CN115260160B - Compound of targeted fibroblast activation protein FAP, preparation method and application thereof - Google Patents

Compound of targeted fibroblast activation protein FAP, preparation method and application thereof Download PDF

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CN115260160B
CN115260160B CN202211006861.1A CN202211006861A CN115260160B CN 115260160 B CN115260160 B CN 115260160B CN 202211006861 A CN202211006861 A CN 202211006861A CN 115260160 B CN115260160 B CN 115260160B
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cancer
compound
fap
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徐鹏飞
陈小元
吴晓明
杨清宝
何田
张静静
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Yantai Lannacheng Biotechnology Co ltd
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Abstract

The invention relates to a compound of a dimer of a target FAP protein, which has the following structural formulas (I) and (II), wherein R 1 、R 2 、R 3 And R is 4 The same or different are independently selected from H or F; z and U are the same or different connecting structures and are respectively and independently selected from-NH-or- (CH) -based 2 ) n -an alternative structure; q is selected from
Figure DDA0003809206260000011
Or alternatively
Figure DDA0003809206260000012
Q' is selected from
Figure DDA0003809206260000013
Or alternatively
Figure DDA0003809206260000014
W is the structure of a chelating species. The invention also provides radionuclide-labeled compounds based on the targeting compounds, and methods of preparing the same and use thereof in diagnosing or treating diseases characterized by overexpression of Fibroblast Activation Protein (FAP).

Description

Compound of targeted fibroblast activation protein FAP, preparation method and application thereof
Technical Field
The present invention relates to the fields of nuclear medicine and molecular imaging, in particular to a compound, a pharmaceutical composition comprising or consisting of said compound, a kit comprising or consisting of said compound or pharmaceutical composition, and the use of said compound or pharmaceutical composition in the diagnosis or treatment of a disease characterized by overexpression of Fibroblast Activation Protein (FAP).
Background
Fibroblast activation protein (Fibroblast activation protein, FAP) is a membrane serine peptidase expressed on the surface of tumor stroma activated fibroblasts and plays an important role in the development and progression of tumors. Previous studies have shown that FAP is not normally expressed in normal human tissues, but is selectively highly expressed on the surface of stromal fibroblasts of more than 90% of epithelial malignancies, including breast cancer, esophageal cancer, thyroid cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, and the like. In view of its broad expression and important role in tumors, FAP has become an important target for tumor imaging and therapy.
Radionuclide-labeled Fibroblast Activation Protein Inhibitors (FAPI) represented by quinolinic acid derivatives have made important progress in the field of tumor precision 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. The FAPI reported so far is rapidly cleared in the blood circulation and simultaneously eluted rapidly at the tumor site. This metabolic profile is very disadvantageous for treatment because rapid metabolism and elution results in lower effective doses at the tumor site, too short retention times, and the need to use high doses or more frequent dosing to meet the therapeutic needs, increasing the likelihood of adverse reactions. Based on multivalent effects, chen Hao et al (J nucleic Med.2022Jun;63 (6): 862-868) developed a dimer based on the FAPI46 structure, the structure of which is as follows:
Figure BDA0003809206240000011
such dimeric FAPI, while capable of increasing tumor uptake and residence time, have been observed to have higher physiological uptake in the kidneys, blood pool, liver, thyroid and pancreas. It is well known in the art that high background uptake in normal organs results in relatively low tumor to background ratios, which can affect the detection efficiency of diseased regions and increase the potential risk of therapeutic applications. Therefore, it is necessary to optimize the structure of dimer FAPI to have appropriate metabolism kinetics, higher tumor uptake dose and longer tumor retention time, and meet nuclide treatment and imaging requirements.
Disclosure of Invention
In view of the above background, a primary object of the present invention is: a new compound structure targeting Fibroblast Activation Protein (FAP) was developed.
Another object of the invention is: methods of preparing the novel compounds are provided.
A further object of the invention is: there is provided the use of said compounds in the diagnosis or treatment of diseases characterized by overexpression of Fibroblast Activation Protein (FAP).
The above object 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, the structure of the compound is shown in the following formula (I):
Figure BDA0003809206240000021
on the basis, the invention also provides a radiolabeled dimer compound targeting FAP protein or pharmaceutically usable salt thereof, the structure of the compound is shown as the following formula (II),
Figure BDA0003809206240000022
in the above formula (I) and formula (II):
R 1 、R 2 、R 3 and R is 4 The same or different are independently selected from H or F;
z and U are the same or different and are independently selected from-NH-or- (CH) -based 2 ) n -an alternative structure wherein n is an integer from 1 to 16, wherein each-CH 2 -with or without-O-, -NH-, - (CO) -, -NH- (CO) -, -CH (NH) alone 2 ) -or- (CO) -NH-substitution,the condition for substitution is that there are no two adjacent-CH 2 -the group is replaced;
in the above formula (I), Q is selected from
Figure BDA0003809206240000023
Or->
Figure BDA0003809206240000024
In the above formula (II), Q' is selected from
Figure BDA0003809206240000025
Or->
Figure BDA0003809206240000026
In the above formula (II), W is a nuclide chelating moiety, is a group derived from a chelating radionuclide of any of 1,4,7, 10-tetraazacyclododecane-N, N ', N, N ' -tetraacetic acid (DOTA), ethylenediamine tetraacetic acid (EDTA), 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N, N, N ', N ', N ' -pentaacetic acid (DTPA), bis- (carboxymethyl imidazole) glycine or 6-hydrazinopyridine-3-carboxylic acid (HYNIC), or is any of the following structures:
Figure BDA0003809206240000031
wherein D is based on- (CH) 2 ) p -an alternative structure wherein p is an integer from 0 to 16, wherein each-CH 2 -with or without-O-, -NH-, - (CO) -, -NH- (CO) -, -CH (NH) alone 2 ) -or- (CO) -NH-substitution, provided that there are no two adjacent-CH 2 -the group is replaced.
In a preferred embodiment of the present invention, the compound of formula (I) has a structure represented by any one of the following formulas (I-1) to (I-3):
Figure BDA0003809206240000032
in a preferred embodiment of the present invention, the compound of formula (II) has a structure represented by any one of the following formulas (II-1) to (II-11):
Figure BDA0003809206240000041
Figure BDA0003809206240000051
Figure BDA0003809206240000061
Figure BDA0003809206240000071
further, the present invention also provides a radionuclide-labeled dimer compound targeting FAP protein, which is a radionuclide-labeled compound represented by the formula (II) of the present invention; the radionuclide may be selected from the group consisting of an alpha-emitting isotope, a beta-emitting isotope, a gamma-emitting isotope, an Auger electron-emitting isotope, an X-ray emitting isotope, etc., e.g 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、 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 Th 199 Any one of Ag; more preferred radionuclides are 18 F、 64 Cu、 68 Ga、 89 Zr、 90 Y、 111 In、 99m Tc、 177 Lu、 188 Re or 225 Ac。
The invention also provides pharmaceutically acceptable tautomers, racemates, hydrates, solvates or salts of said dimer compound targeting FAP protein, said dimer compound targeting FAP protein which can be labeled with a radionuclide, or said dimer compound targeting FAP protein which is 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
Performing amide condensation reaction on carboxyl hydroxyl of 6-hydroxyquinoline-4-carboxylic acid and amino of glycine tert-butyl ester to obtain an intermediate I;
then, the hydroxyl of the intermediate I and N-Boc-bromoethylamine are subjected to condensation reaction to obtain an intermediate II containing protected amino and protected carboxyl;
performing amide condensation reaction on the carboxyl in the intermediate II after deprotection and (S) -pyrrolidine-2-carbonitrile hydrochloride or (S) -4, 4-difluoro pyrrolidine-2-carbonitrile hydrochloride to obtain an intermediate III containing protected amino;
the amino in the intermediate III is deprotected to obtain an intermediate IV, and the amino in the intermediate IV and the carboxyl hydroxyl of the tert-butyloxycarbonyl-L-glutamic acid-5-tert-butyl ester undergo condensation reaction to obtain an intermediate V containing protected amino and protected carboxyl;
(2) synthesis of dimers
Firstly, deprotecting carboxyl of an intermediate V, and carrying out condensation reaction with amino of any intermediate IV obtained in the step (1) to obtain an intermediate VI; the intermediate VI is deprotected to obtain the dimer compound of the target FAP protein;
(3) synthesis of dimeric compounds targeting FAP proteins that can be labeled with radionuclides
Removing the Boc protecting group of the intermediate VI; the chelating moiety W is introduced as a group from any one of 1,4,7, 10-tetraazacyclododecane-N, N ', N ' -tetraacetic acid (DOTA), ethylenediamine tetraacetic acid (EDTA), 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N, N ', N "-pentaacetic acid (DTPA), bis- (carboxymethyl imidazole) glycine or 6-hydrazinopyridine-3-carboxylic acid (HYNIC), or any one of the following structures:
Figure BDA0003809206240000081
wherein D is based on- (CH) 2 ) p -an alternative structure wherein p is an integer from 0 to 16, wherein each-CH 2 -with or without-O-, -NH-, - (CO) -, -NH- (CO) -, -CH (NH) alone 2 ) -or- (CO) -NH-substitution, provided that there are no two adjacent-CH 2 -the group is replaced; obtaining a radionuclide-labeled dimer compound of a target FAP protein, namely a compound shown in a formula (II);
(4) the radionuclide-labeled dimer compound of the target FAP protein obtained in the step (3) reacts with the radionuclide-containing compound according to the existing wet labeling method or freeze-drying labeling method, and the radionuclide-labeled dimer compound of the target FAP protein can be prepared.
In a third aspect, the present invention provides a pharmaceutical composition comprising a dimer compound targeting FAP protein according to 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 that is labeled with a radionuclide, or any tautomer, racemate, hydrate, solvate, or salt thereof that is pharmaceutically acceptable; or consists of the dimer compound targeting FAP protein, which can be labeled by a radionuclide, or any tautomer, racemate, hydrate, solvate or salt thereof which is pharmaceutically acceptable, and any pharmaceutically acceptable carrier and/or excipient.
In a fourth aspect, the present invention provides the use of a dimer compound targeting FAP protein as described in the first aspect, a dimer compound targeting FAP protein which can be labelled with a radionuclide, a dimer compound targeting FAP protein as described in the radionuclide, or a pharmaceutical composition as described in the third aspect, for the manufacture of a medicament for diagnosing or treating a disease characterized by overexpression of Fibroblast Activation Protein (FAP) in an animal or human subject.
In the use of the invention, the diseases characterized by the 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, hypopharynx cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, clear cell renal cancer, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (primary unknown cancer), thymus cancer, glioma, astrocytoma, cervical cancer, or prostate cancer.
In a fifth aspect, the present invention also provides a kit comprising or consisting of a compound of formula (I) according to the present invention, a compound of formula (II) according to the present invention, a radionuclide-labeled FAP protein-targeting dimer compound according to the present invention, or a pharmaceutical composition according to the present invention, and instructions for diagnosing a disease.
In the development of FAPI monomer, researchers have found that piperazine ring in FAPI structure plays an important role in maintaining tumor uptake, so adding piperazine ring into structure becomes a routine or customary means for designing FAPI probes in the art. It can also be seen from Chen Hao et al (J Nucl Med 2022Jun;63 (6): 862-868) that this design has been retained in the development of dimer FAPI. However, in research and development experiments, the inventor finds that the high-performance FAPI probe cannot be obtained by simple dimerization modification based on the existing FAPI monomer, but rather, the high background uptake occurs, so that the ratio of tumor to background in the aspect of organ uptake of the drug is low, and the performance is poor. The inventors have observed through extensive experimentation that the presence of a piperazine ring structure may adversely affect the pharmacokinetic properties of dimer FAPI. Therefore, the piperazine ring structure is not introduced in the synthesis of the dimer FAPI, and experiments prove that the prepared compound structure of the targeting FAP protein and the radiolabeled compound thereof have obviously improved tumor uptake effect compared with the existing dimer FAPI, and are expected to be applied to diagnosis or treatment of diseases characterized by over-expression of Fibroblast Activation Protein (FAP).
Drawings
FIG. 1 is a mass spectrum of Compound 2 in example 1 of the present invention.
FIG. 2 is a mass spectrum of Compound 6 in example 1 of the present invention.
FIG. 3 is a mass spectrum of Compound 8 in example 1 of the present invention.
FIG. 4 is a mass spectrum of compound 10 in example 1 of the present invention.
FIG. 5 is a mass spectrum of compound 11 of formula (I-1) in example 1 of the present invention.
FIG. 6 is a mass spectrum of Compound 12 in example 1 of the present invention.
FIG. 7 is a mass spectrum of Compound 14 in example 2 of the present invention.
FIG. 8 is a mass spectrum of compound 16 in example 2 of the present invention.
FIG. 9 is a mass spectrum of the formula (II-2) in example 3 of the present invention.
FIG. 10 is a mass spectrum of the intermediate of formula (II-3) in example 4 of the present invention.
FIG. 11 is a mass spectrum of the intermediate of formula (II-3) in example 4 of the present invention.
FIG. 12 is a mass spectrum of the formula (II-3) in example 4 of the present invention.
FIG. 13 is a mass spectrum of the formula (II-3) in example 4 of the present invention.
FIG. 14 is a mass spectrum of the formula (II-4) in example 5 of the present invention.
FIG. 15 is a mass spectrum of the formula (II-10) in example 6 of the present invention.
FIG. 16 is a schematic view of the present invention 68 A graph of the results of MicroPET imaging of Ga-labeled complexes of formula (I-1) in HT1080-hFAP tumor-bearing mice.
FIG. 17 shows the present invention 68 MicroPET imaging results of Ga-labeled FAPI46 complex in HT1080-hFAP tumor-bearing mice.
FIG. 18 is a graph showing the results of MicroPET imaging of a 68 Ga-labeled complex of formula (I-1) of the present invention in HT1080-hFAP tumor-bearing mice 1 hour after co-injection with FAPI-04.
FIG. 19 is a diagram of the present invention 68 Ga-labeled complex of formula (I-1) tumor and important organ uptake results statistics (horizontal axis in figure is different organs, and left to right columnar graph in each organ corresponds to each other respectively) after injection of HT1080-hFAP tumor-bearing mice for 1 hr, 2 hr and 4 hr 68 Uptake value of Ga-labeled complex of formula (I-1).
FIG. 20 shows the present invention 68 A graph of MicroPET imaging results of Ga-labeled F2 complexes in HT1080-hFAP tumor bearing mice.
FIG. 21 is a schematic view of an embodiment of the present invention 68 A graph of MicroPET imaging results of Ga-labeled F3 complexes in HT1080-hFAP tumor bearing mice.
Detailed Description
The technical scheme of the invention is further illustrated and described below by the specific embodiments in combination with the accompanying drawings.
Example 1: preparation of Compounds of formula II-1
Synthesis of Compound 2:
into a 100mL flask, compound 1 (6-hydroxyquinoline-4-carboxylic acid, 1.89g,10.0 mmol), tert-butyl glycinate (1.89 g,10.0 mmol), HATU (3.8 g,10.0 mmol) and N, N-diisopropylethylamine (2.6 g,20.0 mmol) were each charged successively to 30mL of N, N-dimethylformamide. The reaction mixture was stirred overnight, and the solvent was distilled off under reduced pressure to give a crude product. Purification over a silica gel column (dichloromethane/methanol=30:1) afforded compound 2 as a white solid in 87% yield. Fig. 1 is a mass spectrum of compound 2.
Synthesis of Compound 3:
in a 100mL flask, compound 2 (1.51 g,5.0 mmol), N-Boc-bromoethylamine (2.25 g,10.0 mmol), and potassium carbonate (1.38 g,10.0 mmol) were successively introduced into 50mL of N, N-dimethylformamide. The system was warmed to 60℃and kept under stirring at 60℃overnight, and the solvent was removed by distillation to give a crude product. Purification on a silica gel column (dichloromethane/methanol=30:1) gave compound 3 as a white solid in 51% yield.
Synthesis of Compound 5:
compound 4 (0.44 g,1.0 mmol) was dissolved in a mixed solution of 10mL of dichloromethane and trifluoroacetic acid (volume ratio: 9:1) under ice bath, the system was warmed to room temperature and reacted for 2 hours, the solvent was distilled off under reduced pressure after the completion of the reaction, 10mL of di-t-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, and the solvent was distilled off under reduced pressure to give a crude product. Purification on a silica gel column (dichloromethane/methanol=10:1) gave compound 5 as a white solid in 77% yield.
Synthesis of Compound 6:
into a 100mL flask, compound 5 (0.39 g,1.0 mmol), (S) -pyrrolidine-2-carbonitrile hydrochloride (0.13 g,10.0 mmol), HATU (0.38 g,1.0 mmol) and N, N-diisopropylethylamine (0.26 g,2.0 mmol) were charged sequentially to 10mL of N, N-dimethylformamide. The reaction mixture was stirred at room temperature until the reaction was completed, and the solvent was distilled off under reduced pressure to obtain a crude product. Purification on a silica gel column (dichloromethane/methanol=30:1) gave compound 6 as a white solid in 82% yield. Fig. 2 is a mass spectrum of compound 6.
Synthesis of Compound 8:
compound 7 (0.55 g,1.0 mmol) and p-toluenesulfonic acid monohydrate (0.27 g,1.5 mmol) were each introduced into a 100mL flask, and then introduced into 10mL of acetonitrile. The reaction system was warmed to 60℃and stirred until the reaction was completed, the solvent was distilled off under reduced pressure, 10mL of N, N-dimethylformamide was dissolved, and tert-butoxycarbonyl-L-glutamic acid-5-tert-butyl ester (0.3 g,1.0 mmol), HATU (0.38 g,1.0 mmol) and N, N-diisopropylethylamine (0.26 g,2.0 mmol) were added, respectively. The reaction mixture was stirred until the reaction was completed, and the solvent was distilled off under reduced pressure to obtain a crude product. Purification on a silica gel column (dichloromethane/methanol=20:1) gave compound 8 as a white solid in 75% yield. FIG. 3 is a mass spectrum of Compound 8.
Synthesis of Compound 10:
compound 8 (0.65 g,1.0 mmol) was dissolved in a mixed solution of 10mL of dichloromethane and trifluoroacetic acid (volume ratio 9:1) under ice bath, the system was warmed up to room temperature for reaction for 2 hours, the solvent was distilled off under reduced pressure after the completion of the reaction, 10mL 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, stirring was carried out at room temperature overnight, after the completion of the reaction, 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 sequentially added to the system, and after the completion of the reaction, the solvent was distilled off under reduced pressure, the crude product was obtained.
Synthesis of Compound 12:
compound 7 (95 mg,0.1 mmol) and p-toluenesulfonic acid monohydrate (0.09 g,0.5 mmol) were each introduced into a 50mL flask, and then introduced into 5mL of acetonitrile. The reaction system was warmed to 60℃and stirred until the reaction was completed, the solvent was distilled off under reduced pressure, 5mL of N, N-dimethylformamide was dissolved, and DOTA-NHS (0.05 g,0.1 mmol) and N, N-diisopropylethylamine (0.04 g,0.3 mmol) were added in this order. The reaction system was stirred at room temperature and was monitored by HPLC until the reaction was completed, and the solvent was distilled off under reduced pressure to obtain a crude product. The crude product was purified by reverse phase column chromatography and freeze-dried to give pure compound 12 in 37% yield. Fig. 5 is a mass spectrum of compound 11, and fig. 6 is a mass spectrum of compound 12.
The synthetic route of the steps is as follows:
Figure BDA0003809206240000121
example 2: preparation of Compounds of formula II-7
Synthesis of Compound 14:
into a 50mL flask were charged compound 11 (85 mg,0.1 mmol), compound 13 (please provide the chemical name 52mg,0.1 mmol), HATU (38 mg,0.1 mmol) and N, N-diisopropylethylamine (39 mg,0.30 mmol) prepared in example 1, respectively. The reaction mixture was stirred overnight. After the completion of the reaction, the solvent was distilled off under reduced pressure to give a crude product. Purification over a silica gel column (dichloromethane/methanol=10:1) afforded compound 14 as a white solid in 54% yield. Fig. 7 is a mass spectrum of compound 14.
Synthesis of Compound 16:
compound 14 (67 mg,0.1 mmol) was dissolved in a mixed solution of 10mL of dichloromethane and trifluoroacetic acid (volume ratio 9:1) under ice bath conditions, the reaction was carried out at room temperature for 2 hours, the solvent was distilled off under reduced pressure after the completion of the reaction to give compound 15, and DOTA-NHS (0.05 g,0.1 mmol) and N, N-diisopropylethylamine (0.04 g,0.3 mmol) were added in this order after compound 15 was dissolved with 5mL of N, N-dimethylformamide. The reaction system was stirred at room temperature and was monitored by HPLC until the reaction was completed, and the solvent was distilled off under reduced pressure to obtain a crude product. The crude product was purified by reverse phase column chromatography and freeze-dried to give pure compound 16 in 39% yield. Fig. 8 is a mass spectrum of compound 16.
The synthetic route of the steps is as follows:
Figure BDA0003809206240000131
the structures of the compounds of examples 3-11 are shown as the formulas (II-2) to (II-6) and the formulas (II-8) to (II-11), respectively, and the preparation methods thereof can refer to the examples 1 and 2, so as to obtain the following corresponding structures:
Figure BDA0003809206240000141
Figure BDA0003809206240000151
Figure BDA0003809206240000161
example 12 preparation of a radioactive Ga-68 labeling complex:
wet process: about 18.5 to 1850 megabellum (MBq) 68 GaCl 3 Hydrochloric acid solution (eluted from gallium germanium generator) was added to a centrifuge tube containing 0.5mL of acetic acid-acetate solution (1.0 g/L) of compound 12 prepared in example 1, and reacted at 37℃for 20 minutes. A C18 separation column was taken, which was slowly rinsed with 10mL absolute ethanol followed by 10mL water. After diluting the labeling solution with 10mL of water, the solution was applied to a separation column, and unlabeled solution was removed with 10mL of water 68 Ga ion, and leaching by 0.3mL of 10mM HCl ethanol solution to obtain 68 Ga-labeled complexes. Diluting the leaching solution with normal saline, and sterile filtering to obtain 68 Ga-labeled injection of the complex of formula (II-1).
And (3) lyophilization: about 18.5 to 1850 megabellum (MBq) 68 GaCl 3 Hydrochloric acid solution (eluted from germanium gallium generator) was added to the lyophilized kit containing the labeled precursor, and reacted at 37 ℃ for 20min after mixing. A C18 separation column was taken, which was slowly rinsed with 10mL absolute ethanol followed by 10mL water. After diluting the labeling solution with 10mL of water, the solution was applied to a separation column, and unlabeled solution was removed with 10mL of water 68 Ga ions are leached by ethanol solution of 0.3ml of 10mM HCl to obtain complex leaching solution. Diluting the leaching solution with normal saline, and sterile filtering to obtain 68 Ga-labeled (II-1) complex injection.
Experimental example application Effect analysis
The complex of formula (II-1) prepared as in example 12 was injected into HT1080-hFAP tumor bearing mice (7.4 MBq) via the tail vein and then administered under isoflurane anesthesia at 0 th to third post-administrationMicroPET imaging was performed for 60 min. Figure 16 shows intravenous injection 68 MicroPET images of HT1080-hFAP tumor bearing mice at various times after the Ga-formula (II-1) complex, fig. 17 shows intravenous injection 68 MicroPET images of HT1080-hFAP tumor bearing mice after 1 hour of Ga-FAPI-46 complex. As can be seen from the view of figure 16, 68 ga-formula (II-1) complex is fast and high-efficiency ingested at tumor site, very low in most normal organs, and is mainly rapidly cleared through kidney. And monomer which has been widely used at present 68 Ga-FAPI46 in dimeric form 68 The uptake of Ga-formula (II-1) complexes in tumors is markedly increased. In addition, the above-mentioned 68 Ga-formula (II-1) complex and FAPI-04 are co-injected into HT1080-hFAP tumor-bearing mice, and the MicroPET imaging result chart is shown in FIG. 18, and co-injection leads to the rapid decrease of tumor uptake, and blocking experiments prove that 68 Ga-formula (II-1) complex can realize tumor specific targeting through FAP protein in vivo. In a biodistribution study, HT1080-hFAP mice were injected with 1.48MBq 68 Ga-formula (II-1) complex, after 1h, 2h and 4h (3 parallel experiments at each time point) after injection, respectively, separated, weighed, and gamma counter measured and analyzed for major organ and tumor radioactivity (counts per minute, cpm), the test results are shown in FIG. 19, consistent with PET imaging results, 68 ga-formula (II-1) complexes exhibit high uptake and high residence time in tumors, while normal organ uptake is very low.
In the development of FAPI monomers, previous studies have shown that piperazine rings in FAPI structures play an important role in maintaining tumor uptake. However, the present inventors have found through experiments that the presence of the piperazine ring structure may have an adverse effect on the pharmacokinetic properties of dimer FAPI, such that a higher background uptake occurs, resulting in a lower tumor to background ratio of the drug in terms of organ uptake. In this experiment, as a control, the researcher will have two piperazine ring structures 68 Ga-F2 (its labeling precursor structure is shown in FIG. 20) and piperazine ring 68 Ga-F3 (its labeled precursor structure is shown in FIG. 21) was injected into HT1080-hFAP tumor bearing mice via the tail vein (7.4 MBq) and subjected to MicroPET imaging. As can be seen from FIGS. 20 and 21, the structure of the piperazine ring is two 68 Ga-F2 has higher uptake in tumors, but also in kidneys; at the same time with a piperazine ring 68 Ga-F3 although the ratio of renal uptake 68 Ga-F2 was somewhat decreased, but higher uptake was observed at the joints. And is connected with 68 Ga-F2 68 Compared with Ga-F3, the dimer probe structure disclosed by the invention does not contain piperazine ring, and as shown in fig. 16 and 19, the dimer FAPI has excellent metabolic dynamics property, high tumor uptake and retention time and application potential.
In view of the above, the present invention has developed a novel fibroblast activation protein FAP targeting compound, which is expected to be applied to diagnosis or treatment of diseases characterized by overexpression of Fibroblast Activation Protein (FAP).
While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. A FAP protein-targeting dimer compound of any one of the following structures or a pharmaceutically acceptable salt thereof:
Figure FDA0004236894240000011
Figure FDA0004236894240000021
Figure FDA0004236894240000031
2. radionuclide-labeled targeting FAP proteinA dimeric compound of formula (II-1) to formula (II-6) according to claim 1, wherein the dimeric compound is obtained by labeling a radionuclide; 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. A process for preparing a compound represented by the formulae (I-1) to (I-3) according to claim 1, comprising:
(1) preparation of monomers
Performing amide condensation reaction on carboxyl hydroxyl of 6-hydroxyquinoline-4-carboxylic acid and amino of glycine tert-butyl ester to obtain an intermediate I;
then, the hydroxyl of the intermediate I and N-Boc-bromoethylamine are subjected to condensation reaction to obtain an intermediate II containing protected amino and protected carboxyl;
performing amide condensation reaction on the carboxyl in the intermediate II after deprotection and (S) -pyrrolidine-2-carbonitrile hydrochloride or (S) -4, 4-difluoro pyrrolidine-2-carbonitrile hydrochloride to obtain an intermediate III containing protected amino;
the amino in the intermediate III is deprotected to obtain an intermediate IV, and the amino in the intermediate IV and the carboxyl hydroxyl of the tert-butyloxycarbonyl-L-glutamic acid-5-tert-butyl ester undergo condensation reaction to obtain an intermediate V containing protected amino and protected carboxyl;
(2) synthesis of dimers
Firstly, deprotecting carboxyl of an intermediate V, and carrying out condensation reaction with amino of any intermediate IV obtained in the step (1) to obtain an intermediate VI; the intermediate VI is deprotected to obtain the dimer compound of the target FAP protein shown in the formulas (I-1) to (I-3).
4. A process for preparing a compound represented by the formulae (II-1) to (II-6) according to claim 1, comprising:
removing the Boc protecting group of intermediate VI obtained in claim 3; introducing a chelating moiety W, W being any one of the following structures:
Figure FDA0004236894240000032
wherein D is-NH-;
to obtain a dimer compound which can be marked by radionuclides and targets FAP protein, namely, the compounds shown in formulas (II-1) to (II-6).
5. A method of preparing the radionuclide-labeled FAP protein-targeted dimer compound of claim 2, comprising: the radionuclide-labeled targeting compound can be prepared by reacting the compounds shown in the formulas (II-1) to (II-6) obtained in the claim 4 with a radionuclide-containing compound according to the existing wet labeling method or freeze-drying labeling method.
6. A pharmaceutical composition characterized by: a dimeric compound comprising any one of the FAP-targeted proteins of claim 1, any one of the radionuclide-labeled FAP-targeted dimeric compounds of claim 2, or a pharmaceutically acceptable salt thereof.
7. Use of a dimer compound targeting FAP protein of any one of formulae (II-1) to (II-6) as defined in claim 1, a dimer compound targeting FAP protein as defined in claim 2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as defined in claim 6, for the manufacture of a medicament for the diagnosis or treatment of a disease characterized by overexpression of Fibroblast Activation Protein (FAP) in an animal or human subject.
8. The use 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 scar disease.
9. The use of claim 8, wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharynx cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, clear cell renal cancer, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (primary unknown cancer), thymus cancer, glioma, astrocytoma, cervical cancer, and prostate cancer.
10. A kit comprising or consisting of: (1) a FAP protein-targeting dimer compound represented by any one of formulas (II-1) to (II-6) of claim 1, a radionuclide-labeled FAP protein-targeting dimer compound of claim 2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 6; (2) instructions for diagnosing a disease.
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