CN115260160B - A compound targeting fibroblast activation protein FAP and its preparation method and application - Google Patents

Abstract

或者

Q'选自

或者

W为可螯合核素的结构。本发明还提供基于所述靶向化合物的放射性核素标记化合物，及其制备方法和诊断或治疗以成纤维细胞激活蛋白(FAP)过度表达为特征的疾病中的应用。

The present invention relates to a compound targeting the dimer of FAP protein, the structure of which is shown in the following formulas (I) and (II), wherein R ₁ , R ₂ , R ₃ and R ₄ are the same or different, and are independently selected from H or F; Z and U are the same or different linking structures, independently selected from -NH- or based on - (CH ₂ ) _n - replacement structure; Q is selected from

or

Q' from

or

W is a structure that can chelate a nuclide. The present invention also provides a radionuclide-labeled compound based on the targeting compound, a preparation method thereof, and an application in diagnosing or treating a disease characterized by overexpression of fibroblast activation protein (FAP).

Description

A compound targeting fibroblast activation protein FAP and its preparation method and application

Technical Field

The present invention relates to the fields of nuclear medicine and molecular imaging, and in particular to a compound, a pharmaceutical composition comprising or consisting of the compound, a kit comprising or consisting of the compound or the pharmaceutical composition, and use of the compound or the pharmaceutical composition in diagnosing or treating a disease characterized by overexpression of fibroblast activation protein (FAP).

Background Art

Fibroblast activation protein (FAP) is a membrane serine peptidase expressed on the surface of activated fibroblasts in tumor stroma and plays an important role in the occurrence and development of tumors. Previous studies have shown that FAP is generally not expressed in normal human tissues, but is selectively highly expressed on the surface of stromal fibroblasts in more than 90% of epithelial malignancies, including breast cancer, esophageal cancer, thyroid cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer, and pancreatic cancer. Given its widespread expression and important role in tumors, FAP has become an important target for tumor imaging and treatment.

Radionuclide-labeled fibroblast activation protein inhibitors (FAPI), represented by quinolinic acid derivatives, have made important progress in the field of precise tumor imaging. For example, PET/CT imaging agents such as FAPI-02 and FAPI-04 have achieved more than 30 different types of tumor-specific imaging. Currently reported FAPI is rapidly cleared in the blood circulation and rapidly eluted at the tumor site. This metabolic characteristic is very unfavorable for treatment, because rapid metabolism and elution result in a low effective dose at the tumor site and a short retention time. High doses or more frequent dosing are required to meet treatment needs, increasing the possibility of adverse reactions. Based on the multivalent effect, Chen Haoyun et al. (J Nucl Med. 2022Jun; 63(6): 862-868) developed a dimer based on the FAPI46 structure, the structure of which is as follows:

Although this type of dimer FAPI can improve tumor uptake and retention time, higher physiological uptake is observed in the kidney, blood pool, liver, thyroid and pancreas. It is well known in the art that high background uptake in normal organs leads to relatively low tumor-to-background ratios, which will affect the detection efficiency of lesions and increase potential risks during therapeutic applications. Therefore, it is necessary to optimize the structure of dimer FAPI so that it has suitable metabolic kinetics, higher tumor uptake dose and longer tumor retention time to meet the needs of radionuclide therapy and imaging.

Summary of the invention

In view of the above background, the primary purpose of the present invention is to develop a new compound structure targeting fibroblast activation protein (FAP).

Another object of the present invention is to provide a method for preparing the novel compound.

Another object of the present invention is to provide use of the compound in diagnosing or treating a disease characterized by overexpression of fibroblast activation protein (FAP).

The above-mentioned purpose of the present invention is achieved by the following technical solutions:

In a first aspect, the present invention provides a dimer compound targeting FAP protein or a pharmaceutically acceptable salt thereof, wherein the compound structure is shown in the following formula (I):

On this basis, the present invention also provides a radiolabeled dimer compound targeting FAP protein or a pharmaceutically acceptable salt thereof, wherein the compound structure is shown in the following formula (II):

In the above formula (I) and formula (II):

R ₁ , R ₂ , R ₃ and R ₄ are the same or different and are independently selected from H or F;

Z and U are the same or different and are independently selected from -NH- or a replacement structure based on -(CH ₂ ) _n -, wherein n is an integer from 1 to 16, wherein each -CH ₂ - is individually replaced with or without -O-, -NH-, -(CO)-, -NH-(CO)-, -CH(NH ₂ )- or -(CO)-NH-, provided that no two adjacent -CH ₂ - groups are replaced;

或者

上述式(II)中，Q'选自

或者

In the above formula (I), Q is selected from

or

In the above formula (II), Q' is selected from

or

In the above formula (II), W is a nuclide chelating group part, which is a group that can chelate radionuclides from any one of 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetraamine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTPA), bis-(carboxymethylimidazole)glycine or 6-hydrazinopyridine-3-carboxylic acid (HYNIC), or any one of the following structures:

wherein D is a replacement structure based on -( _CH2 ) _p- , wherein p is an integer from 0 to 16, wherein each _-CH2- is individually replaced with or without -O-, -NH-, -(CO)-, -NH-(CO)-, -CH( _NH2 )- or -(CO)-NH-, provided that no two adjacent _-CH2- groups are replaced.

In a preferred embodiment of the present invention, the structure of the compound of formula (I) is any one of the following formulas (I-1) to (I-3):

In a preferred embodiment of the present invention, the structure of the compound of formula (II) is any one of the following formulas (II-1) to (II-11):

Further, the present invention also provides a radionuclide-labeled dimer compound targeting FAP protein, which is a compound represented by formula (II) of the present invention labeled with a radionuclide; the radionuclide can be selected from an isotope emitting α rays, an isotope emitting β rays, an isotope emitting γ rays, an isotope emitting Auger electrons, or an isotope emitting X-rays, such as ¹⁸ F, ⁵¹ Cr, ⁶⁷ Ga, ⁶⁸ Ga, ¹¹¹ In, ^99m Tc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹³⁹ La, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁵³ Sm, ¹⁶⁶ Ho, ⁸⁶ Y, ⁹⁰ Y, ¹⁴⁹ Pm, ¹⁶⁵ Dy, ¹⁶⁹ Er, ¹⁷⁷ Lu, ⁴⁷ Sc, ¹⁴² Pr, ¹⁵⁹ Gd, ²¹² Bi, ²¹³ Bi, ⁷² As, ⁷² Se, ⁹⁷ Ru, any one ^{of 109} Pd, ¹⁰⁵ Rh, ^101m Rh, ¹¹⁹ Sb, ¹²⁸ Ba, ¹²³ I, ¹²⁴ I, ¹³¹ I, ¹⁹⁷ Hg, ²¹¹ At, ¹⁵¹ Eu, ¹⁵³ Eu, ¹⁶⁹ Eu, ²⁰¹ Tl, ²⁰³ Pb, ²¹² Pb, ⁶⁴ Cu, ⁶⁷ Cu, ¹⁹⁸ Au, ²²⁵ Ac, ²²⁷ Th or ¹⁹⁹ Ag; more preferably, the radionuclide is ¹⁸ F, ⁶⁴ Cu, ⁶⁸ Ga, ⁸⁹ Zr, ⁹⁰ Y, ¹¹¹ In, ^99m Tc, ¹⁷⁷ Lu, ¹⁸⁸ Re or ²²⁵ Ac.

The present invention also provides the dimer compound targeting FAP protein, the dimer compound targeting FAP protein that can be labeled with a radionuclide, or a pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt of the dimer compound targeting FAP protein labeled with a radionuclide.

In a second aspect, the present invention provides a method for preparing a dimer compound targeting FAP protein represented by formula (I), a compound represented by formula (II) and a radionuclide label thereof, comprising:

① Preparation of monomers

The carboxylic acid hydroxyl group of 6-hydroxyquinoline-4-carboxylic acid and the amino group of glycine tert-butyl ester undergo an amide condensation reaction to obtain intermediate I;

Then, the hydroxyl group of the intermediate I is subjected to a condensation reaction with N-Boc-bromoethylamine to obtain an intermediate II containing a protected amino group and a protected carboxyl group;

After the carboxyl group in the intermediate II is deprotected, an amide condensation reaction is carried out with (S)-pyrrolidine-2-carbonitrile hydrochloride or (S)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride to obtain an intermediate III containing a protected amino group;

The amino group in the intermediate III is deprotected to obtain the intermediate IV, and the amino group of the intermediate IV undergoes a condensation reaction with the carboxyl hydroxyl group of tert-butyloxycarbonyl-L-glutamic acid-5-tert-butyl ester to obtain a class of intermediates V containing a protected amino group and a protected carboxyl group;

② Synthetic dimer

First, the carboxyl group of intermediate V is deprotected, and a condensation reaction is carried out with the amino group of any intermediate IV obtained in ① to obtain intermediate VI; after deprotection, intermediate VI is the dimer compound targeting FAP protein of the present invention;

③Synthesis of radionuclide-labeled dimer compounds targeting FAP protein

The Boc protecting group of the intermediate VI is removed; a chelating group part W is introduced, wherein W is any one of 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetraamine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTPA), bis-(carboxymethylimidazole)glycine or 6-hydrazinopyridine-3-carboxylic acid (HYNIC) that can chelate a radionuclide, or any one of the following structures:

wherein D is a replacement structure based on -(CH ₂ ) _p -, wherein p is an integer from 0 to 16, wherein each -CH ₂ - is individually replaced with or without -O-, -NH-, -(CO)-, -NH-(CO)-, -CH(NH ₂ )- or -(CO)-NH-, and the replacement condition is that no two adjacent -CH ₂ - groups are replaced; and a FAP protein-targeting dimer compound that can be labeled with a radionuclide is obtained, which is the compound represented by formula (II);

④ The radionuclide-labeled dimer compound targeting FAP protein obtained in ③ is reacted with a compound containing a radionuclide according to an existing wet labeling method or a freeze-drying labeling method to prepare a radionuclide-labeled dimer compound targeting FAP protein described in the present invention.

In a third aspect, the present invention provides a pharmaceutical composition, which comprises the dimer compound targeting FAP protein described in the first aspect of the present invention, the dimer compound targeting FAP protein that can be labeled with a radionuclide, the dimer compound targeting FAP protein labeled with a radionuclide, or any pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt thereof; or is composed of the dimer compound targeting FAP protein described in the first aspect of the present invention, the dimer compound targeting FAP protein that can be labeled with a radionuclide, the dimer compound targeting FAP protein labeled with a radionuclide, or any pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt thereof and any pharmaceutically acceptable carrier and/or excipient.

In a fourth aspect, the present invention provides the use of the dimer compound targeting FAP protein described in the first aspect, the dimer compound targeting FAP protein that can be labeled with a radionuclide, the dimer compound targeting FAP protein labeled with a radionuclide, or the pharmaceutical composition described in the third aspect in the preparation of a drug for diagnosing or treating a disease characterized by overexpression of fibroblast activation protein (FAP) in an animal or human individual.

In the application of the present invention, the diseases characterized by overexpression of fibroblast activation protein (FAP) include but are not limited to: cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and scar disease; preferably, the cancer is further selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocarcinoma, clear cell renal carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, CUP (unknown primary cancer), thymic carcinoma, glioma, glioma, astrocytoma, cervical cancer or prostate cancer.

In a fifth aspect, the present invention also provides a kit, which comprises or consists of a compound represented by formula (I) of the present invention, a compound represented by formula (II) of the present invention, a radionuclide-labeled dimer compound targeting FAP protein of the present invention, or a pharmaceutical composition of the present invention, and instructions for diagnosing a disease.

In the past, in the development of FAPI monomers, researchers found that the piperazine ring in the FAPI structure played an important role in maintaining tumor uptake. Therefore, adding a piperazine ring to the structure has become a conventional choice or common means for designing FAPI probes in this field. It can also be seen from the report of Chen Haoyun et al. (J Nucl Med. 2022Jun; 63 (6): 862-868) that this design has also been retained in the development of dimer FAPI. However, the inventors found in the research and development experiments that simple dimerization modification based on the existing FAPI monomers could not obtain high-performance FAPI probes, but instead caused them to have higher background uptake, resulting in a lower tumor-to-background ratio in terms of organ uptake of drugs, and poor performance. After a large number of experimental observations, the inventors believe that the presence of the piperazine ring structure may have an adverse effect on the pharmacokinetic properties of dimer FAPI. Therefore, the present invention does not introduce a piperazine ring structure in the synthesis of the dimer FAPI, and experiments have verified that the prepared compound structure targeting the FAP protein and its radiolabeled compound have significantly improved tumor uptake effect compared with the existing dimer FAPI, and are expected to be used in the diagnosis or treatment of diseases characterized by overexpression of fibroblast activation protein (FAP).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a mass spectrum of compound 2 in Example 1 of the present invention.

FIG2 is a mass spectrum of compound 6 in Example 1 of the present invention.

FIG3 is a mass spectrum of compound 8 in Example 1 of the present invention.

FIG4 is a mass spectrum of compound 10 in Example 1 of the present invention.

FIG5 is a mass spectrum of compound 11 of formula (I-1) in Example 1 of the present invention.

FIG6 is a mass spectrum of compound 12 in Example 1 of the present invention.

FIG7 is a mass spectrum of compound 14 in Example 2 of the present invention.

FIG8 is a mass spectrum of compound 16 in Example 2 of the present invention.

FIG9 is a mass spectrum of formula (II-2) in Example 3 of the present invention.

FIG10 is a mass spectrum of the intermediate of formula (II-3) in Example 4 of the present invention.

FIG11 is a mass spectrum of the intermediate of formula (II-3) in Example 4 of the present invention.

FIG12 is a mass spectrum of formula (II-3) in Example 4 of the present invention.

FIG13 is a mass spectrum of formula (II-3) in Example 4 of the present invention.

FIG14 is a mass spectrum of formula (II-4) in Example 5 of the present invention.

Figure 15 is a mass spectrum of formula (II-10) in Example 6 of the present invention.

FIG. 16 is a diagram showing the MicroPET imaging results of the ⁶⁸ Ga-labeled complex of formula (I-1) in the present invention in HT1080-hFAP tumor-bearing mice.

FIG. 17 is a diagram showing the MicroPET imaging results of the ⁶⁸ Ga-labeled FAPI46 complex of the present invention in HT1080-hFAP tumor-bearing mice.

FIG. 18 is a diagram showing the MicroPET imaging results of the 68Ga-labeled complex of formula (I-1) of the present invention and FAPI-04 co-injected in HT1080-hFAP tumor-bearing mice 1 hour after injection.

Figure 19 is a statistical graph showing the uptake results of the ⁶⁸ Ga-labeled complex of formula (I-1) of the present invention in tumors and important organs 1 hour, 2 hours and 4 hours after injection into HT1080-hFAP tumor-bearing mice (the horizontal axis in the figure represents different organs, and the bar graphs from left to right in each organ correspond to the uptake values of the ⁶⁸ Ga-labeled complex of formula (I-1)).

FIG. 20 is a diagram showing the MicroPET imaging results of the ⁶⁸ Ga-labeled F2 complex of the present invention in HT1080-hFAP tumor-bearing mice.

FIG. 21 is a diagram showing the MicroPET imaging results of the ⁶⁸ Ga-labeled F3 complex of the present invention in HT1080-hFAP tumor-bearing mice.

DETAILED DESCRIPTION

The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

Example 1: Preparation of compound of formula II-1

Synthesis of compound 2:

In a 100mL flask, compound 1 (6-hydroxyquinoline-4-carboxylic acid, 1.89g, 10.0mmol), glycine tert-butyl ester (1.89g, 10.0mmol), HATU (3.8g, 10.0mmol) and N,N-diisopropylethylamine (2.6g, 20.0mmol) were added to 30mL N,N-dimethylformamide. The reaction mixture was stirred overnight, and the solvent was removed by distillation under reduced pressure to obtain a crude product. White solid compound 2 was purified by silica gel column (dichloromethane/methanol=30:1) with a yield of 87%. Figure 1 is the mass spectrum of compound 2.

Synthesis of compound 3:

In a 100mL flask, compound 2 (1.51g, 5.0mmol), N-Boc-bromoethylamine (2.25g, 10.0mmol), and potassium carbonate (1.38g, 10.0mmol) were added to 50mL N,N-dimethylformamide. The system was heated to 60 degrees, and the system was stirred at 60 degrees overnight. The solvent was removed by distillation to obtain a crude product. The white solid compound 3 was purified by silica gel column (dichloromethane/methanol = 30:1) with a yield of 51%.

Synthesis of compound 5:

Under ice bath conditions, compound 4 (0.44 g, 1.0 mmol) was dissolved in a mixed solution of 10 mL of dichloromethane and trifluoroacetic acid (volume ratio 9:1), the system was heated to room temperature for 2 h, the solvent was removed by vacuum distillation after the reaction was completed, 10 mL of N, N-dimethylformamide was used to dissolve, di-tert-butyl dicarbonate (0.22 g, 1.0 mmol) and N, N-diisopropylethylamine (0.39 g, 3.0 mmol) were added respectively, stirred at room temperature overnight, and the solvent was removed by vacuum distillation to obtain a crude product. White solid compound 5 was purified by silica gel column (dichloromethane/methanol = 10:1) with a yield of 77%.

Synthesis of compound 6:

In a 100mL flask, compound 5 (0.39g, 1.0mmol), (S)-pyrrolidine-2-carbonitrile hydrochloride (0.13g, 10.0mmol), HATU (0.38g, 1.0mmol) and N,N-diisopropylethylamine (0.26g, 2.0mmol) were added to 10mL N,N-dimethylformamide. The reaction mixture was stirred at room temperature until the reaction was completed, and the solvent was removed by distillation under reduced pressure to obtain a crude product. White solid compound 6 was purified by silica gel column (dichloromethane/methanol=30:1) with a yield of 82%. Figure 2 is the mass spectrum of compound 6.

Synthesis of compound 8:

In a 100mL flask, compound 7 (0.55g, 1.0mmol) and p-toluenesulfonic acid monohydrate (0.27g, 1.5mmol) were added to 10mL acetonitrile in turn. The reaction system was heated to 60 degrees Celsius and stirred until the reaction was completed. The solvent was removed by distillation under reduced pressure. After 10mL N,N-dimethylformamide was dissolved, tert-butyloxycarbonyl-L-glutamic acid-5-tert-butyl ester (0.3g, 1.0mmol), HATU (0.38g, 1.0mmol) and N,N-diisopropylethylamine (0.26g, 2.0mmol) were added respectively. The reaction mixture was stirred until the reaction was completed, and the solvent was removed by distillation under reduced pressure to obtain a crude product. White solid compound 8 was purified by silica gel column (dichloromethane/methanol = 20:1) with a yield of 75%. Figure 3 is a mass spectrum of compound 8.

Synthesis of compound 10:

Under ice bath conditions, compound 8 (0.65 g, 1.0 mmol) was dissolved in a mixed solution of 10 mL of dichloromethane and trifluoroacetic acid (volume ratio 9:1), and the system was heated to room temperature for 2 h. After the reaction, the solvent was removed by distillation under reduced pressure. After 10 mL of N, N-dimethylformamide was dissolved, di-tert-butyl dicarbonate (0.22 g, 1.0 mmol) and N, N-diisopropylethylamine (0.39 g, 3.0 mmol) were added respectively, and stirred at room temperature overnight. After the reaction was completed, compound 7 (0.37 g, 1.0 mmol), HATU (0.38 g, 1.0 mmol) and N, N-diisopropylethylamine (0.26 g, 2.0 mmol) were added to the system in sequence. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain a crude product. The white solid compound 10 was purified by silica gel column (dichloromethane/methanol = 10:1) with a yield of 43%. FIG4 is a mass spectrum of compound 10.

Synthesis of compound 12:

Compound 7 (95 mg, 0.1 mmol) and p-toluenesulfonic acid monohydrate (0.09 g, 0.5 mmol) were added to 5 mL acetonitrile in a 50 mL flask. The reaction system was heated to 60 degrees Celsius and stirred until the reaction was completed. The solvent was removed by distillation under reduced pressure. After 5 mL N, N-dimethylformamide was dissolved, DOTA-NHS (0.05 g, 0.1 mmol) and N, N-diisopropylethylamine (0.04 g, 0.3 mmol) were added in sequence. The reaction system was stirred at room temperature and desorbed by HPLC until the reaction was completed. The solvent was removed by distillation under reduced pressure to obtain a crude product. The crude product was columned by reverse phase and freeze-dried to obtain pure compound 12 with a yield of 37%. Figure 5 is a mass spectrum of compound 11, and Figure 6 is a mass spectrum of compound 12.

The synthetic route of the above steps is as follows:

Example 2: Preparation of Compound of Formula II-7

Synthesis of compound 14:

In a 50 mL flask, compound 11 (85 mg, 0.1 mmol), compound 13 (please provide chemical name 52 mg, 0.1 mmol), HATU (38 mg, 0.1 mmol) and N, N-diisopropylethylamine (39 mg, 0.30 mmol) prepared in Example 1 were added respectively. The reaction mixture was stirred overnight. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain a crude product. White solid compound 14 was purified by silica gel column (dichloromethane/methanol = 10:1) with a yield of 54%. Figure 7 is the mass spectrum of compound 14.

Synthesis of compound 16:

Under ice bath conditions, compound 14 (67 mg, 0.1 mmol) was dissolved in a mixed solution of 10 mL of dichloromethane and trifluoroacetic acid (volume ratio 9: 1), and the system was heated to room temperature for 2 h. After the reaction, the solvent was removed by reduced pressure distillation to obtain compound 15. After compound 15 was dissolved in 5 mL of N, N-dimethylformamide, DOTA-NHS (0.05 g, 0.1 mmol) and N, N-diisopropylethylamine (0.04 g, 0.3 mmol) were added in sequence. The reaction system was stirred at room temperature for reaction, and the reaction was monitored by HPLC until the reaction was completed. The solvent was removed by reduced pressure distillation to obtain a crude product. The crude product was subjected to reverse phase columnization and freeze-dried to obtain pure compound 16 with a yield of 39%. Figure 8 is a mass spectrum of compound 16.

The synthetic route of the above steps is as follows:

The structures of the compounds of Examples 3-11 are shown in Formula (II-2) to Formula (II-6) and Formula (II-8) to Formula (II-11), respectively. Their preparation methods can refer to Example 1 and Example 2 to obtain the following corresponding structures:

Example 12. Preparation of radioactive Ga-68 labeled complex:

Wet method: Add about 18.5-1850 MBq ⁶⁸ GaCl ₃ hydrochloric acid solution (eluted from a gallium germanium generator) to a centrifuge tube containing 0.5 mL of acetic acid-acetate solution (1.0 g/L) of compound 12 prepared in Example 1, and place at 37°C for reaction for 20 min. Take a C18 separation column, slowly elute with 10 mL of anhydrous ethanol, and then elute with 10 mL of water. Dilute the labeled solution with 10 mL of water, load it onto the separation column, first remove the unlabeled ⁶⁸ Ga ions with 10 mL of water, and then elute with 0.3 mL of 10 mM HCl ethanol solution to obtain the ⁶⁸ Ga-labeled complex. The eluent is diluted with physiological saline and sterile filtered to obtain the injection of ^{the 68} Ga-labeled complex of formula (II-1).

Lyophilization method: Add about 18.5-1850 MBq ⁶⁸ GaCl ₃ hydrochloric acid solution (eluted from the germanium gallium generator) to the freeze-dried medicine box containing the labeled precursor, mix well and react at 37°C for 20 minutes. Take a C18 separation column, slowly elute with 10 mL of anhydrous ethanol, and then elute with 10 mL of water. Dilute the labeled solution with 10 mL of water, load it onto the separation column, first remove the unlabeled ⁶⁸ Ga ions with 10 mL of water, and then elute with 0.3 mL of 10 mM HCl ethanol solution to obtain the complex eluent. The eluent is diluted with physiological saline and sterile filtered to obtain the injection of ^{the 68} Ga labeled (II-1) complex.

Experimental example. Application effect analysis

The complex of formula (II-1) prepared according to the method of Example 12 was injected into HT1080-hFAP tumor-bearing mice (7.4 MBq) via the tail vein, and then MicroPET imaging was performed at 0 to 60 minutes after administration under isoflurane anesthesia. Figure 16 shows the MicroPET images of HT1080-hFAP tumor-bearing mice at different times after intravenous injection of ⁶⁸ Ga-complex (II-1), and Figure 17 shows the MicroPET images of HT1080-hFAP tumor-bearing mice 1 hour after intravenous injection of ⁶⁸ Ga-FAPI-46 complex. It can be seen from Figure 16 that ⁶⁸ Ga-complex (II-1) is rapidly and efficiently taken up at the tumor site, and is very low in most normal organs, and is mainly cleared quickly through the kidneys. Compared with the currently widely used monomer ⁶⁸ Ga-FAPI46, the uptake of the dimer form of ⁶⁸ Ga-complex (II-1) in the tumor is significantly improved. In addition, the above-mentioned ⁶⁸ Ga-formula (II-1) complex and FAPI-04 were co-injected into HT1080-hFAP tumor-bearing mice, and the MicroPET imaging results were shown in Figure 18. Co-injection resulted in a sharp decrease in tumor uptake, and the blocking experiment confirmed that ^{the 68} Ga-formula (II-1) complex can achieve tumor-specific targeting through FAP protein in vivo. In the biodistribution study, HT1080-hFAP mice were injected with 1.48MBq of ⁶⁸ Ga-formula (II-1) complex, which was separated and weighed 1h, 2h and 4h after injection (three parallel experiments at each time point), and the radioactivity of major organs and tumors was measured and analyzed by a γ counter (counts per min, cpm). The test results are shown in Figure 19, which are consistent with the PET imaging results. The ⁶⁸ Ga-formula (II-1) complex showed high uptake and high retention time in tumors, while the uptake of normal organs was extremely low.

In the development of FAPI monomers, previous studies have shown that the piperazine ring in the FAPI structure plays an important role in maintaining tumor uptake. However, the inventors have found through experiments that the presence of the piperazine ring structure may have an adverse effect on the pharmacokinetic properties of the dimer FAPI, causing it to have a higher background uptake, resulting in a lower tumor-to-background ratio in terms of drug organ uptake. In this experiment, as a control, the researchers injected ⁶⁸ Ga-F2 with two piperazine ring structures (its labeled precursor structure is shown in Figure 20) and ⁶⁸ Ga-F3 with one piperazine ring (its labeled precursor structure is shown in Figure 21) into HT1080-hFAP tumor-bearing mice via the tail vein (7.4MBq) and performed MicroPET imaging. As can be seen from Figures 20 and 21, ⁶⁸ Ga-F2 with two piperazine ring structures has a higher uptake in the tumor, but the uptake in the kidney is also high; at the same time, although the kidney uptake of ⁶⁸ Ga-F3 with one piperazine ring is lower than that of ⁶⁸ Ga-F2, a higher uptake is observed in the joints. Compared with ⁶⁸ Ga-F2 and ⁶⁸ Ga-F3, the dimer probe structure of the present invention does not contain a piperazine ring, as shown in FIG. 16 and FIG. 19 . The dimer FAPI has excellent metabolic kinetics, high tumor uptake and retention time, and has application potential.

In summary, the present invention has developed a new fibroblast activation protein FAP targeting compound, which is expected to be used in the diagnosis or treatment of diseases characterized by overexpression of fibroblast activation protein (FAP).

Although the present invention has been described in detail above by general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims (10)

1. A dimer compound targeting FAP protein of any of the following structures or a pharmaceutically available salt thereof:

2. a dimer compound of the targeting FAP protein of radionuclide labeling, it is that the compound shown in formula (II-1)～formula (II-6) described in claim 1 has marked radionuclide to obtain The radionuclide is selected from ¹⁸ F, ⁶⁴ Cu, ⁶⁸ Ga, ⁸⁹ Zr, ⁹⁰ Y, ¹¹¹ In, ^99m Tc, ¹⁷⁷ Lu, ¹⁸⁸ Re or ²²⁵ Ac. 3. the method for preparing the compound shown in formula (I-1)～(I-3) described in claim 1, comprising: ① Preparation of monomer The carboxyl hydroxyl group of 6-hydroxyquinoline-4-carboxylic acid and the amino group of glycine tert-butyl ester undergo amide condensation reaction to obtain intermediate I; Then the hydroxyl group of the intermediate I is condensed with N-Boc-bromoethylamine to obtain the intermediate II containing a protected amino group and a protected carboxyl group; After deprotection of the carboxyl group in intermediate II, there is an amide condensation reaction with (S)-pyrrolidine-2-carbonitrile hydrochloride or (S)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride, Intermediate III containing a protected amino group is obtained; After deprotection of the amino group in intermediate III, intermediate IV is obtained, and the amino group of intermediate IV is condensed with the carboxyl hydroxyl group of tert-butoxycarbonyl-L-glutamic acid-5-tert-butyl ester to obtain a class of protected amino and Protected carboxyl intermediate V; ②Synthesis of dimers First deprotect the carboxyl group of intermediate V, and condense with the amino group of any intermediate IV obtained in ① to obtain intermediate VI; after deprotection of intermediate VI, the formula (I-1)～(I- 3) The indicated dimer compounds targeting FAP protein. 4. the method for preparing the compound shown in formula (II-1)～(II-6) described in claim 1, comprising: Remove the Boc protecting group of the intermediate VI of claim 3 gained; Introduce chelating group part W, W is any one in the following structures:

where D is -NH-; A dimer compound targeting the FAP protein that can be labeled with a radionuclide is obtained, that is, compounds represented by formulas (II-1) to (II-6). 5. prepare the method for the dimer compound of the target FAP protein of radionuclide labeling described in claim 2, comprise: the compound shown in the formula (II-1)～(II-6) that claim 4 gained and The radionuclide-containing compound can be reacted according to the existing wet labeling method or freeze-drying labeling method to prepare the radionuclide-labeled targeting compound. 6. A pharmaceutical composition, characterized in that: comprising any dimer compound targeting FAP protein according to claim 1, any radionuclide-labeled targeting FAP protein according to claim 2 dimer compounds, or their pharmaceutically acceptable salts. 7. any one of the dimer compounds targeting FAP protein shown in formulas (II-1) to (II-6) according to claim 1, any one of the radionuclide-labeled compounds according to claim 2 The dimeric compound targeting FAP protein, or their pharmaceutically acceptable salts, or the pharmaceutical composition described in claim 6 are used in the preparation of fibroblast activation protein (FAP) for diagnosis or treatment of animals or human individuals ) application in medicine for diseases characterized by overexpression. 8. The application according to claim 7, characterized in that: the diseases characterized by the overexpression of fibroblast activation protein (FAP) include: cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and keloids. 9. The application according to claim 8, wherein the cancer is selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, Hypopharyngeal carcinoma, nasopharyngeal carcinoma, laryngeal carcinoma, myeloma cell carcinoma, bladder carcinoma, cholangiocarcinoma, clear cell renal carcinoma, neuroendocrine tumor, carcinogenic osteomalacia, sarcoma, CUP (unknown primary carcinoma), thymic carcinoma , glioma, glioma, astrocytoma, cervical cancer, or prostate cancer. 10. A kit comprising or consisting of: ① any dimer compound targeting FAP protein represented by formulas (II-1) to (II-6) according to claim 1; Any one of the radionuclide-labeled dimer compounds targeting the FAP protein described in 2, or their pharmaceutically acceptable salts, or the pharmaceutical composition described in claim 6; ② instructions for diagnosing diseases .

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