAPI-FUSCC-Tri2, and the specific reaction route is as follows:

[0043]

[0044] Example 3

[0045] Developing agent 177 Labeling conditions and methods of Lu-FAPI-FUSCC-Tri2

[0046] The FAPI trimer compound DOTA-FAPI-FUSCC-Tri2 prepared in Example 1 was dissolved in water to prepare a DOTA-FAPI-FUSCC-Tri2 solution with a concentration of 1 μg / μL. 10 uL of the above DOTA-FAPI-FUSCC-Tri2 solution was taken and added to 2.5 μL of 0.099 GBq / μL 177The original LuCl3 solution (in 0.04M HCl), 2 μL acetic acid-sodium acetate buffer, 10 μL gentisic acid and 90 μL normal saline were mixed, and the pH of the mixed solution was adjusted to 3.6, and then the mixture was reacted at 90°C for 30 min. After the reaction, the labeling rate was directly measured, and the labeling rate could reach 95% without further purification, and 177 Lu-FAPI-FUSCC-Tri2, and the specific reaction route is as shown below:

[0047]

[0048] Example 4

[0049] Stability experiment and cell uptake experiment

[0050] 200 uL of normal saline, PBS and serum were respectively taken into EP tubes (n = 3), and 4 uCi of 68 Ga-FAPI-FUSCC-Tri2 or 177 Lu-FAPI-FUSCC-Tri2 was added into each EP tube, and the mixture was reacted at 37°C. iTLC analysis was performed at 30 min, 60 min and 120 min respectively, so as to obtain the stability of each sample at different time, 68 Ga-FAPI-FUSCC-Tri2 or 177 Lu-FAPI-FUSCC-Tri2, and the results are shown in Figure 3 and Figure 4

[0051] A 24-well plate was taken out and the cells were cultured, and then the culture solution was sucked out, and then 500 μL of PBS was used for washing 3 times, and then 100 μL of 68 Ga-FAPI-FUSCC-Tri2 and DMEM serum-free medium (the 68 Ga-FAPI-FUSCC-Tri2 was diluted to 2 μCi-4 μCi) was placed in an air incubator for incubation for 30 min, 60 min, 120 min and 240 min respectively. Near the incubation time, a pipette was used to take out 68 Ga-FAPI-FUSCC-Tri2. Finally, the cpm value was measured by a γ counter, and the results are shown in Figure 3 d.

[0052] Example 5

[0053] PET and SPECT imaging study

[0054] In order to evaluate the tumor targeting ability and monitor the in vivo pharmacokinetics of the above two imaging agents, PET and SPECT imaging studies were performed in mice bearing HT-1080-FAP tumors. 68 ​PET imaging of Ga-FAPI-FUSCC-Tri2. First, inject the mice with Ga-FAPI-FUSCC-Tri2 68 Ga-FAPI-FUSCC-Tri2, then anesthetize the mice with isoflurane, after the mice are well anesthetized, press the OPEN button of the PET instrument, fix the mice to the machine bed and turn on the heating pad, push the fixed bed into the PET instrument. Take different PET / CT images at different time points to get the required images; in mice bearing HT-1080-FAP tumors 177 SPET imaging of Lu-FAPI-FUSCC-Tri2. First, inject the mice with Lu-FAPI-FUSCC-Tri2 177 Lu-FAPI-FUSCC-Tri2, then anesthetize the mice with isoflurane, after the mice are well anesthetized, press the OPEN button of the SPECT instrument, fix the mice to the machine bed and turn on the heating pad, push the fixed bed into the SPECT instrument. Take different SPET / CT images at different time points to get the required images.

[0055] 68 The tissue distribution of Ga-FAPI-FUSCC-Tri2 is shown in Figure 5 , the PET imaging results are shown in Figure 6 , relative to the blocking group Figure 6 c, the probe showed good tumor accumulation effect, and still maintained a high uptake at 240 min Figure 6 a). Compared with 68 Ga-FAPI-04 Figure 6 b), 68 Ga-FAPI-FUSCC-Tri2 uptake is faster, the probe is faster in tumor site accumulation, while 68 Ga-FAPI-04 has no obvious uptake at 240 min, but 68 Ga-FAPI-FUSCC-Tri2 uptake reached the highest, and in addition to tumor tissue, at 240 min, 68 Ga-FAPI-FUSCC-Tri2 in vivo metabolic tissue radioactivity is significantly weakened.

[0056] 177 SPECT imaging results of Lu-FAPI-FUSCC-Tri2 are shown in Figure 7 , 177 Lu-FAPI-FUSCC-Tri2 has obvious uptake at the tumor site, and the duration is very long, and obvious tumor site uptake can still be seen after 120 h, indicating that the probe has the potential to become a therapeutic probe.

[0057] The above examples are only for illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the present application and to implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made according to the essence of the present application should be covered within the protection scope of the present application.

Claims

1. A FAPI trimeric compound based on a GalNAc linker chain or a pharmaceutically acceptable salt thereof, characterized in that, The FAPI trimer compound has a structure shown in Formula I:

2. The method of producing a FAPI trimer compound according to claim 1, wherein The method comprises the following steps: (1) Compound 1 and Boc propanolamine are dissolved in DMF, HATU and triethylamine are added and reacted for 5 h, water is added to quench the reaction, ethyl acetate is added for extraction three times, and the DMF is removed by washing with brine three times, the solvent is dried and then DCM / TFA mixed solvent is added, stirring is carried out at room temperature for 1 h, the solvent is dried to obtain compound 2; (2) Compound 2 and UAMC-1110 are dissolved in DMF, HATU and triethylamine are added and reacted for 5 h, water is added to quench the reaction, ethyl acetate is added for extraction three times, and the DMF is removed by washing with brine three times, the solvent is dried, MeOH and palladium-carbon are added to the mixture, hydrogen is introduced to remove the protective gas, and compound 3 is obtained; (3) Compound 3 and a DOTA derivative are dissolved in DMF, HATU and triethylamine are added and reacted for 5 h, water is added to quench the reaction, ethyl acetate is added for extraction three times, and the DMF is removed by washing with brine three times, the solvent is dried, DCM / TFA mixed solvent is added, stirring is carried out at room temperature for 1 h, the solvent is dried to obtain the FAPI trimer compound; 3. The production method according to claim 2, wherein The volume ratio of DCM to TFA in the DCM / TFA mixed solvent of steps (1) and (3) is 8:

1.

4. The production method according to claim 2, wherein The mass fraction of palladium in the palladium-carbon of step (2) is 10%.

5. An imaging agent, characterized by, obtained from a diagnostic nuclide-labeled FAPI trimer compound or a pharmaceutically acceptable salt thereof; the FAPI trimer compound is the FAPI trimer compound according to claim 1 or the FAPI trimer compound obtained from the preparation method according to any one of claims 2 to 4; the diagnostic nuclide comprises 68 Ga, 177 Lu, 131 I, 126 I, 191 Ir and 193 Ir.

6. The imaging agent of claim 5, wherein, The diagnostic nuclide is 68 Ga and 177 Lu.

7. A method of preparing a visualizing agent according to any one of claims 5-6, characterized in that, The steps are as follows: The FAPI trimer compound or a pharmaceutically acceptable salt thereof and a solution containing a diagnostic nuclide are mixed, the pH value is adjusted to 3.3-3.6, and then radiolabeling is carried out to obtain the imaging agent.

8. The FAPI trimer compound or a pharmaceutically acceptable salt thereof of claim 1, the FAPI trimer compound obtained by the preparation method of any one of claims 2-4, the imaging agent of any one of claims 5-6, or the imaging agent obtained by the preparation method of claim 7, in PET / SPECT imaging or preparation of a diagnostic drug for FAP-q-expressing tumors.

Images