Cyclic peptide-bioorthogonal group conjugates targeting fap and methods of making and using the same

Abstract

The application discloses a FAP-targeting cyclic peptide-bioorthogonal group conjugate and a preparation method and application thereof, and the conjugate comprises a precursor compound and a radionuclide, and the structural general formula of the precursor compound is T-B-L-Y, wherein T is a fibroblast activation protein targeting cyclic peptide, B is a cycloaddition product group, L is a linker, and Y is a chelating group for complexing the radionuclide; wherein the cycloaddition product group is formed through bioorthogonal reaction of a bioorthogonal group connected to the fibroblast activation protein targeting cyclic peptide and a complementary reactive group connected to the linker. The application can greatly enrich the modification strategy, and through adjusting the linker, the tumor-specific uptake is enhanced, the whole-body normal organ background signal is improved, and the imaging contrast and diagnostic accuracy are improved while the FAP affinity is maintained or enhanced.

Description

Technical Field

[0001] This invention relates to the field of nuclear medicine technology, specifically to cyclic peptide-bioorthogonal group conjugates targeting FAP, their preparation methods, and applications. Background Technology

[0002] Fibroblast activating protein (FAP) is a type II transmembrane serine protease that is highly expressed in more than 90% of fibroblasts in epithelial-derived malignant tumors, but low in normal tissues. It is one of the most promising pan-cancer targets in the field of tumor diagnosis and treatment.

[0003] Currently, the development of radiopharmaceuticals targeting FAP mainly revolves around small-molecule FAP inhibitors (FAPI) with a quinoline backbone and the cyclic peptide FAP-binding peptide FAP2286. FAP2286 is a FAP-binding cyclic peptide coupled to the radionuclide chelator DOTA. A pair of cysteine ​​residues in its cyclic peptide backbone are linked by the linker molecule TBMB (1,3,5-tris(bromomethyl)benzene), maintaining the peptide's stable rigid cyclic conformation and matching its binding pocket to FAP. Clinical studies have shown that radiolabeled FAP2286 has been observed to be taken up by tumors in tumor-bearing mice and cancer patients, and in multiple tumor types, FAP2286 exhibits superior tumor-suppressive effects compared to FAPI.

[0004] Currently, FAP2286-type radiopharmaceuticals still exhibit high non-specific uptake in normal organs throughout the body, and the background signal in normal organs needs improvement to enhance imaging contrast and diagnostic accuracy. However, the linker molecule TBMB used in the cyclic peptide backbone of FAP2286 is only used to maintain a rigid cyclic conformation and connect linkers. This limits its modification strategies, making it difficult to actively regulate the molecule's behavior in vivo by modulating linkers, thereby reducing non-specific uptake in normal organs. Summary of the Invention

[0005] One objective of this invention is to provide a cyclic peptide-bioorthogonal group conjugate targeting FAP, wherein the bioorthogonal group on the fibroblast activation protein-targeting cyclic peptide not only stabilizes the cyclic peptide backbone but also serves as a reaction site, connecting with a linker containing complementary reactive groups via a bioorthogonal reaction. This not only avoids product heterogeneity but also significantly enriches modification strategies. By adjusting the linker, while maintaining or enhancing FAP affinity, tumor-specific uptake is enhanced, and background signals from normal organs throughout the body are improved, thereby improving imaging contrast and diagnostic accuracy.

[0006] This invention is achieved through the following technical solution:

[0007] A cyclic peptide-bioorthogonal group conjugate targeting FAP includes a precursor compound and a radionuclide. The precursor compound has the general structural formula TBLY, where T is a cyclic peptide targeting fibroblast activation protein, B is a cycloaddition product group, L is a linker, and Y is a chelating group for complexing the radionuclide.

[0008] The cycloaddition product group is formed by a bioorthogonal reaction between a bioorthogonal group attached to a fibroblast activation protein targeting cyclic peptide and a complementary reactive group attached to a linker.

[0009] In this technical solution, the cyclic peptide-bioorthogonal group conjugate targeting FAP comprises two parts: a precursor compound and a radionuclide. Specifically, the chelating group Y in the general structural formula TBLY of the precursor compound is linked to the fibroblast activation protein targeting cyclic peptide T via a linker L and a cycloaddition product group B. The chelating group Y forms a stable six- or eight-coordinate chelate complex with the radionuclide through a coordination complexation reaction.

[0010] Depending on the radionuclide it chelates with, FAP-targeting cyclic peptide-bioorthogonal group conjugates can be used for tumor imaging diagnosis or treatment. For example, in one or more embodiments, the radionuclide may be used for diagnostic imaging. 68 Ga、 89 Zr、 64 Cu、 124 Class I radionuclides. For example, in one or more embodiments, the radionuclide may be used for antitumor therapy. 177 Lu、 90 Y、 225 Ac and other radionuclides. In some preferred embodiments, the radioactivity can be selected based on diagnostic or therapeutic purposes. 68 Ga / 177 Lu、 68 Ga / 225 Ac、 89 Zr / 177 Lu、 89 Zr / 225 Ac and other diagnostic and treatment options are available.

[0011] In this technical solution, the chelating group can be any existing chelating group. In one or more embodiments, the chelating group can be a macrocyclic chelating agent such as DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane-1,4,7-triacetic acid-glutaric acid), PCTA (1,4,7,10-tetraazacycloundecane-1,4,7-triacetic acid), Macropa, PYTA, TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), and DOTMA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tetramethyl derivative). In some embodiments, the chelating group may also be some non-macrocyclic chelating agents, such as linear aminocarboxylic acid complexing agent DTPA (diethylenetriaminepentaacetic acid), HBED-CC (N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid-cyclohexanecarboxylic acid), bicyclic [3.3.1]nonane chelating agent Bispidine, octate chelating agent HOPO, trihydroxyoxime acid iron chelating agent DFO, trihydroxyphosphonopropionic acid chelating agent TRAP, etc.

[0012] In this technical solution, the fibroblast activation protein-targeting cyclic peptide T in the precursor compound is similar to the binding cyclic peptide of the existing FAP2286, both having a cyclic peptide backbone. The difference lies in the presence of a bioorthogonal group on the fibroblast activation protein-targeting cyclic peptide prior to its linker connection. This bioorthogonal group directly or indirectly anchors a pair of cysteine ​​residues in the cyclic peptide backbone as the sole chemical anchor. This not only stabilizes the cyclic peptide backbone, enabling the fibroblast activation protein-targeting cyclic peptide to bind with the active pocket of fibroblast activation protein (FAP) with a high affinity (Kd value at the nanomolar level), but more importantly, the bioorthogonal group, as a reaction site, can assemble with complementary reactive groups linked to the linker through a bioorthogonal reaction to form a complete precursor compound. This bioorthogonal reaction, through site-specific modification, effectively avoids product heterogeneity caused by spontaneous coupling, and offers rapid reaction rates and good biocompatibility. Furthermore, this strategy greatly enriches the modification strategies for precursor compounds, allowing for the regulation of the overall physicochemical properties of the conjugate molecule by altering and screening linker parameters. This reduces non-specific uptake by normal organs throughout the body, improves the tumor / background ratio, and achieves clearer imaging contrast and higher diagnostic accuracy.

[0013] In this technical solution, the linker is not merely a spacer between the chelating group and the fibroblast activation protein targeting cyclic peptide, but a crucial component capable of actively regulating the overall performance of the conjugate. By selecting an appropriate linker, the physicochemical properties of the conjugate can be adjusted. For example, using a PEG chain as the linker can increase the hydrophilicity of the conjugate through its hydration effect, reduce non-specific adsorption of plasma proteins, and also reduce uptake by the reticuloendothelial system in organs such as the liver and spleen through a shielding effect, thereby improving the background signal of normal organs throughout the body. Simultaneously, by attaching complementary reactive groups that match the bioorthogonal groups to the linker, the linker can be rapidly and accurately linked to the fibroblast activation protein targeting cyclic peptide, achieving the assembly of the conjugate.

[0014] In this technical solution, the bioorthogonal group of the fibroblast activation protein targeting cyclic peptide matches the complementary reactive group on the linker, thereby enabling the formation of a cycloaddition product group through a bioorthogonal reaction. Depending on the type of bioorthogonal reaction, the cycloaddition product group can be of various types. For example, the bioorthogonal group can be a tetrazine group, and its complementary group can be a dienophile; the two react via an inverse electron-demanding Diels-Alder reaction (iEDDA) to obtain a pyridazine cycloaddition product group. Another example is that the bioorthogonal group can be an azide group, and its complementary group can be a terminal alkyne compound; the two react via a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) to obtain a triazole cycloaddition product group.

[0015] In this technical solution, the cycloaddition product group can further adjust the physicochemical properties of the conjugate. Specifically, the rigid covalent linkage structure of the cycloaddition product can regulate the conformation of adjacent linkers and ensure that the cyclic peptide and FAP bind in an ideal spatial orientation to enhance tumor targeting. Simultaneously, it forms a metabolic barrier to delay protease degradation, prolonging the circulating half-life and tumor retention time. Furthermore, the cycloaddition product group retains highly water-soluble metabolic fragments after degradation, thus allowing for rapid excretion via renal metabolism and urine. In addition, the strong dipole moment of the cycloaddition product group, such as triazole, can enhance the water and shielding effects of linkers like PEG chains, reducing plasma protein adsorption and non-specific uptake, while moderate π-π stacking optimizes hydrophobic balance and improves tumor extravasation efficiency.

[0016] In some preferred embodiments, the FAP-targeting cyclic peptide-bioorthogonal group conjugate has the following structural formula:

[0017]

[0018] In the formula, the substituent R2 modifies the N-terminus of the cyclic peptide with different polarities and can be selected from C1-C6 alkyl, C1-C6 acyl, or sulfonyl groups; Y' is a chelating group containing a radionuclide. It can be seen that a pair of cysteine ​​residues of the fibroblast activation protein targeting the cyclic peptide are linked to the cycloaddition product group via a thiol ether bond. The latter is then linked to the chelating group via a linker, and the desired radionuclide is chelated through the chelating group. When a different radionuclide is needed, a switch from diagnosis to treatment can be achieved without re-optimizing the molecular structure.

[0019] In a preferred embodiment of the present invention, the bioorthogonal group is a tetrazine group, and the complementary reactive group of the tetrazine group is a dienophile or an isonitrile group; or the bioorthogonal group is an azide group, and the complementary reactive group of the azide group is a terminal alkyne compound or a cyclic alkyne compound.

[0020] In this technical solution, the bioorthogonal group can be a tetrazine group, and the corresponding complementary reactive group can be a dienophile. The tetrazine group and the dienophile form a cycloaddition product through a Diels-Alder reaction with reverse electron demand. For example, the dienophile can be bicyclic [6.1.0]nonyne (BCN), and the two react to obtain a pyridazine cycloaddition reactive group. In one or more embodiments, the dienophile can also be selected from other diens capable of undergoing bioorthogonal reactions with the tetrazine group, such as trans-cyclooctene (TCO), norbornene, cyclopropene, etc. When the bioorthogonal group is a tetrazine group, the corresponding complementary group can also be an isonitrile group. The isonitrile group and the tetrazine group form a pyrazole cycloaddition product through a [4+1] cycloaddition reaction. In some preferred embodiments, the linker connected to the isonitrile group has the structure shown in Formula IV:

[0021] Formula IV: ;

[0022] In Formula IV, the group R3 is selected from H or C1~C6 alkyl groups.

[0023] In this technical solution, the bioorthogonal group can also be an azide group indirectly linked to the S of the cyclic peptide, and its corresponding complementary reactive group can be a terminal alkynyl compound. A CuAAC reaction between the azide group and the terminal alkynyl compound yields a triazole cycloaddition product. Alternatively, the complementary reactive group can also be a cyclic alkynyl compound, such as dibenzocyclooctyne (DBCO). A strain-promoted azido-alkynyl cycloaddition reaction (SPAAC) between the azide group and the cyclic alkynyl compound yields a triazole cycloaddition product.

[0024] Furthermore, the fibroblast activation protein targeting cyclic peptide with a bioorthogonal group has the following structural formula:

[0025] Formula I: ;

[0026] Formula II: ;

[0027] In Formula I, group R1 is selected from H, C1~C6 alkyl or phenyl; in Formula I or Formula II, group R2 is selected from C1~C6 alkyl, C1~C6 acyl or sulfonyl.

[0028] In this technical solution, based on the different biological orthogonal groups, the fibroblast activation protein targeting cyclic peptide preferably has the structure of Formula I or Formula II.

[0029] In the structural formula shown in Formula I, the bioorthogonal group is a tetrazine group. The tetrazine group serves as the reaction site for the bioorthogonal reaction and also constitutes the backbone of the FAP-targeting cyclic peptide. Group R1 can be H or a C1-C6 alkyl group, such as methyl, ethyl, isopropyl, tert-butyl, n-pentyl, etc.; group R1 can also be a substituted or unsubstituted phenyl group, preferably an unsubstituted phenyl group. In the structural formula shown in Formula II, the bioorthogonal group is an azide group. The benzene ring attached to the azide group constitutes the backbone of the FAP-targeting cyclic peptide, while the azide group itself serves as the reaction site for the bioorthogonal reaction.

[0030] In some preferred embodiments, the structural formula of Formula I is used as a fibroblast activation protein targeting cyclic peptide with a bioorthogonal group.

[0031] Furthermore, the linker is selected from one or more polyethylene glycol units, C1~C1. 12 Alkylene, or divalent glucose.

[0032] In this technical solution, the physicochemical properties of the conjugate can be adjusted by selecting different linkers. In some embodiments, the linker can be one or more polyethylene glycol units, i.e., PEG chains. PEG chains are hydrophilic flexible chains that, by increasing the hydration radius and reducing plasma protein adsorption, adjust the drug from rapid renal clearance to moderately prolonged circulation without excessive accumulation, thereby increasing the cumulative tumor exposure dose. Simultaneously, the hydrophilic shielding layer of the PEG chain inhibits capture by the hepatic and splenic reticuloendothelial systems, the neutral charge avoids charge-mediated reabsorption in the renal tubules, and the metabolites maintain high water solubility for rapid excretion.

[0033] In one or more embodiments, the PEG chain In the n=1~12. In some preferred embodiments, n=4~8. By adjusting the appropriate PEG chain length, not only can the steric hindrance and decreased affinity caused by excessively short PEG chains be avoided, but the delay in normal tissue clearance caused by excessively long PEG chains can also be prevented.

[0034] In this technical solution, in addition to the PEG chain, the linker can also be selected as a hydrophobic alkylene chain. In one or more embodiments, the linker can be C1~C1. 12 Alkylene. In some embodiments, the linker may be methylene, ethylene, propylene, butylene, pentylene, or hexylene. Alternatively, the linker may be a hydrophilic rigid sugar ring, such as a divalent glucose stalk. In one or more embodiments, the linker may be glucose-1,6-diyl or glucose-1,4-diyl.

[0035] In a preferred embodiment of the present invention, the precursor compound is selected from the following structural formulas:

[0036] ,

[0037] ,

[0038] ,

[0039] ;

[0040] In the formula, group R1 is selected from H, C1~C6 alkyl or phenyl; group R2 is selected from C1~C6 alkyl, C1~C6 acyl or sulfonyl; group R3 is selected from H or C1~C6 alkyl.

[0041] Furthermore, the precursor compound is selected from the following structural formulas:

[0042] ,

[0043] ,

[0044] ,

[0045] ;

[0046] ;

[0047] .

[0048] Another object of the present invention is to provide a method for preparing any of the aforementioned cyclic peptide-bioorthogonal group conjugates targeting FAP, which specifically includes the following steps:

[0049] The fibroblast activation protein targeting cyclic peptide shown in Formula I undergoes an anti-electron-demand Diels-Alder reaction or a [4+1] cycloaddition reaction with a linker connected to a first complementary reactive group and a chelating group to obtain a precursor compound, wherein the first complementary reactive group is a dienophile or an isonitrile group; or

[0050] The fibroblast activation protein targeting cyclic peptide shown in Formula II undergoes a 1,3-dipolar cycloaddition reaction with a linker connected to a second complementary reactive group and a chelating group to obtain a precursor compound, wherein the second complementary reactive group is a terminal alkyne compound or a cyclic alkyne compound.

[0051] The precursor compound undergoes a coordination complexation reaction with a radionuclide to prepare the cyclic peptide-bioorthogonal group conjugate targeting FAP.

[0052] Formula I: ;

[0053] Formula II: ;

[0054] In Formula I, group R1 is selected from H, C1~C6 alkyl or phenyl; in Formula I or Formula II, group R2 is selected from C1~C6 alkyl, C1~C6 acyl or sulfonyl.

[0055] Furthermore, the polypeptide represented by Formula III and dienetetrazine compounds The reaction yields the fibroblast activation protein targeting cyclic peptide shown in Formula I; or

[0056] The polypeptide shown in Formula III was reacted with 1,1'-[5-(azidomethyl)-1,3-phenylene]bis(2-iodoethane-1-one) to prepare the fibroblast activation protein targeting cyclic peptide shown in Formula II.

[0057] Formula III: .

[0058] This invention also provides a tumor imaging agent, which employs any of the aforementioned cyclic peptide-bioorthogonal group conjugates targeting FAP. This is achieved by loading a diagnostic imaging agent... 68 Ga、 89 Zr、 64 Cu、 124 Radionuclides such as I, and their conjugates, can be used to prepare tumor imaging agents.

[0059] This invention also provides an antitumor drug that employs any of the aforementioned cyclic peptide-bioorthogonal group conjugates targeting FAP. This is achieved by incorporating a drug for antitumor therapy. 177 Lu、 90 Y、 225 Radioactive nuclides such as Ac, conjugates of which can be used to prepare antitumor drugs.

[0060] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0061] 1. The FAP-targeting cyclic peptide-bioorthogonal group conjugate of the present invention uses a pair of cysteine ​​residues in the cyclic peptide backbone as the only chemical anchors by using bioorthogonal groups. This not only stabilizes the cyclic peptide backbone by using bioorthogonal groups, enabling the fibroblast activation protein-targeting cyclic peptide to bind to the active pocket of FAP with a high affinity at the nanomolar level, but more importantly, the bioorthogonal groups, as reaction sites, can assemble into complete precursor compounds with complementary reactive groups attached to the linkers through bioorthogonal reactions. Bioorthogonal reactions can effectively avoid product heterogeneity caused by constant coupling through site-specific modification, and have a fast reaction rate and good biocompatibility.

[0062] 2. The cycloaddition product group of the present invention can regulate the conformation of adjacent linkers and enable the cyclic peptide to bind to FAP in an ideal spatial orientation to enhance tumor targeting. Simultaneously, it forms a metabolic barrier to delay protease degradation, prolonging the circulating half-life and tumor retention time. Furthermore, the cycloaddition product group retains highly water-soluble metabolic fragments after degradation, thus allowing it to be metabolized by the kidneys and rapidly excreted in urine.

[0063] 3. This invention greatly enriches the modification strategies for precursor compounds, enabling the regulation of the overall physicochemical properties of conjugate molecules by changing the parameters of the linker and screening the linker parameters. This reduces non-specific uptake by normal organs throughout the body, improves the tumor / background ratio, and achieves clearer imaging contrast and higher diagnostic accuracy. Attached Figure Description

[0064] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0065] Figure 1 This is a flowchart of the preparation method of the conjugate in a specific embodiment of the present invention;

[0066] Figure 2 shows the FAP-targeting cyclic peptide T1 (a) and precursor compound P1 (b) in a specific embodiment of the present invention. 68 Chromatogram of Ga-F2286b-DOTA (c), and mass spectra of FAP-targeted cyclic peptide T1 (d) and precursor compound P1 (e). 68 Radiochromatogram of Ga-F2286b-DOTA(f);

[0067] Figure 3 shows the precursor compound P2(a) in a specific embodiment of the present invention. 68 Chromatogram of Ga-F2286n-DOTA(b), and mass spectrum of precursor compound P2(c). 68 Radiochromatogram of Ga-F2286n-DOTA(d);

[0068] Figure 4 shows the chromatogram (a) and mass spectrum (b) of the precursor compound P5 in a specific embodiment of the present invention. 64 Radiochromatogram of Cu-F2286b-NOTA (c);

[0069] Figure 5 shows the chromatogram (a) and mass spectrum (b) of the precursor compound P6 in a specific embodiment of the present invention. 64 Radiochromatogram of Cu-F2286b-C2-NOTA (c);

[0070] Figure 6 The affinity of the conjugate for human FAP protein is shown in a specific embodiment of the present invention;

[0071] Figure 7 This invention illustrates a specific embodiment equipped with 68 Imaging properties of Ga conjugates in vivo;

[0072] Figure 8 This illustrates the tumor's ability to take up the conjugate in a specific embodiment of the present invention;

[0073] Figure 9 The distribution of the conjugate in vivo in a specific embodiment of the present invention is shown, wherein the horizontal axis represents T-tumor, H-heart, L-liver, K-kidney, M-muscle, B-bone, G-gallbladder, and I-intestine;

[0074] Figure 10 Tumor uptake conjugates are shown in a specific embodiment of the present invention. 64 The ability of Cu-F2286b-NOTA and its metabolic status;

[0075] Figure 11 Tumor uptake conjugates are shown in a specific embodiment of the present invention. 64 The ability of Cu-F2286b-C2-NOTA and its metabolism. Detailed Implementation

[0076] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0077] The sources of all raw materials used in this invention are not particularly limited; they can be purchased commercially or prepared using conventional methods well-known to those skilled in the art. The purity of all raw materials used in this invention is not particularly limited; however, analytical grade or purity requirements commonly used in the nuclear medicine field are preferred. The designations and abbreviations of all raw materials used in this invention are conventional designations and abbreviations in the art, and each designation and abbreviation is clearly defined within its relevant application. Those skilled in the art can obtain these materials from commercial sources or prepare them using conventional methods based on the designation, abbreviation, and corresponding application.

[0078] The present invention does not impose any particular restrictions on the expression of the substituents, and all expressions are well known to those skilled in the art. Based on common sense, those skilled in the art can correctly understand their meaning according to their expression.

[0079] The terms "first," "second," etc., used in this invention (e.g., first complementary reactive group, second complementary reactive group, etc.) are merely for clarity of description and are not intended to limit any order or emphasize importance. Furthermore, the term "link" used in this invention, unless otherwise specified, can refer to a direct link or an indirect link via other groups.

[0080] I. Preparation of FAP-targeting cyclic peptide-bioorthogonal group conjugates

[0081]

Example 1

[0082] 1) Preparation of FAP-targeting cyclic peptide T1 with a tetrazine group

[0083]

[0084] Specifically, 2.0 eq of dienetetrazine was added to the peptide solution (pH=8.0), and the reaction was carried out at a constant temperature of 37°C for 2–5 h. After the reaction, the labeling rate and purification were calculated using the proportion of the product peak area monitored by HPLC at 254 nm. The test results showed that the labeling rate was 98%, and the total yield after purification was 46%, as shown in Figure 2. The purity of T1 was >95% as characterized by LC-MS.

[0085] In one or more embodiments, the amount of dienetetrazine used is 1.2 to 3.0 eq.

[0086] 2) Preparation of DOTA-PEG5-BCN

[0087] DOTA was dissolved in anhydrous DMF. Under an inert gas atmosphere, EDC·HCl (1.0 eq), N-hydroxysuccinimide (NHS, 1.0 eq), and N,N-diisopropylethylamine (DIPEA, 1.5 eq) were added sequentially, and the mixture was stirred at room temperature to activate the carboxyl groups. The activated solution was then slowly added dropwise to an anhydrous N,N-dimethylformamide solution (DMF, 0.2 eq) of NH2-PEG5-NH2 while cooling in an ice-water bath. After the addition was complete, the ice bath was removed, and the mixture was allowed to rise naturally to room temperature before stirring continued. After the reaction was complete, the mixture was concentrated under reduced pressure below 40°C. The residue was poured into cold diethyl ether to precipitate the solid, which was then collected and purified by preparative high-performance liquid chromatography (HPLC). Finally, the solid was freeze-dried to obtain DOTA-PEG5-NH2, with a yield of 88%.

[0088]

[0089] In one or more embodiments, the amount of anhydrous DMF used is 0.05~0.1 M.

[0090] In one or more embodiments, the reaction was stirred at room temperature for 2 to 4 hours to complete the activation of the carboxyl group.

[0091] In one or more embodiments, the reaction was allowed to rise naturally to room temperature and then stirred for 12 to 24 hours.

[0092]

[0093] Raw material I (1.0 eq) and DOTA-PEG5-NH2 (1.5 eq) were dissolved in 2 mL of acetonitrile / dimethyl sulfoxide (0.05 M, 1:1, v / v) mixed solvent, and triethylamine (about 4.0 eq) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, water was added to quench the reaction, and the mixture was purified by preparative high performance liquid chromatography to obtain a white solid with a yield of 80%.

[0094] 3) Preparation of precursor compound P1

[0095]

[0096] The reactants FAP-targeting cyclic peptide T1 and DOTA-PEG5-BCN (1.5 eq) were dissolved in a mixture of acetonitrile and water (1 mM / mL) and incubated at 37°C for 1–3 hours. After the reaction, the product was identified and purified by LC-MS, with a yield of 77%.

[0097] 4) Preparation of conjugates 68 Ga-F2286b-DOTA

[0098]

[0099] Add 20 μL of an aqueous solution of precursor compound P1 (2 μg / μL) to 150 μL of NaOAc (1M) solution, mix well, and then add... 68 A high-purity GaCl3 hydrochloric acid solution (3 mCi, 1 mL) was heated to 90°C and reacted for 10 minutes. The solution was then passed through a C18 Lighting reversed-phase column, washed with physiological saline, and collected as waste. The reversed-phase column was then washed with 75% medical alcohol (0.1 mL) and 0.9% physiological saline (1 mL). The alcohol wash and physiological saline wash were collected, and the radiochemical purity was measured by high-performance liquid chromatography (HPLC). The radiochemical purity was >95%.

[0100]

Example 2

[0101] 1) Preparation of DOTA-PEG5-NC

[0102]

[0103] Raw material II (1.0 eq) and DOTA-PEG5-NH2 (approximately 1.5 eq) were dissolved in an acetonitrile / dimethyl sulfoxide (0.05 M, 1:1, v / v) mixed solvent, and triethylamine (approximately 4.0 eq) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, water was added to quench the reaction, and the mixture was purified by preparative LC-MS to obtain a white solid with a yield of 77%.

[0104] 2) Preparation of precursor compound P2

[0105]

[0106] The reactants FAP-targeting cyclic peptide T1 (1.0 eq) and DOTA-PEG5-BCN (1.5 eq) were dissolved in acetonitrile and DMF (1 mM / mL) and stirred at 37°C for 24 hours. After the reaction was completed, the product was identified and purified by LC-MS, with a yield of 55%.

[0107] 3) Preparation of conjugates 68 Ga-F2286n-DOTA

[0108]

[0109] Add 20 μL of an aqueous solution of precursor compound P2 (2 μg / μL) to 150 μL of NaOAc (1M) solution, mix well, and then add... 68High-purity GaCl3 hydrochloric acid solution (3 mCi, 1 mL) was heated to 90℃ and reacted for 10 minutes. The solution was then passed through a C18 Lighting reversed-phase column, washed with physiological saline, and collected as waste liquid. The reversed-phase column was then washed with 75% medical alcohol (0.1 mL) and 0.9% physiological saline (1 mL). The alcohol wash and physiological saline wash were collected, and the radiochemical purity was measured by high-performance liquid chromatography (HPLC). The radiochemical purity was >95%.

[0110]

Example 3

[0111] 1) Preparation of FAP-targeting cyclic peptide T2 with a tetrazine group

[0112]

[0113] The peptide was dissolved in 1M 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) (pH=8.0), and a DMSO solution of 1,1'-[5-(azidomethyl)-1,3-phenylene]bis(2-iodoethane-1-one) was added. The reaction was carried out at room temperature. After the reaction was completed, the peptide was purified by HPLC, with a yield of 70%.

[0114] 2) Preparation of alkyne-

[0115] DOTA was dissolved in anhydrous DMF, and EDC·HCl (1.0 eq), NHS (1.0 eq), and DIPEA (1.5 eq) were added sequentially under an inert gas atmosphere. The reaction was stirred at room temperature to complete the carboxyl activation. Subsequently, the activated solution was slowly added dropwise to acetylene under an ice-water bath cooling. Anhydrous DMF solution (0.2 eq) was added dropwise, the ice bath was removed, and the mixture was allowed to rise naturally to room temperature before stirring was continued. After the reaction was completed, the mixture was concentrated under reduced pressure below 40°C. The residue was poured into cold diethyl ether to precipitate the solid, which was then collected and purified by preparative high-performance liquid chromatography. Finally, the solid was freeze-dried to obtain the acetylene. The yield was 75%.

[0116]

[0117] In one or more embodiments, the amount of anhydrous DMF used is 0.05~0.1 M.

[0118] In one or more embodiments, the reaction was stirred at room temperature for 2 to 4 hours to complete the activation of the carboxyl group.

[0119] In one or more embodiments, the reaction was allowed to rise naturally to room temperature and then stirred for 12 to 24 hours.

[0120] 3) Preparation of precursor compound P3

[0121]

[0122] Under nitrogen protection, the azide-modified FAP-targeting cyclic peptide T2 and alkyne were... Dissolve in PBS buffer (pH 7.4) that has been pre-degassed by bubbling with nitrogen or argon for 15-20 minutes, then add tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), CuSO4 solution, and sodium ascorbate in sequence. After thorough mixing, stir the reaction system at 25 °C in the dark for 2-4 hours. After the reaction is completed, add EDTA to chelate the residual Cu(II) ions to terminate the catalytic reaction.

[0123] The reaction solution was purified by preparative reversed-phase HPLC, the target peak was collected, and the product was lyophilized to obtain a white powder product with a yield of 53%.

[0124] In one or more embodiments, FAP targets cyclic peptide T2 and acetylene. The molar ratio is 1:1 to 1:5.

[0125] In one or more embodiments, the working concentration of THPTA is 1 to 10 mM.

[0126] In one or more embodiments, The working concentration of the solution is 0.1~5 mM.

[0127] In one or more embodiments, the working concentration of sodium ascorbate is 5-100 mM.

[0128] In one or more embodiments, the final concentration of EDTA is 10-20 mM.

[0129] In some preferred embodiments, the molar ratio of Cu to THPTA is 1:2 to 1:5, and more preferably, the molar ratio is 1:5.

[0130] 4) Preparation of conjugates

[0131]

[0132] Add 20 μL of an aqueous solution of precursor compound P3 (2 μg / μL) to 150 μL of NaOAc (1M) solution, mix well, and then add... 68 A high-purity GaCl3 hydrochloric acid solution (3 mCi, 1 mL) was heated to 90°C and reacted for 10 minutes. The solution was then passed through a C18 Lighting reversed-phase column, washed with physiological saline, and collected as waste. The reversed-phase column was then washed with 75% medical alcohol (0.1 mL) and 0.9% physiological saline (1 mL). The alcohol wash and physiological saline wash were collected, and the radiochemical purity was measured by high-performance liquid chromatography (HPLC). The radiochemical purity was >95%.

[0133]

Example 4

[0134] 1) Preparation

[0135]

[0136] Mix raw material III (1.0 eq) with (1.5 eq) was dissolved in a mixed solvent of acetonitrile / dimethyl sulfoxide (0.05 M, 1:1, v / v), and triethylamine (about 4.0 eq) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, water was added to quench the reaction, and the mixture was purified by preparative high performance liquid chromatography to obtain a white solid with a yield of 73%.

[0137] 2) Preparation of precursor compound P4

[0138]

[0139] Dissolve the FAP-targeting cyclic peptide T2 in degassed PBS buffer (pH 7.4), then add... The reaction was carried out at room temperature in the dark. During the reaction, the reaction progress was monitored by reversed-phase HPLC (C18 column, 0.1% TFA water / acetonitrile gradient elution, detection wavelength 220 nm or 280 nm) until the azide raw material peak basically disappeared. After the reaction was completed, the reaction solution was filtered through a 0.22 μm filter membrane and purified by preparative HPLC (C18 preparative column, 0.1% TFA water / acetonitrile linear gradient elution). The target product fraction was collected, lyophilized, and a white powder was obtained with a yield of 67%.

[0140] In one or more embodiments, the concentration of FAP-targeting cyclic peptide T2 is 0.5–5 mM.

[0141] In one or more embodiments, a molar ratio of 1.5:1 to 3:1 relative to the FAP-targeting cyclic peptide T2 is added. .

[0142] In one or more embodiments, the reaction was carried out at room temperature in the dark for 12 to 24 hours.

[0143] 3) Preparation of conjugates

[0144]

[0145] Add 20 μL of an aqueous solution of precursor compound P4 (2 μg / μL) to 150 μL of NaOAc (1M) solution, mix well, and then add... High-purity hydrochloric acid solution (3 mCi, 1 mL) was heated to 90°C and reacted for 10 minutes. The solution was then passed through a C18 Lighting reversed-phase column, washed with physiological saline, and collected as waste. The reversed-phase column was then washed with 75% medical alcohol (0.1 mL) and 0.9% physiological saline (1 mL). The alcohol wash and physiological saline wash were collected, and the radiochemical purity was measured by high-performance liquid chromatography (HPLC). The radiochemical purity was >95%.

[0146]

Example 5

[0147] 1) Preparation of Nota-PEG4-BCN

[0148]

[0149] Starting material V (30 mg, 0.078 mmol) and starting material VI (31.5 mg, 0.093 mmol) were added to 4 mL of acetonitrile / dimethyl sulfoxide (DMSO) in a 1:1 ratio. Triethylamine (43 μL, 0.31 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was quenched with water, identified by LC-MS, and purified to obtain 22.8 mg of a white solid (56% yield). The product was added to 2 mL of a 30% TFA / DCM mixture, stirred at room temperature for 1 hour, and the reaction was monitored by MS. After the reaction was complete, the solvent was removed by rotary evaporation. This process was repeated several times to obtain the final product, which did not require purification and was used directly in the next step. The HPLC preparation separation conditions were: Anglientan C18 column (250 mm x 9.4 mm x 5 μm), flow rate: 3.0 mL / min, column temperature: room temperature. The gradient was acetonitrile:water (0.1% TFA), where acetonitrile increased from 10% to 100% within 20 minutes. Observe its 210 nm absorption peak.

[0150]

[0151] Starting material VII (10 mg, 0.036 mmol) and compound VIII (28.7 mg, 0.055 mmol) prepared in the previous step were added to 2 mL of acetonitrile / dimethyl sulfoxide (DMSO) in a 1:1 ratio. Triethylamine (20 μL, 0.15 mmol) was then added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was quenched with water, identified by LC-MS, and purified to give 13.0 mg of a white solid, with a yield of 52%.

[0152] The HPLC preparative separation conditions were as follows: Angilent C18 column (250 mm x 9.4 mm x 5 μm), flow rate: 3.0 mL / min, column temperature: room temperature. The gradient was acetonitrile:water (0.1% TFA), in which acetonitrile increased from 10% to 100% over 20 minutes. The 210 nm absorption peak was observed.

[0153] 2) Preparation of precursor compound P5

[0154]

[0155] The reactants FAP-targeting cyclic peptide T1 (2 mg, 0.0019 mmol) and NOA-PEG4-BCN (1.60 mg, 0.0023 mmol) were dissolved in acetonitrile (1 ml) and water (1 ml) and incubated at 37 °C for 1 hour. After the reaction was completed, the product was identified and purified by LC-MS, with a yield of 54%.

[0156] The HPLC preparative separation conditions were as follows: Angilent C18 column (250 mm x 9.4 mm x 5 μm), flow rate: 3.0 mL / min, column temperature: room temperature. The gradient was acetonitrile:water (0.1% TFA), in which acetonitrile increased from 10% to 100% over 25 minutes. The absorption peak at 254 nm was observed.

[0157] 3) Preparation of conjugates 64 Cu-F2286b-NOTA

[0158]

[0159] Add 20 μL of an aqueous solution of precursor compound P5 (2 μg / μL) to 240 μL of NaOAc (1M) solution, mix well, and then add... 64 A high-purity CuCl2 hydrochloric acid solution (5 mCi, 0.6 mL) was heated to 90°C and reacted for 10 minutes. The solution was then passed through a C18 Lighting reversed-phase column, washed with physiological saline, and collected as waste. The reversed-phase column was then washed with 75% medical alcohol (0.1 mL) and 0.9% physiological saline (1 mL). The alcohol wash and physiological saline wash were collected, and the radiochemical purity was measured by high-performance liquid chromatography (acetonitrile / water, acetonitrile 10% to 90% within 15 minutes, water and acetonitrile both containing 0.1% trifluoroacetic acid). The radiochemical purity was >95%.

[0160] HPLC conditions were as follows: Angilent C18 column (250 mm x 4.6 mm x 3.5 μm), flow rate: 1.0 ml / min, column temperature: room temperature. The gradient was acetonitrile:water (0.1% TFA), in which acetonitrile increased from 10% to 90% within 12 minutes.

[0161]

Example 6

[0162] 1) Preparation of Nota-C2-BCN

[0163]

[0164] Starting material V (30 mg, 0.078 mmol) and starting material IX (22 mg, 0.055 mmol) were added to 4 mL of acetonitrile / dimethyl sulfoxide (DMSO) in a 1:1 ratio. Triethylamine (43 μL, 0.31 mmol) was then added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was quenched with water, identified by LC-MS, and purified to give 23.9 mg of a white solid, with a yield of 59%.

[0165] The HPLC preparative separation conditions were as follows: Angilent C18 column (250 mm x 9.4 mm x 5 μm), flow rate: 3.0 mL / min, column temperature: room temperature. The gradient was acetonitrile:water (0.1% TFA), in which acetonitrile increased from 10% to 100% over 20 minutes. The 210 nm absorption peak was observed.

[0166] 2) Preparation of precursor compound P6

[0167]

[0168] The reactants FAP-targeting cyclic peptide T1 (2 mg, 0.0019 mmol) and NOA-C2-BCN (1.19 mg, 0.0023 mmol) were dissolved in acetonitrile (1 ml) and water (1 ml) and incubated at 37 °C for 1 hour. After the reaction, the product was identified and purified by LC-MS, yield: 56%.

[0169] The HPLC preparative separation conditions were as follows: Angilent C18 column (250 mm x 9.4 mm x 5 μm), flow rate: 3.0 mL / min, column temperature: room temperature. The gradient was acetonitrile:water (0.1% TFA), in which acetonitrile increased from 10% to 100% over 25 minutes. The absorption peak at 254 nm was observed.

[0170] 3) Preparation of conjugates 64 Cu-F2286b-C2-NOTA

[0171]

[0172] Add 20 μL of an aqueous solution of precursor compound P6 (2 μg / μL) to 240 μL of NaOAc (1M) solution, mix well, and then add... 64A high-purity CuCl2 hydrochloric acid solution (5 mCi, 0.6 mL) was heated to 90℃ and reacted for 10 minutes. The solution was then passed through a C18 Lighting reversed-phase column, washed with physiological saline, and collected as waste. The reversed-phase column was then washed with 75% medical alcohol (0.1 mL) and 0.9% physiological saline (1 mL). The alcohol wash and physiological saline wash were collected, and the radiochemical purity was measured by high-performance liquid chromatography (acetonitrile / water, acetonitrile 10% to 90% within 15 minutes, water and acetonitrile both containing 0.1% trifluoroacetic acid). The radiochemical purity was >95%.

[0173] HPLC conditions were as follows: Angilent C18 column (250 mm x 4.6 mm x 3.5 μm), flow rate: 1.0 ml / min, column temperature: room temperature. The gradient was acetonitrile:water (0.1% TFA), in which acetonitrile increased from 10% to 90% within 12 minutes.

[0174] II. Performance Testing of FAP-Targeting Cyclic Peptide-Bioorthogonal Group Conjugates

[0175]

Example 7

[0176] In this embodiment, the stability and bioorthogonal kinetics of FAP-targeting cyclic peptide T1 were tested.

[0177] Specifically, 10 μL of FAP-targeting cyclic peptide T1 (20 mM) was dissolved in three 290 μL vials of fetal bovine serum (FBS) and incubated at 37°C for 0, 4, 8, 12, 24, and 48 hours. At each time point, 40 μL of sample was added to 100 μL of acetonitrile, vortexed to precipitate, centrifuged, and 100 μL of the supernatant was collected. The change in the absorption peak area at 254 nm was determined by HPLC. The experiment showed that the sample maintained a retention rate of 85.6% after 48 hours. This indicates that FAP-targeting cyclic peptide T1 has good stability in FBS.

[0178] Subsequently, the second-order rate constant of the bioorthogonal reaction between FAP-targeting cyclic peptide T1 (final concentration 50 μM) and trans-cyclic olefin TCO (final concentration 50 μM) was determined by UV-Vis spectroscopy at 37°C in PBS buffer. The absorbance was measured at specified time intervals, and the concentration of tetrazine was calculated based on the change in absorption peak intensity at 520 nm over time. The k2 value was then determined based on the slope of (1 / c - 1 / c0) over time, where c0 is the concentration of the tetrazine analog at time zero, and c is the corresponding concentration at the specified time. The data were then analyzed, and the k2 value was found to be 981.22 ± 11.2 M. -1 s -1 .

[0179]

Example 8

[0180] In this embodiment, the affinity of two precursor compounds, P1 and P2, for human FAP protein (hFAP) was tested.

[0181] FAP protein was diluted to 0.4 μg / mL using assay buffer (25 mM Tris, 250 mM NaCl, pH 7.4). The substrate Gly-Pro-AMC was diluted to 40 μM using assay buffer. The test samples were serially diluted with assay buffer to 40 μM, 4 μM, 400 nM, 40 nM, 4 nM, 400 pM, 40 pM, and 4 pM. Then, equal volumes (25 μL) of the inhibitor and substrate Gly-Pro-AMC were added sequentially to a 96-well plate, followed by 50 μL of protein solution. After incubation at 37°C for 1 hour, the enzyme activity of hFAP against substrate Z-Gly-Pro-AMC was measured using a microplate reader under the following conditions: excitation wavelength 360 nm, emission wavelength 465 nm. Figure 6 As shown, precursor compounds P1 (F2286b-DOTA) and P2 (F2286n-DOTA) both exhibited nM-level inhibitory effects on the hFAP target, demonstrating that the tetrazine-based precursor compounds retained the high affinity for FAP-2286. Furthermore, the addition of a PEG chain as a linker further increased the hydrophilicity of precursor compounds P1 and P2, which is beneficial for reducing the accumulation of FAP-2286 in non-target organs.

[0182]

Example 9

[0183] In this embodiment, the following tests were conducted. 68 Ga-F2286b-DOTA and 68 Performance of Ga-F2286n-DOTA in animal PET / CT imaging experiments.

[0184] Specifically, HEK293-FAP cells were seeded in the axilla of the right forelimb of BALB / c nu / nu mice and housed in an SPF-grade animal facility for approximately 1-2 weeks. The tumor mass was approximately 200-300 mm in size. 3 Approximately 100 µL of a dose of approximately 160 µCi was administered via tail vein injection. 68 Ga-F2286b-DOTA, 68 Ga-F2286n-DOTA and 68 Ga-FAP-2286 (comparative example) is a highly radiochemically pure drug (radiochemical purity >95%).

[0185] The test results are as follows Figure 7 and Figure 8As shown, in HEK293-FAP model mice overexpressing FAP, 68Ga-F2286b-DOTA and 68Ga-F2286n-DOTA achieved high signal-to-noise ratio imaging results. Compared with 68Ga-FAP2286, 68Ga-F2286n-DOTA showed higher tumor uptake and better tumor targeting, while 68Ga-F2286b-DOTA also exhibited good tumor uptake.

[0186] Furthermore, such as Figure 9 As shown, both 68Ga-F2286b-DOTA and 68Ga-F2286n-DOTA exhibited low nonspecific accumulation in normal tissues, particularly in the gallbladder and intestines, where accumulation was significantly lower than that of the current 68Ga-FAP2286, which is beneficial in reducing radiation damage to non-target organs. Furthermore, the higher accumulation in the kidneys reflects the easier metabolism of 68Ga-F2286b-DOTA and 68Ga-F2286n-DOTA by the kidneys. This is partly due to the ease with which the cyclic peptide structure based on bioorthogonal groups is metabolized in the kidneys, and partly due to the significant increase in hydrophilicity of the conjugates due to the modification of the PEG chain.

[0187] In this embodiment, testing was also performed. 64 Cu-F2286b-NOTA and 64 Performance of Cu-F2286b-C2-NOTA in animal PET / CT imaging experiments.

[0188] Specifically, HEK293-FAP cells were seeded in the axilla of the right forelimb of BALB / c nu / nu mice and housed in an SPF-grade animal facility for approximately 1-2 weeks. The tumor mass was approximately 200-300 mm in size. 3 Approximately 100 µL of a dose of approximately 200 µCi was administered via tail vein injection. 64 Cu-F2286b-NOTA is a highly radiochemically pure reagent (radiochemical purity >95%). Imaging was performed at 1, 3, 12, 24, and 40 hours.

[0189] Similarly, HEK293-FAP cells were seeded in the axilla of the right forelimb of BALB / c nu / nu mice and housed in an SPF-grade animal facility for approximately 1–2 weeks. The tumor mass was approximately 200–300 mm in size. 3 Approximately 100 µL of a dose of approximately 190 µCi was administered via tail vein injection. 64 Cu-F2286b-C2-NOTA is a highly radiochemically pure reagent (radiochemical purity >95%). Imaging was performed at 1, 3, 12, 24, and 40 hours.

[0190] like Figure 10 and Figure 11 As shown,64 Cu-F2286b-NOTA and 64 Both Cu-F2286b-C2-NOTA exhibit excellent tumor uptake and tumor targeting, enabling stable, accurate, and clear imaging of tumors within 24 hours, and completing metabolism within approximately 40 hours.

[0191]

Example 10

[0192] In this embodiment, the following tests were conducted. 68 Ga-F2286b-DOTA and 68 The hydrophilicity of Ga-F2286n-DOTA.

[0193] Specifically, the partition coefficient (logP) of each radiotracer was measured in octanol and phosphate-buffered saline (PBS) to assess the lipophilicity of the radioligands. 50 μL of radiotracer was added to 450 μL of PBS, followed by 500 μL of octanol. The mixture was thoroughly shaken at room temperature and centrifuged at 3000 rcf for 2 minutes. Subsequently, 50 μL of each layer was taken, and the radioactivity was measured using a gamma counter. The partition coefficient logP was determined by the following formula: logP = Log [(γ-count in octanol) / (γ-count in PBS)].

[0194] The experimental results are shown in Table 1, indicating that... 68 Ga-F2286b-DOTA and 68 Ga-F2286n-DOTA has compared to 68 Ga-FAP-2286 has better hydrophilicity.

[0195] Table 1:

[0196]

[0197] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A cyclic peptide-bioorthogonal group conjugate targeting FAP, characterized in that, It includes a precursor compound and a radionuclide. The general structural formula of the precursor compound is TBLY, where T is a fibroblast activation protein targeting cyclic peptide, B is a cycloaddition product group, L is a linker, and Y is a chelating group for complexing the radionuclide. The cycloaddition product group is formed by a bioorthogonal reaction between a bioorthogonal group attached to a fibroblast activation protein targeting cyclic peptide and a complementary reactive group attached to a linker.

2. The cyclic peptide-bioorthogonal group conjugate targeting FAP according to claim 1, characterized in that, The bioorthogonal group is a tetrazine group, and the complementary reactive group of the tetrazine group is a dienophile or an isonitrile group; or The bioorthogonal group is an azide group, and the complementary reactive group of the azide group is a terminal alkyne compound or a cyclic alkyne compound.

3. The cyclic peptide-bioorthogonal group conjugate targeting FAP according to claim 2, characterized in that, The fibroblast activation protein targeting cyclic peptide with a bioorthogonal group has the following structural formula: Formula I: ; Formula II: ; In Formula I, group R1 is selected from H, C1~C6 alkyl or phenyl; in Formula I or Formula II, group R2 is selected from C1~C6 alkyl, C1~C6 acyl or sulfonyl.

4. The cyclic peptide-bioorthogonal group conjugate targeting FAP according to claim 1, characterized in that, The linker is selected from one or more polyethylene glycol units, C1~C1. 12 Alkylene, or divalent glucose.

5. A cyclic peptide-bioorthogonal group conjugate targeting FAP according to any one of claims 1 to 4, characterized in that, The precursor compound is selected from the following structural formulas: 、 、 、 ； In the formula, group R1 is selected from H, C1~C6 alkyl or phenyl; group R2 is selected from C1~C6 alkyl, C1~C6 acyl or sulfonyl; group R3 is selected from H or C1~C6 alkyl; n=4~8.

6. The cyclic peptide-bioorthogonal group conjugate targeting FAP according to claim 5, characterized in that, The precursor compound is selected from the following structural formulas: 、 、 、 ； ； 。 7. A method for preparing a cyclic peptide-bioorthogonal group conjugate targeting FAP, characterized in that, Includes the following steps: The fibroblast activation protein targeting cyclic peptide shown in Formula I undergoes an anti-electron-demand Diels-Alder reaction or a [4+1] cycloaddition reaction with a linker connected to a first complementary reactive group and a chelating group to obtain a precursor compound, wherein the first complementary reactive group is a dienophile or an isonitrile group; or The fibroblast activation protein targeting cyclic peptide shown in Formula II undergoes a 1,3-dipolar cycloaddition reaction with a linker connected to a second complementary reactive group and a chelating group to obtain a precursor compound, wherein the second complementary reactive group is a terminal alkyne compound or a cyclic alkyne compound. The precursor compound undergoes a coordination complexation reaction with a radionuclide to prepare the cyclic peptide-bioorthogonal group conjugate targeting FAP. Formula I: ; Formula II: ; In Formula I, group R1 is selected from H, C1~C6 alkyl or phenyl; in Formula I or Formula II, group R2 is selected from C1~C6 alkyl, C1~C6 acyl or sulfonyl.

8. The method for preparing a cyclic peptide-bioorthogonal group conjugate targeting FAP according to claim 7, characterized in that, The polypeptide represented by Formula III and diene tetrazine compounds The reaction yielded the fibroblast activation protein targeting cyclic peptide shown in Formula I; or The polypeptide shown in Formula III was reacted with 1,1'-[5-(azidomethyl)-1,3-phenylene]bis(2-iodoethane-1-one) to prepare the fibroblast activation protein targeting cyclic peptide shown in Formula II. Formula III: .

9. A tumor imaging agent, characterized in that, The tumor imaging agent includes a cyclic peptide-bioorthogonal group conjugate targeting FAP according to any one of claims 1 to 6.

10. An antitumor drug, characterized in that, The antitumor drug includes a cyclic peptide-bioorthogonal group conjugate targeting FAP as described in any one of claims 1 to 6.

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