Novel trifunctional compound and application thereof

Abstract

A new trifunctional compound comprising at least one target T, at least one payload P, in addition to covalent warheads that may be carried by the target T itself, the tri-functional compounds further comprise at least one covalent warhead C that can form a reversible or irreversible covalent linkage with a protein or tissue (e.g., an FAP protein or other biomolecule in vivo). The invention also relates to a pharmaceutical composition and a kit containing the trifunctional compound, and application of the trifunctional compound in diagnosis or treatment of diseases.

Description

A new trifunctional compound and its use Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a trifunctional compound containing a covalent warhead, a pharmaceutical composition thereof, and use thereof for diagnosing or treating diseases. Background Art

[0002] Radiation therapy, surgery, and chemotherapy are traditionally the three main first-line treatments for cancer. Radiopharmaceuticals (abbreviated as "radiopharmaceuticals") can be considered a form of internal irradiation in radiotherapy. Compared with traditional radiotherapy, they have the advantage of a so-called powerful "crossfire" and are one of the few treatment options for patients with advanced cancer and systemic metastasis. Mechanistically, radiopharmaceuticals use high-energy ionizing radiation generated by the decay of medical isotopes to break DNA chains and induce cancer cell death. This requires them to have extremely high tumor selectivity / targeting and a certain degree of tumor uptake and retention, so as to deliver a sufficient dose of radiation only to the tumor target while limiting damage to surrounding normal tissues.

[0003] Radionuclide Ligand Conjugate (RLC) is a major drug form of radiopharmaceuticals, which are generally binary conjugates of chelator-linker-targeting ligand. The ligand part can be a small molecule, peptide, nucleic acid aptamer, antibody, etc. The linker mainly acts as a spacer to prevent the coupled chelator from greatly reducing the affinity of the target molecule and has a certain effect on regulating pharmacokinetic properties. The chelator part can be coordinated with different diagnostic / therapeutic radionuclides by replacing the chelator according to different diagnosis and treatment needs. It has the advantage of "integrated diagnosis and treatment". Therefore, some molecules with average targeting and safety but high tumor uptake still have the potential to be used in molecular imaging diagnosis. Diagnostic radionuclides can be divided into two categories, including positron-emitting radionuclides that can be traced by positron emission tomography (PET-CT) (such as 18 F, 68 Ga, 64 Cu, 86 Y, 89 Zr, etc.), and nuclides with gamma radiation that can be traced by single photon emission tomography (SPECT-CT) (such as 99m Tc, 133 Xe, 123 I, 91m Kr, etc.) Therapeutic radionuclides can be divided into β-emitters (β-emitters, such as 90 Y, 124 I, 151 Tb, 177 Lu, etc.) and α-emitters (α-emitter, such as 211 At,212 Pb, 213 Bi, 223 Ra, 225 In addition, the chelating agent can be replaced with an optical dye that absorbs and emits at different wavelengths to form an optical probe, which is also used for optical imaging at the cell and living body levels.

[0004] In recent years, FDA and EU approvals for prostate-specific membrane antigen (PSMA) and somatostatin receptor (SSTR)-based diagnostic and therapeutic approaches for RLC have encouraged more RLC drugs to enter clinical research. Because PSMA and SSTR are expressed only in prostate cancer and neuroendocrine tumors, respectively, and lack broad tumor coverage, researchers worldwide are pursuing the development of RLC drugs with a broader spectrum of activity.

[0005] Fibroblast activation protein (FAP-α) is a type II membrane-bound glycoprotein and a member of the serine protease family. It is overexpressed in cancer-associated fibroblasts (CAFs) and activated fibroblasts in wound healing / inflammatory sites. As a broad-spectrum cancer target, it is expressed in more than 90% of epithelial tumor microenvironments, including pancreatic, colon, breast, ENT (ear, nose and throat) cancers. In 2019, Kratochwil et al. clinically confirmed the efficacy of quinoline-containing 68 The Ga-FAPI-04 small molecule radiotherapy drug can detect up to 28 different cancers (J. Nucl. Med. 2019, 60, 801). Due to its low expression in normal tissues, FAP-α not only serves as a biomarker for tumor diagnosis and prognosis, but also as a promising target for radiopharmaceutical therapy. Since Lindner et al. first introduced FAPI-04 into clinical practice in 2018 (J. Nucl. Med. 2018, 59, 1415), FAP-α inhibitor (FAPI)-based PET imaging and radiotherapy have been investigated for a variety of cancers and non-cancerous conditions.

[0006] Covalent inhibitors are a class of inhibitors that exert their biological functions by irreversibly binding to target protein residues through covalent bonds. Most of these inhibitors are small molecule kinase inhibitors (Eur. J. Med. Chem. 2017, 138, 96). Generally speaking, covalent inhibition is a two-step process (Figure 1): First, the inhibitor reversibly binds to the target enzyme, bringing the small molecule warhead into close proximity with the active residue in the enzyme. Second, a bonding reaction occurs between the inhibitor warhead and the residue, forming a covalent bond. Common nucleophilic amino acid residues that can undergo covalent reactions include cysteine, serine, tyrosine, lysine, arginine, and glutamate. Common covalent warheads are generally electrophiles, which can be divided into two categories based on whether they form reversible or irreversible covalent bonds. Warheads that generally form irreversible covalent bonds, such as acrylamide, epoxy, chloroacetyl, and sulfonyl fluoride, and warheads that generally form reversible covalent bonds, such as cyano and ketocarbonyl groups, are one type. Covalent warheads that can be used in vivo and ultimately developed as drugs require a balance between activity and stability. A promising class of covalent warheads are "latent electrophiles," which are activated only upon binding to specific proteins to accelerate covalent bond formation, thereby minimizing off-target toxicity associated with warhead activity. In 2014, Sharpless et al. developed a new class of "latent electrophiles"—fluorosulfates—based on hexavalent sulfur click chemistry (SuFEx) (Angew. Chem. Int. Ed. 2014, 53, 9430). Compared to the most common acrylamide-based covalent warheads that react only with cysteine ​​residues, it not only has better in vivo stability but also can covalently link to a wider range of adjacent lysine, histidine, tyrosine, and serine residues after binding to the target protein, making it a promising new class of drug covalent warheads. Similarly, promising covalent warheads include phosphorus (pentavalent)-fluorine covalent warheads, such as (hetero)aryl phosphoramidofluoridate.

[0007] Summary of the Invention

[0008] The present invention, based on the existing technology, has conducted innovative research. By introducing a covalent warhead comprising a (hetero)aryl-substituted halosulfate or (hetero)aryl-substituted phosphoramide fluoride into a drug comprising a target and a payload, the following effects are unexpectedly achieved: the pharmacokinetics of the drug are regulated, so that the drug uptake and retention in the target organ (target organ), such as a tumor, are greatly improved, while the uptake and retention in non-target organs are still relatively low, for example, the uptake in the liver, kidney and / or blood pool can be sustainably reduced. Therefore, compared with traditional drug modification strategies, the drugs engineered by the strategy of the present invention have a higher target-to-target ratio, and the performance of probe drugs, including sensitivity and / or specificity, can be optimized in diagnosis; in treatment, the safe treatment window can be expanded and bioavailability can be increased, thereby improving the therapeutic effect while ensuring safety.

[0009] One aspect of the present disclosure provides a trifunctional compound, and a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein the trifunctional compound comprises at least one target T and at least one payload P. In addition to the covalent warhead that may be carried by the target T itself, the trifunctional compound comprises at least one covalent warhead C that can form a reversible or irreversible covalent bond with a protein or tissue targeted by the target T (e.g., a FAP protein or other biological molecules in vivo). The covalent warhead C is preferably a covalent warhead C comprising a (hetero)aryl-substituted halogenated sulfate or a (hetero)aryl-substituted phosphoramidite fluoride.

[0010] By using trifunctional compounds containing the three functional groups described above, the present invention improves uptake and / or retention in target organs (e.g., tumors), enhancing the sensitivity of diagnostic probes and the efficacy of therapeutic drugs. Simultaneously, binding to plasma albumin is not significantly enhanced, thereby maintaining low uptake and retention in vital organs such as the blood pool, liver, and kidneys, ensuring diagnostic probe specificity and manageable toxicity. Ultimately, compounds with high diagnostic performance or a wide therapeutic window are provided.

[0011] Preferably, the present invention uses a trifunctional compound containing a covalent warhead C, so that the compound retains a certain degree of targeting (ensuring drug safety) while significantly improving its uptake and retention in target sites such as tumor tissues (improving drug effectiveness).

[0012] More preferably, by adjusting or controlling the linker chain length and conformation, the trifunctional compound of the present invention can achieve a more excellent target-to-substance ratio.

[0013] More preferably, the present invention has higher detection sensitivity of targets such as tumor tissues when the payload P is a diagnostic group, and better achieves drug effectiveness when the payload P is a therapeutic group.

[0014] On the other hand, the present invention introduces covalent warheads at other modification sites, such as introducing additional covalent warheads on the payload P, to achieve better pharmacokinetic effects.

[0015] In one aspect, the present invention relates to a trifunctional compound, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound comprises at least one target T and at least one payload P, wherein, in addition to the covalent warhead that may be carried by the target T, the trifunctional compound comprises at least one covalent warhead C as follows:

[0016] The payload P is any optical functional group, radiolabeled functional group, radiolabeled functional group, or small molecule cytotoxic drug functional group that can be used for optical imaging, positron emission tomography imaging, single photon emission computed tomography imaging, chemotherapy or radiotherapy;

[0017] The target T comprises a targeting group that can target a protein or tissue (such as FAP protein or other biological molecules in vivo), and the targeting group is a small molecule;

[0018] The covalent warhead C can form a reversible or irreversible covalent bond with the protein or tissue targeted by the target T;

[0019] wherein the covalent warhead C is selected from the group consisting of:

[0020] in:

[0021] Y is selected from O, S and NR 1 the group formed;

[0022] LG is a leaving group that can be replaced by a protein residue, preferably a halogen or Ots, and Ts is p-toluenesulfonyl;

[0023] R, R' and R" are independently selected from the group consisting of hydrogen, halogen, nitro, cyano, optionally substituted mercapto, optionally substituted seleno, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted amino;

[0024] R 1 is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl;

[0025] Het is an optionally substituted heterocyclic group or heteroaryl group, wherein when the heteroaryl group contains a N atom, the N atom may be present in a free form or in the form of an onium salt;

[0026] Hal is a halogen, preferably F or Cl;

[0027] Each p is independently an integer between 0 and 12;

[0028] The at least one target T, the at least one payload P, and the at least one covalent warhead C are connected via a linker moiety.

[0029] In certain embodiments of the present invention, the covalent warhead C in the trifunctional compound can also be selected from

[0030] In certain embodiments, Hal can also be other leaving groups, such as OH or NH2.

[0031] In a preferred embodiment of the present invention, the covalent warhead C in the trifunctional compound is selected from the group consisting of:

[0032] Wherein: Het is an optionally substituted C4-C 12 Heterocyclyl, or optionally substituted C5-C 12 Heteroaryl groups, wherein when the heteroaryl group contains a nitrogen atom, the nitrogen atom may be present in a free form or in the form of an onium salt;

[0033] Hal is F;

[0034] Each p is independently an integer between 0 and 6;

[0035] R 1 is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl.

[0036] In a preferred embodiment of the present invention, C in the trifunctional compound is selected from the group consisting of:

[0037] in:

[0038] Het is a C4, C5 or C6 heterocyclic group, or a C5 or C6 heteroaryl group, wherein the heterocyclic group or heteroaryl group contains one or two heteroatoms selected from N, O or S, wherein when the heteroaryl group contains a N atom, the N atom may exist in a free form or in the form of an onium salt, preferably in a free form, more preferably Het is an azetidinyl, pyridyl, pyrrolyl or N-oxypyridyl group;

[0039] Hal is F;

[0040] Each p is independently an integer between 0 and 4.

[0041] In a preferred embodiment of the present invention, the trifunctional compound comprises one to three, for example one or two, payloads P, and one to three, for example one or two, targeting agents T, and in addition to the covalent warhead that may be carried by the targeting agent T itself, the trifunctional compound comprises one to three, for example one or two, covalent warheads C, and the linker moieties each independently comprise 0 to 6 linker units, and the linker units can be divalent linkers, such as single bonds, or trivalent linkers.

[0042] In a preferred embodiment of the present invention, the trifunctional compound has a structure of formula (I), formula (II) or formula (III),

[0043] in:

[0044] The payloads P and P' are each independently the same or different groups containing a radionuclide, or chelating groups that can chelate a radionuclide, optical dye groups, or small molecule cytotoxic drug groups;

[0045] Targeting agents T and T' are each independently the same or different targeting groups that can target proteins or tissues (such as FAP protein or other biological molecules in vivo), and the targeting groups are small molecules;

[0046] In general formula (I), general formula (II) and general formula (III), in addition to the covalent warhead that may be carried by the target T itself, there are one, two or three identical or different covalent warheads C, each of which is independently connected to P, T, L1, T' or P' shown in the general formula through the same or different linker moiety L4;

[0047] Each of L1, L2, and L3 is a linker moiety, each independently comprising 0 to 6 linker units.

[0048] In a preferred embodiment of the present invention, the trifunctional compound has a structure of formula (Ia), formula (Ib), formula (Ic), formula (IIa) or formula (IIIa),

[0049] in:

[0050] Each L4, L5, L6, L7, L8, L9, L 10 、L 11 、L 12 、L 13 、L 14 、L 15 、L 16 and L 17 are independently linker moieties, each independently comprising 0 to 6 linker units;

[0051] a, b, c, d, e and f are each independently 0 or 1, wherein when it is 0, it means that the group in the bracket marked with the number does not exist, wherein the sum of a, b and c is at least 1, and the sum of c, d and e is at least 1;

[0052] C, C' and C" each independently have the meanings defined above.

[0053] In a preferred embodiment of the present invention, the targeting group in the trifunctional compound targets fibroblast activation protein-α (FAP-α).

[0054] In a preferred embodiment of the present invention, the targeting group in the trifunctional compound has the general formula (IV), preferably has the general formula (IVa), more preferably has the general formula (IVc), the general formula (IVd), the general formula (IVe) or the general formula (IVf),

[0055] Wherein in the general formula (IV), A is selected from O, S, NR A , R A Selected from H, C1-C6 alkyl,

[0056] There are multiple R on the tetrahydropyrrole ring shown f Group, each R f The groups are each independently selected from H, F, Cl, -CN, -B(OH)2, C1-C6 alkyl, α-chloroketo, and optionally two R f The groups may be linked to each other to form a cycloalkyl group, preferably a C3-C7 cycloalkyl group,

[0057] B1 and B2 are independently selected from O or S,

[0058] R f1 and R f2 independently selected from H, D or C1-C4 alkyl,

[0059] Ar is a C6-C containing a nitrogen atom 10 heteroaryl;

[0060] Wherein in the general formula (IVa), A is selected from O, S, NR A , R A Selected from H, C1-C6 alkyl,

[0061] There are multiple R on the tetrahydropyrrole ring shown f Group, each R f The groups are each independently selected from H, F, Cl, -CN, C1-C6 alkyl, α-chloroketo, optionally two R on adjacent carbons fThe groups may be linked to each other to form a cycloalkyl group, preferably a C3-C7 cycloalkyl group;

[0062] Wherein in general formula (IVb), (IVc), (IVd) or general formula (IVe), A is selected from O, S, NR A , R A Selected from H, methyl, ethyl, n-propyl, isopropyl.

[0063] In a preferred embodiment of the present invention, the targeting group in the trifunctional compound has the general formula (V),

[0064] The peptide sequences are drawn from left to right in the direction of N-terminus to C-terminus.

[0065] Xaa1 is the residue of an amino acid of the general formula (VI),

[0066] in:

[0067] R 1a Yes-NH-R 1a’ , R 1a’ is a protecting group or an amino acid, preferably the protecting group is R 1a” -C(O)- or R 1a” -S(O2)-, and R 1a” is C1-C6 alkyl, wherein optionally one -CH2- group is replaced by -S- or -O-,

[0068] R 1b is H or methyl,

[0069] g is 0 or 1,

[0070] The carbonyl group of Xaa1 is covalently linked to the nitrogen of Xaa2.

[0071] and the sulfur atom of Xaa1 is covalently linked to Yc as a thioether;

[0072] Xaa2 and Xaa3 are independently selected from the residues of amino acids of formula (VII) or (VIII),

[0073] in,

[0074] h is 0, 1, or 2,

[0075] i is 1 or 2,

[0076] j is 1, 2, or 3,

[0077] and the amino acid of formula (VII) may be substituted at ring positions 3 and 4 as shown by one or two substituents selected from methyl, OH, NH2 and F;

[0078] Xaa4 is the residue of an amino acid of the general formula (IX),

[0079] in,

[0080] R 4a is selected from H, OH, COOH and CONH2,

[0081] q is 1, 2 or 3, wherein optionally, one or two hydrogen atoms of said 1, 2 or 3 CH2 groups are each and independently replaced by a methyl group or an ethyl group,

[0082] R 4b is H or methyl;

[0083] Xaa5 is the residue of an amino acid of the general formula (X),

[0084] in:

[0085] R 5a is OH or NH2,

[0086] m is 1, 2 or 3,

[0087] R 5b is H or methyl;

[0088] Xaa6 is an amino acid selected from aromatic La-amino acids, preferably the residue of an amino acid of the general formula (XI),

[0089] in,

[0090] R 6a and R 6b are each independently selected from H, methyl, ethyl, propyl and isopropyl, preferably H,

[0091] R 6c represents 0 to 3 substituents, each such substituent being independently selected from F, Cl, Br, NO2, NH2, CN, CHF3, OH, OR 6d and C1-C4 alkyl,

[0092] R 6d is selected from methyl, ethyl, propyl and isopropyl,

[0093] l is 0 or 1, preferably 0,

[0094] The CR shown 6a R 6b (CH2)l PhR 6c The moiety may constitute part of the covalent warhead C in formula (I), (II) or (III);

[0095] Xaa7 is an aminothiol or amino acid residue of the general formula (XII),

[0096] in,

[0097] R 7a is H, -COOH, -CONH2 or CH2OH,

[0098] n is 1 or 2;

[0099] Yc is a structure of the general formula (XIII),

[0100] It connects the S atom of Xaa1 and the S atom of Xaa7 while forming two thioether bonds, thereby forming a cyclic structure of the general formula (XIV),

[0101] Wherein, the substitution pattern of the aromatic group in the general formula (XIII) is ortho, para or meta,

[0102] g is 0 or 1,

[0103] n is 1 or 2,

[0104] Y 1 is CH or N,

[0105] Y 2 It's CR L1 ,

[0106] R L1 is a linker unit connected to other parts in general formula (I);

[0107] The targeting group preferably has formula (Va),

[0108] In a preferred embodiment of the present invention, the payload P in the trifunctional compound comprises at least one group selected from the following group:

[0109] The first group, groups with at least one radionuclide, are selected from:

[0110] where R 2 、R 3 、R 4 and R 5are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl,

[0111] X is selected from 18 F. 123 I. 124 I. 125 I. 131 I and 211 At or its non-radioactive isotopes,

[0112] q and r are independently integers between 0 and 4;

[0113] The N on the triazole ring at the attachment site shown here may not be present on the payload P but rather on the linker unit;

[0114] The second group, chelating groups that can chelate radionuclides, are selected from:

[0115] The third group, optical dye groups, is selected from:

[0116] The fourth group, small molecule cytotoxic drug group, is selected from:

[0117] In a preferred embodiment of the present invention, in the trifunctional compound,

[0118] When L1 is a trivalent linking group, i.e., when the covalent warhead C is connected, or L4 is independently selected from the following groups:

[0119] L1 to L 17 The divalent linking group units contained in are independently selected from the following groups:

[0120] single bond,

[0121] The methylene groups in the listed divalent linking units may be replaced by one or more -COR L2 or -COOR L2 substituted by a group, wherein each R L2 Independently selected from H, C1-C6 alkyl, C6-C 10 Aryl, C6-C 10 Aryl-substituted C1-C6 alkyl;

[0122] L 18is a single bond, -CH2-, -NHCH2-, or

[0123] Cy is selected from

[0124] u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, v and o are independently 1, 2, 3, 4 or 5,

[0125] Each R 6 and R 7 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl,

[0126] * is the site for connecting the target T,

[0127] ** is the site for connecting the covalent warhead C,

[0128] The condition is that the 17 In the present invention, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S.

[0129] In certain embodiments of the present invention, the L1 to L 17 The divalent linking unit contained in can also be selected from And / or said L4 can also be selected from and / or u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, u12, v and o can also independently be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0130] In a preferred embodiment of the present invention, the trifunctional compound has a structure of formula (Ia), L4 is

[0131] L4 preferably has the following structure:

[0132] where R 6 、R 7 、L 18 , Cy, o and u have the same definitions as described above.

[0133] In a preferred embodiment of the present invention, the trifunctional compound has a structure of formula (Ia) or formula (Ib):

[0134] in:

[0135] L4 is shown as a trivalent linker moiety;

[0136] Each of L5, L6, L7 and L8 shown is independently a divalent linker moiety, each independently comprising 0 to 6, preferably 0 to 3 linker units;

[0137] The C shown is a covalent warhead selected from the group consisting of:

[0138] Het is an optionally substituted C4-C 12 Heterocyclyl, or optionally substituted C5-C 12 Heteroaryl groups, wherein when the heteroaryl group contains a nitrogen atom, the nitrogen atom may be present in a free form or in the form of an onium salt;

[0139] Hal is F or Cl;

[0140] Y is selected from O, S and NR 1 the group formed;

[0141] Each p is independently an integer between 0 and 6;

[0142] Each R 1 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl;

[0143] each R is independently selected from the group consisting of hydrogen, halogen, nitro, cyano, optionally substituted thiol, optionally substituted seleno, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted amino;

[0144] T shown is a targeting group targeting fibroblast activation protein-α (FAP-α);

[0145] P is a chelating group capable of chelating a radionuclide.

[0146] In a preferred embodiment of the present invention, the trifunctional compound has a structure of formula (Ia) or formula (Ib),

[0147] in:

[0148] The L4 is a trivalent linking group selected from the following:

[0149] Each of said L5, L6, L7 and L8 is independently a divalent linker moiety, each of which independently comprises 0 to 3, preferably 0 or 1 linker units, said linker units being independently selected from the following groups:

[0150] single bond,

[0151] The methylene groups in the listed divalent linking units may be replaced by one or more -COR L2 or -COOR L2 substituted by a group, wherein each R L2 Independently selected from H, C1-C6 alkyl, C6-C 10 Aryl, C6-C 10 Aryl-substituted C1-C6 alkyl;

[0152] L 18 is a single bond, -CH2-, -NHCH2-, or

[0153] Cy is selected from

[0154] u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, u12, v and o are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1, 2, 3, 4 or 5,

[0155] Each R 6 and R 7 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl,

[0156] * is the site for connecting T,

[0157] ** is the site for connecting C,

[0158] Provided that in L5, L6, L7 and L8, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0159] C is selected from the group consisting of:

[0160] in:

[0161] Het is a C4, C5 or C6 heterocyclic group, or a C5 or C6 heteroaryl group, wherein the heterocyclic group or heteroaryl group contains one or two heteroatoms selected from N, O or S. When the heteroaryl group contains a N atom, the N atom may exist in a free form or in the form of an onium salt, preferably in a free form; preferably, Het is a C4, C5 or C6 heterocyclic group, or a C5 or C6 heteroaryl group, wherein the heterocyclic group or heteroaryl group contains one heteroatom selected from N, O or S, preferably N; more preferably, Het is an azetidinyl group, a pyridyl group, a pyrrolyl group, or an N-oxypyridyl group;

[0162] Hal is F;

[0163] Each p is independently an integer between 0 and 4;

[0164] T shown is a targeting group targeting fibroblast activation protein-α (FAP-α);

[0165] The P shown is a chelating group that can chelate radionuclides, which is selected from the following groups:

[0166] In a preferred embodiment of the present invention, the trifunctional compound has a structure of formula (Ia),

[0167] in:

[0168] C is selected from the group consisting of:

[0169] in:

[0170] Het is a C4, C5 or C6 heterocyclic group, or a C5 or C6 heteroaryl group, wherein the heterocyclic group or heteroaryl group contains one or two heteroatoms selected from N, O or S. When the heteroaryl group contains a N atom, the N atom may exist in a free form or in the form of an onium salt, preferably in a free form; preferably, Het is a C4, C5 or C6 heterocyclic group, or a C5 or C6 heteroaryl group, wherein the heterocyclic group or heteroaryl group contains one heteroatom selected from N, O or S, preferably N; more preferably, Het is an azetidinyl group, a pyridyl group, a pyrrolyl group, or an N-oxypyridyl group;

[0171] Hal is F;

[0172] Each p is independently an integer between 0 and 4;

[0173] The P shown is a chelating group that can chelate radionuclides, which is selected from the following groups:

[0174] L4 has the following structure:

[0175] Each of said L5 and L6 is independently the following group:

[0176] single bond,

[0177] wherein u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, u12, v and o are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1, 2, 3, 4 or 5,

[0178] Each R6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4,

[0179] * is the site for connecting T,

[0180] ** is the site for connecting C,

[0181] Provided that in L5 and L6, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0182] T is the general formula (IV), preferably has the general formula (IVa), more preferably has the general formula (IVc), the general formula (IVd), the general formula (IVe) or the general formula (IVf),

[0183] Wherein in the general formula (IV), A is selected from O, S, NR A , R A Selected from H, C1-C6 alkyl,

[0184] There are multiple R on the tetrahydropyrrole ring shown f Group, each R f The groups are each independently selected from H, F, Cl, -CN, -B(OH)2, C1-C6 alkyl, optionally two R f The groups may be linked to each other to form a cycloalkyl group, preferably a C3-C7 cycloalkyl group,

[0185] B1 and B2 are independently selected from O or S,

[0186] R f1 and R f2 independently selected from H, D or C1-C4 alkyl,

[0187] Ar is a C6-C containing a nitrogen atom 10 heteroaryl;

[0188] Wherein in the general formula (IVa), A is selected from O, S, NR A , R A Selected from H, C1-C6 alkyl,

[0189] There are multiple R on the tetrahydropyrrole ring shown f Group, each R f The groups are each independently selected from H, F, Cl, -CN, C1-C6 alkyl, optionally two R f The groups may be linked to each other to form a cycloalkyl group, preferably a C3-C7 cycloalkyl group;

[0190] Wherein in general formula (IVb), (IVc), (IVd) or general formula (IVe), A is selected from O, S, NR A , R A Selected from H, methyl, ethyl, n-propyl, isopropyl.

[0191] In a preferred embodiment of the present invention, the trifunctional compound has a structure of formula (Ib),

[0192] in:

[0193] C in the general formula (Ib) is selected from the group consisting of:

[0194] in:

[0195] Het is a C4, C5 or C6 heterocyclic group, or a C5 or C6 heteroaryl group, wherein the heterocyclic group or heteroaryl group contains one or two heteroatoms selected from N, O or S. When the heteroaryl group contains a N atom, the N atom may exist in a free form or in the form of an onium salt, preferably in a free form; preferably, Het is a C4, C5 or C6 heterocyclic group, or a C5 or C6 heteroaryl group, wherein the heterocyclic group or heteroaryl group contains one heteroatom selected from N, O or S, preferably N; more preferably, Het is an azetidinyl group, a pyridyl group, a pyrrolyl group, or an N-oxypyridyl group;

[0196] Hal is F;

[0197] Each p is independently an integer between 0 and 4;

[0198] The P shown is a chelating group that can chelate radionuclides, which is selected from the following groups:

[0199] Each of L7 and L8 is independently the following group:

[0200] single bond,

[0201] wherein u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, u12, v and o are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1, 2, 3, 4 or 5,

[0202] Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4,

[0203] Provided that in L7 and L8, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0204] T has the general formula (V),

[0205] The peptide sequences are drawn from left to right in the direction of N-terminus to C-terminus.

[0206] Xaa1 is the residue of an amino acid of the general formula (VI),

[0207] in:

[0208] R 1a Yes-NH-R 1a’ , R 1a’ is a protecting group or an amino acid, preferably the protecting group is R 1a” -C(O)- or R 1a” -S(O2)-, and R 1a” is C1-C6 alkyl, wherein optionally one -CH2- group is replaced by -S- or -O-,

[0209] R 1b is H or methyl,

[0210] g is 0 or 1,

[0211] The carbonyl group of Xaa1 is covalently linked to the nitrogen of Xaa2.

[0212] and the sulfur atom of Xaa1 is covalently linked to Yc as a thioether;

[0213] Xaa2 and Xaa3 are independently selected from the residues of amino acids of formula (VII) or (VIII),

[0214] in,

[0215] h is 0, 1, or 2,

[0216] i is 1 or 2,

[0217] j is 1, 2, or 3,

[0218] and the amino acid of formula (VII) may be substituted at ring positions 3 and 4 as shown by one or two substituents selected from methyl, OH, NH2 and F;

[0219] Xaa4 is the residue of an amino acid of the general formula (IX),

[0220] in,

[0221] R 4a is selected from H, OH, COOH and CONH2,

[0222] q is 1, 2 or 3, wherein optionally, one or two hydrogen atoms of said 1, 2 or 3 CH2 groups are each and independently replaced by a methyl group or an ethyl group,

[0223] R 4b is H or methyl;

[0224] Xaa5 is the residue of an amino acid of the general formula (X),

[0225] in:

[0226] R 5a is OH or NH2,

[0227] m is 1, 2 or 3,

[0228] R 5b is H or methyl;

[0229] Xaa6 is an amino acid selected from aromatic La-amino acids, preferably the residue of an amino acid of the general formula (XI),

[0230] in,

[0231] R 6a and R 6b are each independently selected from H, methyl, ethyl, propyl and isopropyl, preferably H,

[0232] R 6c represents 0 to 3 substituents, each such substituent being independently selected from F, Cl, Br, NO2, NH2, CN, CHF3, OH, OR 6d and C1-C4 alkyl,

[0233] R 6d is selected from methyl, ethyl, propyl and isopropyl,

[0234] l is 0 or 1, preferably 0,

[0235] The CR shown 6a R 6b (CH2) l PhR 6c The moiety may constitute a part of C in the general formula (Ib);

[0236] Xaa7 is an aminothiol or amino acid residue of the general formula (XII),

[0237] in,

[0238] R 7a is H, -COOH, -CONH2 or CH2OH,

[0239] n is 1 or 2;

[0240] Yc is a structure of the general formula (XIII),

[0241] It connects the S atom of Xaa1 and the S atom of Xaa7 while forming two thioether bonds, thereby forming a cyclic structure of the general formula (XIV),

[0242] Wherein, the substitution pattern of the aromatic group in the general formula (XIII) is ortho, para or meta,

[0243] g is 0 or 1,

[0244] n is 1 or 2,

[0245] Y 1 is CH or N,

[0246] Y 2 It's CR L1 ,

[0247] R L1 is a linker unit connected to other parts in general formula (Ib).

[0248] In a preferred embodiment of the present invention, the trifunctional compound has a structure of formula (Ib),

[0249] in:

[0250] Each of L7 and L8 is independently the following group:

[0251] single bond,

[0252] wherein u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, u12, v and o are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1, 2, 3, 4 or 5,

[0253] Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4,

[0254] Provided that in L7 and L8, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0255] C is selected from the group consisting of:

[0256] in:

[0257] Het is a C4, C5 or C6 heterocyclic group, or a C5 or C6 heteroaryl group, wherein the heterocyclic group or heteroaryl group contains one or two heteroatoms selected from N, O or S. When the heteroaryl group contains a N atom, the N atom may exist in a free form or in the form of an onium salt, preferably in a free form; preferably, Het is a C4, C5 or C6 heterocyclic group, or a C5 or C6 heteroaryl group, wherein the heterocyclic group or heteroaryl group contains one heteroatom selected from N, O or S, preferably N; more preferably, Het is an azetidinyl group, a pyridyl group, a pyrrolyl group, or an N-oxypyridyl group;

[0258] Hal is F;

[0259] Each p is independently an integer between 0 and 4;

[0260] The P shown is a chelating group that can chelate radionuclides, which is selected from the following groups:

[0261] T has the general formula (V),

[0262] The peptide sequences are drawn from left to right in the direction of N-terminus to C-terminus.

[0263] Xaa1 is the residue of an amino acid of the general formula (VI),

[0264] in:

[0265] R 1a Yes-NH-R 1a’ , R 1a’ is a protecting group or an amino acid, preferably the protecting group is R 1a” -C(O)- or R 1a” -S(O2)-, and R 1a” is C1-C6 alkyl, wherein optionally one -CH2- group is replaced by -S- or -O-,

[0266] R 1b is H or methyl,

[0267] g is 0 or 1,

[0268] The carbonyl group of Xaa1 is covalently linked to the nitrogen of Xaa2.

[0269] and the sulfur atom of Xaa1 is covalently linked to Yc as a thioether;

[0270] Xaa2 and Xaa3 are independently selected from the residues of amino acids of formula (VII) or (VIII),

[0271] in,

[0272] h is 0, 1, or 2,

[0273] i is 1 or 2,

[0274] j is 1, 2, or 3,

[0275] and the amino acid of formula (VII) may be substituted at ring positions 3 and 4 as shown by one or two substituents selected from methyl, OH, NH2 and F;

[0276] Xaa4 is the residue of an amino acid of the general formula (IX),

[0277] in,

[0278] R 4a is selected from H, OH, COOH and CONH2,

[0279] q is 1, 2 or 3, wherein optionally, one or two hydrogen atoms of said 1, 2 or 3 CH2 groups are each and independently replaced by a methyl group or an ethyl group,

[0280] R 4b is H or methyl;

[0281] Xaa5 is the residue of an amino acid of the general formula (X),

[0282] in:

[0283] R 5a is OH or NH2,

[0284] m is 1, 2 or 3,

[0285] R 5b is H or methyl;

[0286] Xaa6 is the residue of an amino acid of the general formula (XI),

[0287] in,

[0288] R 6a and R 6b are each independently selected from H, methyl, ethyl, propyl and isopropyl, preferably H,

[0289] R 6c represents 0 to 1 substituents, each such substituent being independently selected from F, Cl, NO2, NH2, CN, CHF3, OH and C1-C4 alkyl,

[0290] R 6d is selected from methyl, ethyl, propyl and isopropyl,

[0291] l is 0 or 1, preferably 0,

[0292] The CR shown 6a R 6b (CH2) l PhR 6c The moiety may constitute a part of C in the general formula (Ib);

[0293] Xaa7 is an aminothiol or amino acid residue of the general formula (XII),

[0294] in,

[0295] R 7a is H or -COOH,

[0296] n is 1 or 2;

[0297] Yc is a structure of the general formula (XIII),

[0298] It connects the S atom of Xaa1 and the S atom of Xaa7 while forming two thioether bonds, thereby forming a cyclic structure of the general formula (XIV),

[0299] Wherein, the substitution pattern of the aromatic group in the general formula (XIII) is ortho, para or meta,

[0300] g is 0 or 1,

[0301] n is 1 or 2,

[0302] Y 1 is CH or N,

[0303] Y 2 It's CR L1 ,

[0304] R L1 is a linker unit connected to other parts in general formula (Ib);

[0305] Said T preferably has the formula (Va),

[0306] In a preferred embodiment of the present invention, the trifunctional compound has one of the following structures:

[0307] Another aspect of the present invention relates to the trifunctional compound as described above, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the payload P is a chelating group capable of chelating a radionuclide, which chelates a radionuclide.

[0308] In a preferred embodiment of this aspect, the radionuclide carried or chelated by the trifunctional compound is a positron-emitting nuclide, a beta-emitter, an alpha-emitter, an Auger electron-emitting isotope, an X-ray-emitting isotope, a fluorescent isotope, or a stable metal / non-metal element coordinated with a radionuclide, preferably 11 C. 13 N. 15 O. 18 F and its coordination elements, 47 Sc, 51 Cr, 67 Ga, 68 Ga, 86 Y. 90 Y. 64 Cu, 67 Cu, 72 As、 72 Se, 89 Zr, 97 Such as 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 123 I. 124 I. 131 I. 142 Pr, 151 Eu, 153 Eu, 169 Eu, 159 Gd, 161 Tb, 177 Lu, 198 Au, 199 Ag, 201 Tl, 211 At 203 Pb, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 99m Tc, 111 In, 149 Pm, 153 Sm, 165 Dy, 169 Second, 186 Re、 188 Re、 197 Hg, 227 Th, 67 Ga, 68 Ga, 86 Y. 90 Y.55 Co、 139 La, 140 La, 149 Tb, 152 Tb, 155 Tb, 166 Ho, 175 Yb, 226 Th, 223 Ra and 230 U.

[0309] Another aspect of the present invention relates to a pharmaceutical composition comprising the aforementioned trifunctional compound, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, and a pharmaceutically acceptable carrier.

[0310] Yet another aspect of the present invention relates to a kit comprising or consisting of the trifunctional compound as described above, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, or the pharmaceutical composition as described above, and instructions for use in diagnosing a disease.

[0311] Another aspect of the present invention relates to a method for diagnosing or treating a disease, preferably a disease involving FAP, comprising administering to a subject a therapeutically effective amount of the trifunctional compound as described above, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the disease is preferably a central nervous system disease, a metabolic disease, preferably a cardiovascular metabolic disease, or cancer.

[0312] In a preferred embodiment of the present invention, in the method of diagnosing or treating a disease, preferably a disease involving FAP, the cancer is selected from prostate cancer, breast cancer, pancreatic cancer, liver cancer, lung cancer, gastric cancer, kidney cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck cancer, thymic cancer, cervical cancer, endometrial cancer, neuroendocrine tumors, thyroid cancer, intestinal cancer, brain glioma and bone metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0313] Figure 1 shows the general mechanism of action of covalent inhibitors.

[0314] Figure 2 shows 68 Radioactive purity spectrum of Ga-FAPI-CB-30, where the left picture is the quality control chart after labeling, the middle picture is the stability monitoring chart after 1 hour of serum co-incubation, and the right picture is the stability monitoring chart after 2 hours of serum co-incubation.

[0315] The left, middle and right figures of FIG3 respectively show 68 Ga-FAPI-CB-31, 68 Ga-FAPI-CB-50、Al 18 RadioHPLC spectrum of F-FAPI-CB-36.

[0316] Figure 4 shows the protein co-incubation - gel electrophoresis - radioautography - Coomassie blue staining after the obtained 177 The covalent attachment efficiency of Lu-FAPI-CB-30 to the FAP target protein. The left panel shows a control incubated at pH 7.4 for 1 hour, and the right panel shows a sample treated with strong acid after 1 hour of incubation.

[0317] Figure 5 shows the protein co-incubation - gel electrophoresis - radioautography - Coomassie blue staining after the obtained 177 The covalent bonding ratio of Lu-FAPI-CB-30 molecules to human FAP recombinant protein. The left image is an autoradiogram, and the right image shows the grayscale values ​​of the bands quantified using image quantification tools such as ImageJ, corresponding to the covalent bonding ratio.

[0318] Figure 6 shows the protein co-incubation - gel electrophoresis - radioautography - Coomassie blue staining after the obtained 177 The covalent bonding ratio of Lu-FAPI-CB series molecules to FAP human recombinant protein. The lanes in the figure from left to right are 177 Lu-labeled FAPI-CB-22, FAPI-CB-28, FAPI-CB-31, FAPI-CB-44, FAPI-CB-45, FAPI-CB-47, FAPI-CB-50.

[0319] FIG7 shows 68 Ga-FAPI-CB-30 and 68 Ga-FAPI-CB-02 and 68 PET / CT contrast imaging of Ga-FAPI-04 in the same HT-1080-FAP mouse model 90 minutes after injection. The left image shows the structure of FAPI-CB-02; the middle image shows 68 Ga-FAPI-CB-30 and 68 PET-CT images of Ga-FAPI-CB-02, SUV is the standard uptake value; the right image shows 68 Ga-FAPI-CB-30 and 68 PET-CT images of Ga-FAPI-04.

[0320] FIG8 shows 68 Ga-FAPI-CB-31 and 68 Comparison of tumor SUV values ​​of Ga-FAPI-04 in HT-1080-FAP tumor-bearing mouse model.

[0321] Figure 9 shows Al 18 F-FAPI-CB-36 and 68PET / CT contrast imaging of Ga-FAPI-04 in the HT-1080-FAP tumor-bearing mouse model.

[0322] FIG10 shows 68 Ga-FAPI-CB-50 and 68 PET / CT contrast imaging of Ga-FAPI-04 in the HT-1080-FAP tumor-bearing mouse model.

[0323] FIG11 shows 68 Ga-FAP-2286 and 68 PET / CT contrast imaging of Ga-FAPI-CB-45 in HT-1080-FAP tumor-bearing mouse model.

[0324] FIG12 shows 68 Ga-FAPI-CB-53 and 68 PET / CT contrast imaging of Ga-FAPI-04 in the HT-1080-FAP tumor-bearing mouse model.

[0325] FIG13 shows 68 PET / CT contrast imaging of Ga-FAPI-CB-59 at different time points in the HT-1080-FAP tumor-bearing mouse model.

[0326] FIG. 14 shows Al 18 F-FAPI-CB-77 and Al 18 PET / CT contrast imaging of F-FAPI-74 in the HT-1080-FAP tumor-bearing mouse model.

[0327] FIG15 shows 68 Ga-FAPI-CB-86 and 68 PET / CT contrast imaging of Ga-FAPI-04 in the HT-1080-FAP tumor-bearing mouse model.

[0328] FIG16 shows 68 Ga-FAPI-CB-93 and 68 PET / CT contrast imaging of Ga-FAPI-04 in the HT-1080-FAP tumor-bearing mouse model.

[0329] FIG. 17 shows 68 Ga-FAPI-CB-94 and 68 PET / CT contrast imaging of Ga-FAPI-04 in the HT-1080-FAP tumor-bearing mouse model.

[0330] FIG. 18 shows Al 18 F-FAPI-CB-95 and Al 18PET / CT contrast imaging of F-FAPI-74 in the HT-1080-FAP tumor-bearing mouse model.

[0331] FIG19 shows 68 Ga-FAPI-CB-30 and 68 Comparison of tumor SUV values ​​60 minutes after injection of Ga-FAPI-04 in the HT-1080-FAP mouse model.

[0332] FIG20 shows that the ratio of the standardized uptake value of tumor to blood pool of the compounds of the present invention FAPI-CB-28 and FAPI-CB-30 in the HT-1080-FAP tumor mouse model is significantly increased compared with the control (FAPI-04).

[0333] Figure 21 shows that there was no significant difference in blood pool uptake of the FAPI-CB-31 molecule compared to FAPI-04 at the 1 hour time point, but tumor uptake was higher.

[0334] FIG22 shows that the blood pool uptake of FAPI-CB-47 molecules decreased rapidly at the 0.5 and 2 hour time points, while the tumor uptake continued to increase and was statistically significantly higher than that of FAPI-04 tumor uptake.

[0335] FIG23 shows a diagram of a cancer patient's 68 Ga-FAPI-CB-50 PET / CT imaging.

[0336] FIG24 shows a diagram of a cancer patient's 177 Lu-FAPI-CB-30 SPECT / CT imaging.

[0337] Figure 25 shows the tumor model mice 177 Lu-FAPI-CB-86 SPECT / CT imaging.

[0338] Figure 26 shows the tumor model mice 177 Lu-FAPI-CB-30 treatment effect.

[0339] FIG27 shows that in renal fibrosis model mice, 68 Ga-FAPI-CB-30 and 68 PET / CT contrast imaging of Ga-FAPI-04. DETAILED DESCRIPTION

[0340] The present invention obtains a trifunctional compound by introducing a covalent warhead into a drug. The uptake and retention of the trifunctional compound at target sites, such as tumors, are greatly improved, while the uptake at non-target organs is low, thereby significantly reducing toxicity. The trifunctional compound of the present invention also has excellent pharmacokinetic properties.

[0341] Before further describing the present invention, the following sections collect certain terms used in the specification, examples, and appended claims. The definitions listed herein should be read and understood by those skilled in the art in light of the remainder of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

[0342] definition

[0343] Unless otherwise indicated, when any type of range is disclosed or claimed, it is intended to disclose or claim individually every possible value that the range may reasonably encompass, including any subranges encompassed therein. For example, a radical number of 1 to 6 indicates an integer within the range, where 1-6 is understood to include 1, 2, 3, 4, 5, 6, and also includes subranges of 1-5, 1-4, and 1-3.

[0344] The description of the present disclosure should be interpreted in accordance with the laws and principles of chemical bonding.In some cases, it may be possible to remove a hydrogen atom in order to accommodate a substituent at a given position.

[0345] As used in this disclosure, words such as "include," "comprising," or "including" mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unlisted elements. The terms "comprising" or "including" as used herein may be open, semi-closed, or closed. In other words, the term also includes "consisting essentially of" or "consisting of."

[0346] The term "pharmaceutically acceptable" as used herein means that the compound or composition is chemically and / or toxicologically compatible with the other ingredients constituting the formulation and / or with humans or mammals for the prevention or treatment of a disease or condition.

[0347] The term "subject" or "patient" as used herein includes humans and mammals. In some embodiments, a "patient" is an individual who has been diagnosed with, is suspected of having, or is at risk of having or developing a disease, wherein the disease is a disease described herein, preferably a disease involving FAP.

[0348] In the context of this application, the term "treatment" may also include prophylaxis, unless specifically stated to the contrary.

[0349] The term "alkyl" refers to a saturated straight or branched carbon chain. Preferably, the chain contains 1 to 10 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, preferably 1 to 6 carbon atoms, most preferably 1 to 3 carbon atoms. The alkyl group is, for example, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, amyl or octyl. The alkyl group is optionally substituted.

[0350] The term "heteroalkyl" refers to a saturated, linear or branched carbon chain. Preferably, the chain comprises 1 to 9 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 carbon atoms, preferably 1 to 6 carbon atoms, most preferably 1 to 3 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, amyl, octyl, which is interrupted once or more than once, for example 1, 2, 3, 4, 5 times, by identical or different heteroatoms. Preferably, the heteroatom is selected from O, S and N, for example, -O-CH3, -S-CH3, -CH2-O-CH3, -CH2-O-C2H5, -CH2-S-CH3, -CH2-S-C2H5, -C2H4-O-CH3, -C2H4-O-C2H5, -C2H4-S-CH3, -C2H4-S-C2H5, etc. The heteroalkyl group is optionally substituted.

[0351] Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl" by themselves or in combination with other terms refer to cyclic forms of "alkyl" and "heteroalkyl", respectively, preferably with 3, 4, 5, 6, 7, 8, 9 or 10 atoms forming the ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. The terms "cycloalkyl" and "heterocycloalkyl" are also intended to include bicyclic, tricyclic and polycyclic forms thereof. The term "heterocycloalkyl" preferably refers to a four-membered saturated ring wherein at least one ring member is a N atom; a five-membered saturated ring wherein at least one ring member is a N, O or S atom, and which optionally contains one additional O or one additional N; a six-membered saturated ring wherein at least one ring member is a N, O or S atom, and optionally contains one additional O or one additional N or two additional N atoms; or a nine- or ten-membered saturated bicyclic ring wherein at least one ring member is a N, O or S atom, and optionally contains one, two or three additional N atoms. "Cycloalkyl" and "heterocycloalkyl" are optionally substituted, wherein the meaning of optionally substituted also includes that a ring carbon atom on the ring is replaced by a carbonyl (C=O). Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, spiro[3,5]nonyl, spiro[5,3]nonyl, spiro[3,6]decyl, spiro[6,3]decyl, spiro[4,5]decyl, spiro[5,4]decyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, adamantyl, and the like. Examples of heterocycloalkyl groups include azetidinyl, 1-(1,2,5,6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, 1,4-diazabicyclo[2.2.2]octan-2-yl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

[0352] As used herein, the term "heterocyclyl" or "heterocycle" refers to a non-aromatic 5-, 6- or 7-membered ring or polycyclic group, including but not limited to a bicyclic or tricyclic group comprising a fused, spirocyclic or bridged three-, four-, five-, six- or seven-membered ring, wherein at least one carbon atom of one of the rings is replaced by a heteroatom. Each heteroatom is independently selected from an atom of oxygen, sulfur (including sulfoxides and sulfones) and / or nitrogen (which may be oxidized or quaternized). The term "heterocyclyl" or "heterocycle" is intended to include groups in which -CH2- in the ring is replaced by -C(=O)-, such as cyclic urea groups (e.g., 2-imidazolidinone) and cyclic amide groups (e.g., β-lactam, γ-lactam, δ-lactam, and ε-lactam) and piperazin-2-one; wherein (i) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms can be optionally oxidized, (iii) the nitrogen heteroatom can be selectively quaternized, and (iv) any of the above heterocycles can be fused to an aryl or heteroaryl ring, such as a benzene ring. Representative heterocycles include, but are not limited to, azetidinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuranyl.

[0353] The term "aryl" preferably refers to an aromatic monocyclic ring containing 6 carbon atoms, an aromatic bicyclic ring system containing 10 carbon atoms or an aromatic tricyclic ring system containing 14 carbon atoms. Examples are phenyl, naphthyl or anthracenyl. Aryl is optionally substituted.

[0354] The term "heteroaryl" preferably refers to a five- or six-membered aromatic monocyclic ring in which at least one carbon atom is replaced by 1, 2, 3 or 4 (for a five-membered ring) or 1, 2, 3, 4 or 5 (for a six-membered ring) identical or different heteroatoms, preferably selected from O, N and S; an aromatic bicyclic ring system in which 1, 2, 3, 4, 5 or 6 of the 8, 9, 10, 11 or 12 carbon atoms are replaced by identical or different heteroatoms, preferably selected from O, N and S; or an aromatic tricyclic ring system in which 1, 2, 3, 4, 5 or 6 of the 13, 14, 15 or 16 carbon atoms are replaced by identical or different heteroatoms, preferably selected from O, N and S. The N atom present in the heteroaryl group may be present in the form of an onium group, for example, an alkyl group or an oxygen group is attached to the trivalent aromatic N to form an onium group, i.e., "heteroaryl" in the context of the present application includes heteroaryl groups in free form (N atom not in salt form) and heteroaryl groups in the form of onium salts. Examples are pyridyl, N-oxypyridyl, N-alkylpyridyl, oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,3-oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, 1,2,5-thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1-phenylenediazole ... benzofuranyl, 2-benzofuranyl, indolyl, isoindolyl, benzothiophenyl, 2-benzothiophenyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoloxazinyl, 2,1-benzoxazolyl, benzothiazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, benzotriazolyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, quinolyl, 1,2,3-benzotriazinyl or 1,2,4-benzotriazinyl.

[0355] As used herein, the term "linker" refers to any chemically suitable linker. Preferably, the linker does not break or only breaks slowly under physiological conditions. When multiple substituents or multiple linking groups are described by writing their conventional chemical formulas from left to right, the multiple substituents or multiple linking groups also encompass chemically identical substituents generated by the structure written from right to left, for example: -CH2O- is equivalent to -OCH2-; -C(=O)O- is equivalent to -OC(=O)-; -OC(=O)NR- is equivalent to -NRC(=O)O-, etc.

[0356] When the term "independently selected" is used, the substituents referred to (eg, multiple R groups, such as groups R1, R2, etc., or variables such as "m" and "n") may be the same or different.

[0357] The term "protecting group" refers to chemical moieties that block some or all reactive moieties of a compound and prevent them from participating in chemical reactions until the protecting group is removed, such as those listed and described in TW Greene, PGM Wuts, Protecting Groups in Organic Synthesis, 3rd ed., John Wiley & Sons (1999). When different protecting groups are employed, it can be advantageous if each can be removed in a different manner. Protecting groups that cleave under distinct reaction conditions allow for differential removal of these protecting groups. For example, protecting groups can be removed by acid, base, and hydrogenolysis. For example, trityl, dimethoxytrityl, acetal, and tert-butyldimethylsilyl groups are acid-labile and can be used to protect carboxyl and hydroxyl reactive moieties in the presence of Cbz groups, which are removable by hydrogenolysis, and amino groups protected by Fmoc groups, which are base-labile. Carboxylic acid and hydroxy reactive moieties can be blocked by base-labile groups such as, but not limited to, methyl, ethyl, and acetyl groups in the presence of amines blocked by acid-labile groups such as t-butyl carbamate, or by acid- and base-stable but hydrolytically removable carbamates.

[0358] The expression "optionally substituted" means that one, two, three or more hydrogen atoms in the group may be replaced by respective substituents independently of one another. The substituents may be selected from C 1-6 Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, halogen, cyano, amino, nitro, -OH, -COOH.

[0359] As used herein, a "radionuclide" is a radioactive isotope of an element that emits alpha particles, beta particles, and / or gamma rays. Such radionuclides include, but are not limited to, the following: 18 F. 51 Cr, 67 Ga, 68 Ga, 89 Zr, 111 In, 99m Tc, 186 Re、 188 Re、 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 86 Y. 88 Y. 90 Y. 149 Pm, 161 Tb, 165 Dy, 169 Second, 177 Lu,47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As、 72 Se, 97 Such as 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, 188 Re、 186 Re、 198 Au, 225 Ac, 227 Th and 199 Ag.

[0360] The term "radiopharmaceutical" as used in the context of the present invention refers to a biologically active compound modified with a radioactive isotope or radionuclide.

[0361] The terms "chelator" or "chelate" are used interchangeably in the context of the present invention and refer to a molecule, typically an organic molecule, often a Lewis base, having two or more unshared electron pairs that can be donated to a metal ion. The metal ion is typically coordinated to the chelate via two or more electron pairs. The terms "bidentate chelator," "tridentate chelator," and "quadridentate chelator" refer to chelates having two, three, and four electron pairs, respectively, that are readily donated simultaneously to the metal ion coordinated by the chelate. Typically, the electron pairs of the chelate form coordination bonds with a single metal ion. However, in certain instances, a chelate can form coordination bonds with more than one metal ion, and a variety of binding patterns are possible. The term "chelate group" refers to a group formed by removing one or more hydrogen atoms from a "chelator" or "chelate."

[0362] The term "optical dye" refers to a compound that emits visible light or infrared light after being excited by electromagnetic radiation of a shorter and appropriate wavelength. It will be understood by those skilled in the art that each optical dye has a predetermined excitation wavelength.

[0363] The term "nuclear drug molecule" or "nuclear drug" refers to a molecule or compound that carries or chelates a radionuclide.

[0364] The structure of the term "targeted fibroblast activation protein-α" refers to a molecular fragment derived from a fibroblast activation protein inhibitor, such as a molecular fragment formed by a compound disclosed in WO2019154886A1.

[0365] The term "pharmaceutically acceptable salts" refers to relatively non-toxic addition salts of the compounds of the present disclosure. See, for example, SM Berge et al. "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19.

[0366] Suitable pharmaceutically acceptable salts of the disclosed compounds may be acid addition salts of the disclosed compounds having a sufficiently basic nature, for example, those carrying a nitrogen atom in a chain or ring, such as those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, or with organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethyl sulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, or thiocyanic acid.

[0367] In addition, another suitable pharmaceutically acceptable salt of the compound of the present invention having sufficient acidity is an alkali metal salt such as a sodium salt or a potassium salt, an alkaline earth metal salt such as a calcium salt or a magnesium salt, an ammonium salt, or a salt formed with an organic base which provides a physiologically acceptable cation, for example, a salt formed with the following substances: N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1,6-hexanediamine, ethanolamine, glucosamine, sarcosine, serinol, trishydroxymethylaminomethane, aminopropylene glycol, 1-amino-2,3,4-butanetriol. In addition, basic nitrogen-containing groups can be quaternized using reagents such as lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates, such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate and diamyl sulfate; long chain halides, such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides, such as benzyl and phenethyl bromides, etc.

[0368] Those skilled in the art will also recognize that acid addition salts of the claimed compounds can be prepared by reacting the compounds with a suitable inorganic or organic acid by any of a variety of known methods. Alternatively, alkali metal and alkaline earth metal salts of the acidic compounds of the present disclosure can be prepared by reacting them with a suitable base by various known methods.

[0369] The present invention includes all possible salts of the disclosed compounds, either as a single salt or as any mixture of such salts in any ratio.

[0370] The term "solvate" is a substance formed by combining, physically combining and / or solvating the compounds of the present invention with solvent molecules, such as a disolvate, a monosolvate or a hemisolvate, wherein the ratio of solvent molecules to the ratio of the compounds of the present invention is about 2:1, about 1:1 or about 1:2, respectively. This physical combination involves ionization and covalent bonding (including hydrogen bonding) to varying degrees. In some cases (for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid), the solvate can be separated. Therefore, solvates include solution phases and separable solvates. The compounds of the present invention can be in solvated form with pharmaceutically acceptable solvents (such as water, methanol and ethanol), and the application is intended to cover solvated and non-solvated forms of the compounds of the present invention. A solvate is a hydrate.

[0371] The compounds of the present disclosure may contain one or more asymmetric centers, depending on the position and properties of the various substituents desired. Asymmetric carbon atoms can exist in the (R) or (S) configuration, resulting in racemic mixtures in the case of one asymmetric center and diastereomeric mixtures in the case of multiple asymmetric centers. In some cases, asymmetry may also exist due to hindered rotation about a particular bond, such as where the central bond connects two substituted aromatic rings of a particular compound.

[0372] Preferred compounds are those that produce more desirable biological activity. Isolation, purification or partial purification of isomers and stereoisomers, or racemic mixtures or diastereomeric mixtures of the disclosed compounds are included within the scope of the present invention. Purification and separation of such substances can be achieved by standard techniques known in the art.

[0373] As used herein, the term "pharmaceutical composition" refers to a substance and / or combination of substances used to identify, prevent, or treat a tissue condition or disease. A pharmaceutical composition is formulated to be suitable for administration to a patient to diagnose, prevent, and / or treat a disease. Additionally, a pharmaceutical composition refers to a combination of an active agent and an inert or active carrier that renders the composition suitable for therapeutic use.

[0374] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more specifically in humans.

[0375] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions, as well as aqueous dextrose and glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, and the like. If desired, the composition may also contain a small amount of a wetting agent or emulsifier or a pH buffer. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0376] The term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0377] The term "optionally" means that the situation may or may not occur.

[0378] The term "not directly connected via a covalent bond" means that there is at least one carbon atom between the two, and the carbon atom may be in the form of C, CH, CH2 or C=O.

[0379] As used herein, a disease involving FAP is a disease in which cells (including but not limited to fibroblasts) expressing (preferably in an upregulated manner) FAP and tissues expressing FAP or containing or comprising cells (e.g., fibroblasts) expressing FAP, preferably in an upregulated manner, are the cause of the disease and / or symptoms of the disease, respectively, or are part of the underlying pathology of the disease. Preferred FAP-expressing cells are cancer-associated fibroblasts (CAFs). In embodiments of the disease, preferably when used in conjunction with the treatment, management, and / or therapy of the disease, the cells, tissues, and pathology, respectively, are affected, resulting in a cure, treatment, or amelioration of the disease and / or symptoms of the disease. In embodiments of the disease, preferably when used in conjunction with the diagnosis and / or performing a diagnosis of the disease, labeling of FAP-expressing cells and / or FAP-expressing tissues allows for the differentiation or distinction of the cells and / or tissues from healthy or non-FAP-expressing cells and / or healthy or non-FAP-expressing tissues. More preferably, this differentiation or distinction forms the basis of the diagnosis and performing a diagnosis, respectively. In an embodiment thereof, labeling refers to the direct or indirect interaction of a detectable label with a FAP-expressing cell and / or a FAP-expressing tissue or a tissue containing such a FAP-expressing cell; more preferably, such interaction involves or is based on the interaction of the label or a compound carrying such a label with FAP.

[0380] As used herein, a tumor is an abnormal new growth of cells. The cells in a tumor grow faster than normal cells and will continue to grow if not treated. Tumors can be benign or malignant.

[0381] As used herein, a tumor is a mass of disease that can be benign or malignant.

[0382] As used herein, cancer is a malignant tumor.

[0383] The amino acid sequences of peptides as provided herein are described in a typical peptide sequence format. For example, the three letter codes for conventional amino acids, or the codes for unconventional amino acids, or the abbreviations for other components represent that the amino acid or component is present at a specific position in the peptide sequence. The code for each amino acid or component is connected to the code for the next and / or previous amino acid or component in the sequence by a hyphen (usually representing an amide bond).

[0384] In case an amino acid contains more than one amino and / or carboxyl group, all orientations of this amino acid are in principle possible, but in α-amino acids, preference is given to utilizing the α-amino and α-carboxyl groups, other preferred orientations being explicitly indicated.

[0385] For amino acids, in their abbreviations, the first letter indicates the stereochemistry of the C-α-atom (if applicable). For example, an uppercase first letter indicates the presence of the L-form of the amino acid in the peptide sequence, while a lowercase first letter indicates the presence of the D-form of the corresponding amino acid in the peptide sequence.

[0386] As used herein, an aromatic L-α-amino acid is any type of L-α-amino acid that contains an aryl group.

[0387] As used herein, a heteroaromatic L-α-amino acid is any species of L-α-amino acid that contains a heteroaryl group.

[0388] Unless indicated to the contrary, amino acid sequences are presented herein in N-terminal to C-terminal direction.

[0389] The amino acids of the present invention may be conventional amino acids (also called natural amino acids), or non-natural amino acids (any type of non-oligomeric compound containing an amino group and a carboxyl group and which is not a conventional amino acid).

[0390] The radionuclide of the present invention has a half-life that allows for diagnostic and / or therapeutic medical use. Specifically, the half-life is from 1 minute to 100 days.

[0391] In preferred embodiments of the present invention, the radionuclide has a decay energy that allows for diagnostic and / or therapeutic medical use. Specifically, for gamma-emitting isotopes, for diagnostic use, the decay energy is 0.004 to 10 MeV, preferably 0.05 to 4 MeV. For positron-emitting isotopes, for diagnostic use, the decay energy is 0.6 to 13.2 MeV, preferably 1 to 6 MeV. For particle-emitting isotopes, for therapeutic use, the decay energy is 0.039 to 10 MeV, preferably 0.4 to 6.5 MeV.

[0392] In a preferred embodiment of the present invention, the radionuclide is industrially produced for medical use. In particular, the radionuclide is of GMP quality.

[0393] In a preferred embodiment of the present invention, the daughter nuclides after radioactive decay of the radionuclide are compatible with diagnostic and / or therapeutic medical uses. In addition, the daughter nuclides are stable or further decay in a manner that does not interfere with or even supports diagnostic and / or therapeutic medical uses.

[0394] Example

[0395] Reagents and models used

[0396] The starting materials of the embodiments are commercially available and / or can be prepared by a variety of methods known to those skilled in the art of organic synthesis. Those skilled in the art of organic synthesis will appropriately select reaction conditions (including solvent, reaction atmosphere, reaction temperature, duration of experiment and aftertreatment) in the following synthetic methods. Those skilled in the art of organic synthesis will appreciate that the functional groups present in each part of the molecule should be compatible with the proposed reagents and reactions.

[0397] All reagents and compounds used in the synthesis were purchased from general commercial sources in China (excluding Hong Kong, Macao, and Taiwan) from suppliers including Shanghai Bid Pharmaceutical Technology Co., Ltd., J&K (Beijing, China), Inno-chem (Beijing, China), and Energy Chemical (Shanghai, China). Unless otherwise stated, they were not further purified. SO₂F₂ gas was purchased from Shanghai Fluoro Corporation (Shanghai, China). All solvents used in the synthesis were purchased from Tongguang (Beijing, China), and HPLC-grade solvents were purchased from Fisher Scientific (Loughborough, UK).

[0398] Nuclide: 68 GaCl3 is obtained by eluting with 0.6M hydrochloric acid 68 Ge- 68 Ga generator (iThemba LABS, South Africa). 177 LuCl3 was dissolved in 0.1 M hydrochloric acid and purchased from ITG (Germany).

[0399] Recombinant protein: Human recombinant protein FAP-His tag was purchased from Jinan Protein Co., Ltd. (China).

[0400] Cell model: The FAP-overexpressing human fibrosarcoma cell line (HT-1080-FAP) was constructed by WuXi Biologics (China) by transfecting the FAP plasmid into the human fibrosarcoma cell line (HT-1080).

[0401] Mouse model: Nu / Nu mice (SPF grade) were purchased from Beijing Weitonglihua Company (China) and injected subcutaneously with HT-1080-FAP cells to construct a FAP-overexpressing tumor model.

[0402] Instruments used

[0403] Compound preparation and identification: high performance liquid chromatography (Waters), high performance liquid chromatography radioactivity detector (Eckert & Ziegler Group), ultra-high performance liquid chromatography-mass spectrometry (Waters), high-resolution mass spectrometer (Orbitrap Fusion Lumos or Bruker Solarix XR), nuclear magnetic resonance (NMR) spectrometer (Bruker 400 / 500 / 600 MHz), Amersham Typhoon imaging system (Amersham Typhoon RGB).

[0404] Animal experiments: Small animal PET / CT or SPECT / CT (Mediso PET122S or Yongxin Medical lnliView-3000B).

[0405] Example 1 Synthesis of Compounds

[0406] The compound FAPI-CB-30 of the present invention can be synthesized through the above synthesis route; some FAP-targeting compounds in the patent can also be synthesized through similar synthesis routes.

[0407] Example 1a. Synthesis of FAPI-CB-30

[0408] The initial intermediate int 1a was prepared using the procedure described in Xu, M. et al., Albumin Binder–Conjugated Fibroblast Activation Protein Inhibitor Radiopharmaceuticals for Cancer Therapy (J. Nucl. Med. 2021, jnumed. 121.262533), replacing the protected lysine used in the reference with protected (S)-2,3-diaminopropionic acid. To a solution of int 1a (450 mg, 0.5 mmol, 1.0 eq) in acetonitrile (10.0 ml) was added NHEt2 (973.0 mg, 13.34 mmol, 1.05 ml, 26.7 eq), and the reaction was stirred at 25°C for 30 minutes. The reaction solution was concentrated under reduced pressure to give a crude product, which was purified by HPLC [eluent A: H2O (0.1% trifluoroacetic acid); eluent B: acetonitrile, from 10% B (at 2 minutes) to 95% B (at 20 minutes)] to give int 2 (268.8 mg, 80%) as a white solid.

[0409] To a solution of int 2a (336 mg, 0.5 mmol, 1.0 eq) in DMF (5.0 mL) were added DOTA-Tris(tBu) (0.5 g, 0.9 mmol, 1.8 eq), HATU (365 mg, 1.0 mmol, 2.0 eq) and DIEA (78 mg, 0.6 mmol, 105 μL, 1.2 eq) and the reaction was stirred at 25° C. for 12 h. The reaction was poured into methyl tert-butyl ether (200 mL) and stirred for 30 min to give a precipitate.

[0410] The precipitate was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (3 mL) was added to the solution, and the reaction was carried out at 20° C. for 30 minutes. The crude product was purified by HPLC [eluent A: H 2 O (0.1% trifluoroacetic acid); eluent B: ACN, from 20% B (at 2 minutes) to 95% B (at 20 minutes)] to give int 3a (0.366 g, 65% yield) as a white solid.

[0411] Int 3a (11.3 mg, 10 μmol, 1.0 eq) was dissolved in DMF (200 μL), and commercially available 1a (1.7 mg, 12.2 μmol, 1.22 eq), DIPEA (3.9 mg, 30 μmol, 5.3 μL, 3.0 eq), and HBTU (9.9 mg, 12.0 μmol, 1.2 eq) were added. The reaction mixture was stirred at 25°C for 30 minutes. Intermediate int 4a (10.32 mg, 80%) was subsequently isolated by HPLC and dissolved in acetonitrile (200 μL). The atmosphere in the container was replaced with SO₂F₂, and 4.0 eq of triethylamine was added and the reaction was allowed to react for 12 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent, and then 1.0 mL of TFA was added. The mixture was reacted at 25°C for 3 hours. The crude product was purified by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (2nd minute) to 60% B (20th minute)] to obtain FAPI-CB-30 (7.87 mg, 82%) as a white solid. UPLC-MS characterization conditions: separation column, ACQUITY UPLC BEH C 18 1.7 μm, 2.1×50 mm. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A. Calculated m / z is [M+H] + : 1162, retention time: 1.56 minutes.

[0412] Example 1b. Synthesis of FAPI-CB-31

[0413] Compound int 1b was prepared using the procedure described in Xu, M. et al., Albumin Binder–Conjugated Fibroblast Activation Protein Inhibitor Radiopharmaceuticals for Cancer Therapy (J. Nucl. Med. 2021, jnumed. 121.262533). To a solution of int 1b (600 mg, 641.0 μmol, 1.0 eq) in acetonitrile (5.0 mL) was added NHEt2 (1249.0 mg, 17.1 mmol, 1.343 mL, 26.7 eq), and the reaction was stirred at 25°C for 30 minutes. The reaction solution was concentrated under reduced pressure to give a crude product, which was purified by HPLC [eluent A: H2O (0.1% trifluoroacetic acid); eluent B: acetonitrile, from 10% B (at 2 min) to 95% B (at 20 min)] to give int 2b (370.7 mg, 81% yield) as a white solid.

[0414] To a solution of int 2b (357 mg, 0.5 mmol, 1.0 eq) in DMF (5.0 mL) were added DOTA-Tris(tBu) (0.5 g, 0.9 mmol, 1.8 eq), HATU (365 mg, 1.0 mmol, 2.0 eq) and DIEA (78 mg, 0.6 mmol, 105 μL, 1.2 eq) and the reaction was stirred at 25° C. for 12 h. The reaction was poured into methyl tert-butyl ether (200 mL) and stirred for 30 min to give a precipitate.

[0415] The precipitate was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (3 mL) was added to the solution, and the reaction was carried out at 20° C. for 30 minutes. The crude product was purified by HPLC [eluent A: H 2 O (0.1% trifluoroacetic acid); eluent B: ACN, from 20% B (at 2 minutes) to 95% B (at 20 minutes)] to give int 3b (0.362 g, 62% yield) as a white solid.

[0416] Int 3b (11.7 mg, 10.0 μmol, 1.0 eq) was dissolved in DMF (200 μL), and 1b (3.9 mg, 12.0 μmol, 1.2 eq), which was readily obtained by oxidation of commercially available 1a with 35% hydrogen peroxide, DIPEA (3.9 mg, 30.0 μmol, 5.6 μL, 3.0 eq), and HBTU (4.6 mg, 12.0 μmol, 1.2 eq) were added to the reaction mixture and stirred at 25°C for 30 minutes. The intermediate int 4b (8.8 mg, 67%) was subsequently isolated by HPLC and dissolved in acetonitrile (200 μL). The atmosphere in the container was replaced with SO2F2 gas, and 4.0 eq of triethylamine was added and the reaction mixture was allowed to react for 12 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent, followed by the addition of 1.0 mL of TFA and reaction at 25°C for 3 hours. The crude product was purified by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (at 2nd minute) to 60% B (at 20th minute)] to yield FAPI-CB-31 (6.5 mg) as a white solid. UPLC-MS characterization conditions: ACQUITY UPLC BEH C18 1.7 μm 2.1×50 mm column. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A. Calculated m / z is [M+H] + : 1220, retention time: 1.32 minutes.

[0417] Example 1c. Synthesis of FAPI-CB-44

[0418] Compound int 1c was prepared using the steps described in Millul, J. et al., An ultra-high-affinity small organic ligand of fibroblast activation protein for tumor-targeting applications (PNAS 2021, 118(16)e2101852118).

[0419] To a solution of int 1c (501 mg, 1.0 mmol, 1.0 eq) in DMF (10.0 mL) were added commercially available 3A (468 mg, 1.0 mmol, 1.0 eq), DIPEA (390 mg, 3.0 mmol, 527 μL, 3.0 eq) and HATU (456.8 mg, 1.2 mmol, 1.2 eq). The reaction mixture was stirred at 20° C. for 60 min. UPLC-MS revealed the detection of a major peak with the desired mass ([M+H] + =952). The reaction mixture was diluted with 20.0 mL of H2O and extracted with ethyl acetate (20.0 mL / cycle, 3 cycles). The combined organic layers were washed with 20.0 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The crude product was used in the next step without further purification. Crude int 2c (813 mg) was obtained as a yellow solid.

[0420] To a solution of int 2c (1.0 mmol, 1.0 eq) in DMF (10.0 mL) was added NHEt2 (1950.2 mg, 26.7 mmol, 2.097 mL, 26.7 eq) and the reaction was stirred at 20°C for 60 min. The reaction was concentrated under reduced pressure to give a crude product, which was purified by preparative HPLC [eluent A: H2O (0.1% trifluoroacetic acid); eluent B: acetonitrile, from 10% B (at 2 min) to 95% B (at 20 min)] to give int 3c (511.7 mg, 70.2% yield) as a white solid.

[0421] To a solution of int 3c (364 mg, 0.5 mmol, 1.0 eq) in DMF (5.0 mL) were added DOTA-Tris(tBu) (0.5 g, 0.9 mmol, 1.8 eq), HATU (365 mg, 1.0 mmol, 2.0 eq) and DIEA (78 mg, 0.6 mmol, 105 μL, 1.2 eq) and the reaction was stirred at 25° C. for 12 h. The reaction was poured into methyl tert-butyl ether (200 mL) and stirred for 30 min to give a precipitate.

[0422] The precipitate was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (3 mL) was added to the solution, and the reaction was carried out at 20° C. for 30 minutes. The crude product was purified by HPLC [eluent A: H 2 O (0.1% trifluoroacetic acid); eluent B: ACN, from 20% B (at 2 minutes) to 95% B (at 20 minutes)] to give int 4c (0.385 g, 65%) as a white solid.

[0423] Int 4c (11.8 mg, 10.0 μmol, 1.0 eq) was dissolved in DMF (200 μL), and commercially available 1a (3.9 mg, 12.0 μmol, 1.2 eq), DIPEA (3.9 mg, 30.0 μmol, 5.6 μL, 3.0 eq) and HBTU (4.6 mg, 12.0 μmol, 1.2 eq) were added to the reaction mixture and stirred at 25°C for 30 minutes. The intermediate int 5c (9.7 mg, 75%) was subsequently isolated by HPLC and dissolved in acetonitrile (500 μL). The gas in the container was replaced with SO2F2 gas, and 4.0 equivalents of triethylamine were added and the reaction was allowed to react for 12 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent, followed by the addition of 1.0 mL of TFA and reaction at 25°C for 3 hours. The crude product was purified by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (at 2nd minute) to 60% B (at 20th minute)] to yield FAPI-CB-44 (7.4 mg, 81%) as a white solid. UPLC-MS characterization conditions: separation column, ACQUITY UPLC BEH C18 1.7 μm, 2.1×50 mm. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A. Calculated m / z is [M+H] + : 1219, retention time: 1.43 minutes.

[0424] Example 1d. Synthesis of FAPI-CB-45

[0425] Compound int 1d was prepared by solid phase synthesis with reference to the steps described in patent WO 2021 / 005125 A1 applied by 3B PHARMACEUTICALS GMBH.

[0426] To a mixture of compound int 1d (200 mg, 174.53 mmol, 1.0 eq) and DOTA-NHS (131.3 mg, 261.80 mmol, 1.5 eq) in DMF (5 ml) was added DIEA (112.8 mg, 872.7 mmol, 152.0 μl, 5.0 eq) in one portion at 25° C., and the mixture was stirred at 25° C. for 0.5 hours. The main peak [M+H] was confirmed by LCMS. += 1486.6. The mixture was purified by preparative HPLC (TFA conditions: A: 0.075% TFA in H2O, B: ACN) to give int 2d (143.3 mg, 119.22 umol, 51% yield).

[0427] Int 2d (14.9 mg, 10.0 μmol) was dissolved in water (500 μL) and DCM (500 μL) was added. The atmosphere was then replaced with SO₂F₂, and 4.0 equivalents of triethylamine were added. The reaction mixture was allowed to react for 6 h. The solvent was removed by concentration under reduced pressure, and the crude product was purified by HPLC (eluent A: H₂O (0.1% TFA); eluent B: ACN, from 10% B (at 2 min) to 60% B (at 20 min)) to yield FAPI-CB-45 (13.5 mg, 86%) as a white solid. UPLC-MS characterization conditions: ACQUITY UPLC BEH C₁₈ column, 1.7 μm, 2.1×50 mm. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A. Calculated m / z is [M+H] + : 1569, retention time: 1.71 minutes.

[0428] Example 1e. Synthesis of FAPI-CB-22

[0429] Compound FAPI-CB-22 was obtained from the readily available intermediate int 2e via the method described in Example 1a (10.5 mg, 27% yield over multiple steps) and purified by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (at 2 minutes) to 60% B (at 20 minutes)]. UPLC-MS characterization conditions: separation column, ACQUITY UPLC BEH C18 1.7 μm, 2.1×50 mm. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A. Calculated m / z is [M+H] + : 1204, retention time: 1.65 minutes.

[0430] Example 1f. Synthesis of FAPI-CB-28

[0431] Compound FAPI-CB-28 was obtained from the readily available intermediate int 2f via the method described in Example 1a (9.7 mg, 30% yield over multiple steps) and purified by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (at 2 minutes) to 60% B (at 20 minutes)]. UPLC-MS characterization conditions: separation column, ACQUITY UPLC BEH C18 1.7 μm, 2.1×50 mm. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A. Calculated m / z is [M+H] + : 1191, retention time: 1.62 minutes.

[0432] Example 1g. Synthesis of FAPI-CB-47

[0433] Compound FAPI-CB-47 was obtained by the method of Example 1a from the readily available intermediates int 2e and int 2g (8.0 mg, multi-step yield 21%). Int 2g was prepared according to the method of Taijie Guo et al. (A New Portal to SuFEx Click Chemistry: A Stable Fluorosulfuryl Imidazolium Salt Emerging as an "F-SO2 + "The method is easily available in Donor of Unprecedented Reactivity, Selectivity, and Scope and Profiling Sulfur(VI)Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome (ACS Chem. Biol. 2023, 18, 2, 285) published by Katharine E. Gilbert et al.

[0434] Purification by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (2nd minute) to 60% B (20th minute)]. UPLC-MS characterization conditions: Separation column, ACQUITY UPLC BEH C18 1.7μm 2.1×50mm. Method, flow rate 0.5mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 minutes, 95% A; 0.2-3.0 minutes, 95% to 5% A, curve 6; 3.0-4.0 minutes, 5% A; 4.0-4.5 minutes, 5% to 95% A, curve 6; 4.5-5.0 minutes, 95% A. Calculated m / z is [M+H] + : 1177, retention time: 1.54 minutes.

[0435] Example 1h. Synthesis of FAPI-CB-50

[0436] Compound FAPI-CB-50 was obtained by the method of Example 1a from the readily available intermediates int 2e and int 2h. Int 2h was prepared by referring to A New Portal to SuFEx Click Chemistry: A Stable Fluorosulfuryl Imidazolium Salt Emerging as an "F-SO2 + "Donor of Unprecedented Reactivity, Selectivity, and Scope" was readily obtained (8.5 mg, 24% yield over multiple steps).

[0437] Purification by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (2nd minute) to 60% B (20th minute)]. UPLC-MS characterization conditions: Separation column, ACQUITY UPLC BEH C18 1.7 μm 2.1×50 mm. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A. Calculated m / z is [M+H] + : 1167, retention time: 1.43 minutes.

[0438] Example 1i. Synthesis of FAPI-CB-36

[0439] Compound FAPI-CB-36 was readily obtained from the readily available intermediate int2i by a similar method as in Example 1a (6.2 mg, multi-step yield 20%).

[0440] Purification by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (2nd minute) to 60% B (20th minute)]. UPLC-MS characterization conditions: Separation column, ACQUITY UPLC BEH C18 1.7μm 2.1×50mm. Method, flow rate 0.5mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 minutes, 95% A; 0.2-3.0 minutes, 95% to 5% A, curve 6; 3.0-4.0 minutes, 5% A; 4.0-4.5 minutes, 5% to 95% A, curve 6; 4.5-5.0 minutes, 95% A. Calculated m / z is [M+H] + : 1061, retention time: 1.67 minutes.

[0441] Example 1j. Synthesis of FAPI-CB-53

[0442] Compound int 1j (5.00 g, 17.8 mmol, 1.00 eq) was dissolved in H2O (65.0 mL) and the pH was adjusted to 9-10 with NaOH (4 mol / L, 50 mL, 11.2 eq). The mixture was heated to 65°C in an oil bath. Sodium nitroprusside (9.57 g, 32.1 mmol, 5.56 mL, 1.80 eq) was added portionwise over 1 hour while maintaining the pH of the reaction mixture between 9-10 with NaOH (4.00 mol / L). The resulting mixture was heated for an additional 3 hours while occasionally adding aqueous NaOH (4.00 mol / L) to maintain the pH between 9-10. The mixture was filtered and extracted three times with ethyl acetate (250 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by preparative HPLC (0.1% TFA condition) to afford compound int 2j (2.90 g, 10.3 mmol, 57.8% yield) as a yellow oil.

[0443] Compound int 2j (10.3 mmol, 1.00 eq) was dissolved in ACN, K2CO3 (30.9 mmol, 3.00 eq) and BnBr (15.4 mmol, 1.50 eq) were added, and the reaction mixture was stirred at 25°C for 12 hours. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography to obtain compound int 3j (2.10 g, 5.65 mmol, 54.8% yield).

[0444] DIAD (1.43 g, 7.07 mmol, 1.37 ml, 1.25 eq) was dissolved in THF (25.0 ml), and PPh3 (1.85 g, 7.07 mmol, 1.25 eq) was added at 0°C. The mixture was stirred for 1 hour, and then compound 3j (2.10 g, 5.65 mmol, 1.00 eq) and AcSH (537 mg, 7.07 mmol, 505 μl, 1.25 eq) were added at 0°C. The mixture was stirred at 20°C for 11 hours. The mixture was concentrated under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1 to 0:1, petroleum ether:ethyl acetate = 5:1, Rf = 0.15). Compound int 4j (2.00 g, 4.66 mmol, 82.3% yield) was obtained as a colorless oil.

[0445] Compound int 4j (1.50 g, 3.49 mmol, 1.00 eq) was dissolved in glacial acetic acid (50.0 mL) and water (5.0 mL), and NCS (1.40 g, 10.4 mmol, 3.00 eq) was added. The reaction mixture was stirred at 25°C for 1 hour. The reaction mixture was poured into water (50 mL) and extracted with dichloromethane (100 mL). The organic layer was washed three times with water (100 mL) and brine (100 mL), dried over Na2SO4, and concentrated to give compound int 5j (1.50 g, crude) as a colorless oil.

[0446] Compound Int 5j (1.50 g, 3.30 mmol, 1.00 eq) was dissolved in acetonitrile (42.0 mL) and water (21.0 mL), and KHF2 (1.03 g, 13.2 mmol, 435 μL, 4.00 eq) was added. The mixture was stirred at 25°C for 12 hours. The mixture was extracted three times with ethyl acetate (150 mL). The combined organic layers were concentrated to a residue. The crude product was purified by preparative HPLC. Compound Int 6j (1.10 g, 2.51 mmol, 76.0% yield) was obtained as a colorless oil.

[0447] Compound Int 6j (1.10 g, 2.51 mmol, 1.00 eq) was dissolved in THF (20.0 mL) and Pd / C (220 mg, 206 μmol, 10% purity, 0.2 eq) was added under a nitrogen atmosphere. The suspension was degassed and purged with H₂ three times. The mixture was stirred at 25°C under H₂ (15 psi) for 12 hours. The reaction mixture was filtered and concentrated to afford Compound Int 7j (1.10 g, 2.51 mmol, 76.0% yield) as a white solid.

[0448] Compound int 7j (100 mg, 468 μmol, 1.00 eq) was dissolved in THF (2.0 ml) and water (2.0 ml), and NaHCO 3 (39.4 mg, 468 μmol, 18.2 μL, 1.00 eq) and FmocOSu (158 mg, 468 μmol, 1.00 eq) were added. The mixture was stirred at 25° C. for 1 hour. The pH of the mixture was adjusted to 5 and extracted with ethyl acetate (100 ml). The organic layer was concentrated. The crude product was purified by preparative HPLC. Compound int 8j (120 mg, 275 μmol, 58.7% yield) was obtained as a white solid.

[0449] To a solution of compound 9a (130 mg, 221.61 μmol, 1.00 eq) and acetonitrile (10.0 mL) was added p-toluenesulfonic acid (133 mg, 775 μmol, 3.50 eq). The mixture was stirred at 40°C for 1 hour. The mixture was concentrated under vacuum to give compound 9b (100 mg, crude product) as a yellow solid.

[0450] To a stirred solution containing compound 9b (95.5 mg, 196 μmol, 0.95 eq) and DMF (5.0 ml), compound int 8j (90.0 mg, 206 μmol, 1.00 eq), DIEA (53.4 mg, 413 μmol, 72.0 μL, 2.00 eq), HOBt (41.8 mg, 310 μmol, 1.50 eq) and EDCI (41.6 mg, 217 μmol, 1.05 eq) were added sequentially, and the reaction mixture was stirred at 25° C. for 12 hours. The reaction mixture was concentrated under vacuum, and the crude product was purified by preparative high-performance liquid chromatography (0.1% TFA conditions) to give compound int 9j (40.0 mg, 44.2 μmol, 21.4% yield) as a white solid.

[0451] To a solution containing compound int 9j (40.0 mg, 44.2 μmol, 1.00 eq) and acetonitrile (16.0 ml) was added diethylamine (2.84 g, 38.8 mmol, 4.00 ml, 877 eq), and the mixture was stirred at 25°C for 1 hour. The mixture was adjusted to pH 5 with citric acid (0.5 ml) and extracted with ethyl acetate (10.0 ml). The aqueous layer was freeze-dried to give a crude product. The crude product was purified by preparative high performance liquid chromatography (0.1% TFA conditions) to give compound int 10j (10.0 mg, 14.6 μmol, 33.1% yield) as a colorless oil.

[0452] To a solution of compound int 10j (10.0 mg, 14.6 μmol, 1.00 equiv) and DOTA-OSu (10.0 mg, 19.9 μmol, 1.36 equiv) in acetonitrile (1.0 mL) was added DIEA (4.74 mg, 36.6 μmol, 6.39 μL, 2.50 equiv), and the mixture was stirred at 25°C for 3 hours. The mixture was concentrated under a nitrogen atmosphere, and the crude product was purified by preparative HPLC (0.1% NH4HCO3) to afford FAPI-FS-53 (6.0 mg, 5.09 μmol, 34.7% yield, 98.6% purity) as a white solid. [Eluent A: H2O (0.1% TFA); Eluent B: ACN, from 10% B (2nd minute) to 60% B (20th minute)]. UPLC-MS characterization conditions: Separation column, ACQUITY UPLC BEH C18 1.7 μm 2.1 × 50 mm. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A. Calculated m / z is [M+H] + : 1069, retention time: 1.53 minutes.

[0453] Example 1k. Synthesis of FAPI-CB-59

[0454] Compound Int 1k (5.30 g, 14.5 mmol, 1.00 eq) and Compound 1a (1.94 g, 16.0 mmol, 1.10 eq) were dissolved in DMF (50.0 mL) and KCO (2.01 g, 14.5 mmol, 1.00 eq) was added. The mixture was stirred at 25°C for 1 hour. The reaction was diluted with ethyl acetate (200 mL). The combined organic layers were washed three times with water (100 mL), dried over NaSO, filtered, and concentrated under reduced pressure to afford Int 2k (5.80 g, 14.3 mmol, 98.0% yield, 99.4% purity) as a yellow oil.

[0455] Compound int 2k (5.80 g, 14.3 mmol, 1.00 eq) was dissolved in DCM (10.0 mL) and TFA (35.4 g, 310 mmol, 23.1 mL, 21.8 eq) was added. The mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to a residue. The residue was diluted with DCM (100 mL), and the organic layer was adjusted to pH 7 with saturated aqueous sodium bicarbonate solution, dried over Na2SO4, filtered, and concentrated under reduced pressure to afford compound int 3k (4.00 g, 13.1 mmol, 92.2% yield) as a yellow oil.

[0456] Compound int 4k (4.00 g, 21.2 mmol, 1.00 equiv) and BnBr (3.62 g, 21.2 mmol, 2.51 mL, 1.00 equiv) were dissolved in DMF (200 mL), and KHCO₃ (3.18 g, 31.7 mmol, 1.50 equiv) was added. The mixture was stirred at 40°C for 1 hour. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The filtrate was diluted with ethyl acetate (500 mL), and the organic phase was washed three times with water (200 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain compound int 5k (4.60 g, 15.8 mmol, 74.8% yield, 96.0% purity) as a brown solid.

[0457] Compound int 5k (4.60 g, 15.8 mmol, 1.00 eq) and compound 5a (9.58 g, 47.4 mmol, 4.84 mL, 3.00 eq) were dissolved in DMF (50.0 mL) and Cs2CO3 (10.3 g, 31.6 mmol, 2.00 eq) was added. The mixture was stirred at 60°C for 1 hour. The reaction mixture was filtered and the filtrate was diluted with ethyl acetate (200 mL). The organic layer was washed three times with water (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC to obtain compound int 6k (4.00 g, 9.68 mmol, 61.2% yield, 96.9% purity) as a red oil.

[0458] Compound int 6k (1.14 g, 3.75 mmol, 1.50 eq) was dissolved in ACN (50.0 mL), and K2CO3 (691 mg, 5.00 mmol, 2.00 eq) and KI (207 mg, 1.25 mmol, 0.50 eq) were added. The mixture was stirred at 70°C for 48 hours. The two reaction mixtures were combined and processed. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC to obtain compound int 7k (2.00 g, 3.08 mmol, 61.7% yield, 96.1% purity) as a red oil.

[0459] Compound int 7k (1.80 g, 2.77 mmol, 1.00 eq) was dissolved in DMF (2.00 ml), and Pd(PPh3)4 (480 mg, 415 μmol, 0.15 eq) and N-methylaniline (594 mg, 5.54 mmol, 602 μL, 2.00 eq) were added. Under N2 protection, the mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC to obtain compound int 8k (1.00 g, 1.60 mmol, 57.70% yield, 93.3% purity) as a yellow solid.

[0460] Compound int 8k (750 mg, 1.20 mmol, 1.00 eq) and compound 8a (240.16 mg, 1.20 mmol, 1 eq) were dissolved in DCM (20.0 ml), and pyridine (474 ​​mg, 6.00 mmol, 484 μL, 5.00 eq) and POCl3 (221 mg, 1.44 mmol, 134 μL, 1.20 eq) were added at 0°C. The mixture was stirred at 25°C for 1 hour. The organic layer was washed three times with water (10.0 ml), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC to give compound int 9k (500 mg, 646 μL, 53.8% yield, 98.9% purity) as a yellow oil.

[0461] Compound int 9k (200 mg, 258 μmol, 1.00 eq) was dissolved in methanol (2.00 mL) and Pd / C (55.0 mg, 51.7 μmol, 10% purity, 0.20 eq) was added under N2 protection. The suspension was debubbled under vacuum and purged with H2 several times. The mixture was stirred at 25°C under H2 (15 psi) for 3 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to provide compound int 10k (50.0 mg, crude) as a colorless oil.

[0462] 50.0 mg (111 μmol, 1.00 eq) of compound int 10k and DOTA-OSu (74.2 mg, 111 μmol, 1.00 eq) were dissolved in DMF (1.00 ml), and DIEA (14.3 mg, 1115 μmol, 19.3 μL, 1.00 eq) was added. The mixture was stirred at 25 ° C for 1 hour. The reaction mixture was added to stirred MTBE (100 ml) and then centrifuged to obtain compound int 11k (50.0 mg, 49.69 μmol, 44.9% yield, 100% purity) as a yellow oil.

[0463] Compound int 11k (50.0 mg, 49.7 μmol, 1.00 eq) was dissolved in DMF (2.00 ml), and HBTU (28.3 mg, 74.5 μmol, 1.50 eq), HOBT (10.1 mg, 74.5 μmol, 1.50 eq), TEA (25.1 mg, 248 μmol, 34.6 μL, 5.00 eq) and compound 11a (47.0 mg, 74.5 μmol, 1.50 eq) were added. The mixture was stirred at 25° C. for 12 hours. The reaction mixture was purified by preparative HPLC (column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water (FA)-acetonitrile]; gradient: 14%-44% B, lasting 10 minutes) to give compound int 12k (25.0 mg, 18.5 μmol, 37.3% yield, 87.3% purity) as a white solid.

[0464] 20.0 mg (14.8 μmol, 1.00 eq) of compound int 12k was dissolved in DCM (5.00 ml) and TFA (6.14 g, 53.9 mmol, 4.00 ml, 3631 eq) and TIS (9.39 mg, 59.3 μmol, 12.2 μL, 4.00 eq) were added. The mixture was stirred at 25 ° C for 4 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (TFA conditions) to obtain compound int 13k (14.2 mg, 9.49 μmol, 64.0% yield, 99.4% purity, 4TFA) as a white solid.

[0465] Compound FAPI-CB-59 was obtained from 14.0 mg of compound 13k by a similar method as described in Example 1d (11.8 mg, 78% yield, 99.5% purity). It was purified by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (at 2nd minute) to 60% B (at 20th minute)]. UPLC-MS characterization conditions: separation column, ACQUITY UPLC BEH C18 1.7 μm 2.1×50 mm. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A. Calculated m / z is [M+H] + : 1092, retention time: 1.76 minutes.

[0466] Example 11. Synthesis of FAPI-CB-77

[0467] Compound int 21 was readily prepared via peptide solid-phase synthesis. Compound FAPI-CB-77 was purified from compound int 31 (5.5 mg, 32.6% yield, 98.2% purity) by HPLC using a similar route to Example 1a [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (at 2nd minute) to 60% B (at 20th minute)]. UPLC-MS characterization conditions: separation column, ACQUITY UPLC BEH C18 1.7 μm, 2.1×50 mm. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A. Calculated m / z is [M+H] + : 1577, retention time: 1.71 minutes.

[0468] Example 1m. Synthesis of FAPI-CB-86

[0469] Compound FAPI-CB-86 was obtained from compound int 1m by a similar method as described in Example 1k (3.5 mg, 12.0% yield, 97.2% purity). It was purified by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (2nd minute) to 60% B (20th minute)]. UPLC-MS characterization conditions: separation column, ACQUITY UPLC BEH C18 1.7 μm 2.1×50 mm. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A.

[0470] The calculated m / z is [M+H] + : 1221, retention time: 1.90 minutes.

[0471] Example 1n. Synthesis of FAPI-CB-93

[0472] Compound FAPI-CB-93 was obtained from int 1n by a similar method as described in Example 1k (3.5 mg, 12.0% yield, 97.2% purity). It was purified by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (2nd minute) to 60% B (20th minute)]. UPLC-MS characterization conditions: separation column, ACQUITY UPLC BEH C18 1.7 μm 2.1×50 mm. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A. Calculated m / z is [M+H] + : 1164, retention time: 1.85 minutes.

[0473] Example 1o. Synthesis of FAPI-CB-94

[0474] Compound int 5o was prepared with reference to the steps described in Sun, S. et al., Phosphorus fluoride exchange: Multidimensional catalytic click chemistry from phosphorus connective hubs (Chem 2023, 9, 2128–2143).

[0475] Compound Int 1o (6.0 g, 37.0 mmol) was dissolved in acetone (150 ml) and stirred. KF (17.1 g, 296 mmol) was added to the solution. The mixture was stirred at room temperature for 3 hours and then filtered through Celite. The solvent was evaporated under reduced pressure to obtain a sufficient amount of phosphoramide difluoro compound Int 2o in a single yield.

[0476] The reaction mixture of int 2o and commercially available int 3o was stirred at room temperature for 1 hour. Ethyl acetate (200 mL) was then added to dilute the reaction mixture, and the organic phase was washed sequentially with water (200 mL) and brine (200 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure to afford the crude product, which was then purified by column chromatography (silica gel, Hexane:EA = 4:1) to afford int 3o (4.6 g, 60.9%) as a colorless oil.

[0477] 100 mg of 10% Pd / C was added to a 50 ml round-bottom flask, followed by 5 ml of methanol under an inert atmosphere (argon). Compound int 4o (1.0 g, 3.0 mmol) was dissolved in methanol (5 ml) and added to the flask, which was then sealed with a rubber stopper. The round-bottom flask was vacuum-evacuated and filled with hydrogen using a hydrogen balloon. The mixture was stirred vigorously under hydrogen for 1 hour, then filtered through diatomaceous earth. The solvent was removed under reduced pressure to give compound int 5o (627 mg, 84.6%) as a white solid.

[0478] Compound Int 5o (120 mg, 0.81 mmol, 1 eq), Compound 5 (115 mg, 0.97 mmol, 1.2 eq), and EDCI (191 mg, 0.97 mmol, 1.2 eq) were dissolved in DCM (5 mL) and stirred at room temperature for 1 hour. LCMS analysis indicated incomplete reaction. DMAP (9 mg, 0.08 mmol, 0.1 eq) was then added. Stirring was continued for 2 hours, when LCMS indicated completion. Petroleum arginine was added for recrystallization to afford Int 6o (140 mg, 50% yield) as a white solid.

[0479] Compound int 7o was obtained from int 3a by a similar method as in Example 1d (yield 80.1%). Compound FAPI-CB-94 was purified from int 7o by a similar method as in Example 1a by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (second minute) to 60% B (second minute) (3.5 mg, yield 77.6%, purity 98.2%). UPLC-MS characterization conditions: separation column, ACQUITY UPLC BEH C18 1.7 μm 2.1×50 mm. Method, flow rate 0.5 mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 min, 95% A; 0.2-3.0 min, 95% to 5% A, curve 6; 3.0-4.0 min, 5% A; 4.0-4.5 min, 5% to 95% A, curve 6; 4.5-5.0 min, 95% A. Calculated m / z is [M+H] + : 1190, retention time: 1.53 minutes.

[0480] Example 1p. Synthesis of FAPI-CB-95

[0481] Compound FAPI-CB-95 was obtained by peptide solid phase synthesis using a similar method as in Example 1a to easily obtain the intermediate compound int 1p (3.6 mg, yield 11.9%, purity 98.5%).

[0482] Purification by HPLC [eluent A: H2O (0.1% TFA); eluent B: ACN, from 10% B (2nd minute) to 60% B (20th minute)]. UPLC-MS characterization conditions: Separation column, ACQUITY UPLC BEH C18 1.7μm 2.1×50mm. Method, flow rate 0.5mL / min, eluent A: H2O (0.1% TFA); eluent B: MeOH; 0-0.2 minutes, 95% A; 0.2-3.0 minutes, 95% to 5% A, curve 6; 3.0-4.0 minutes, 5% A; 4.0-4.5 minutes, 5% to 95% A, curve 6; 4.5-5.0 minutes, 95% A. Calculated m / z is [M+H] + : 1203, retention time: 1.61 minutes.

[0483] Example 2 Preparation of Nuclear Drug Molecules from Nuclide-Labeled Precursor Compounds

[0484] Generally, 68 The radiolabeling of Ga was carried out by incubation with 10 nmol of FAPI-CB series precursors at pH 4.0 and 90°C for 10 minutes. 18 The protein was purified by a Waters column, and its radiochemical purity was determined by high performance liquid chromatography (radio-HPLC) equipped with a radioactivity detector. The stability in phosphate buffer (0.01 M, pH 7.4) and human serum was also tested at 37°C (acetonitrile precipitation was required to remove proteins in human serum).

[0485] Generally, 177 Radiolabeling of Lu was performed by incubation with several FAPI-CB series precursors at pH 4.5, 50 to 90°C for 10 minutes. 18 The protein was purified by a Waters column, and its radiochemical purity was determined by high performance liquid chromatography (radio-HPLC) equipped with a radioactivity detector. The stability in phosphate buffer (0.01 M, pH 7.4) and human serum was also tested at 37°C (acetonitrile precipitation was required to remove proteins in human serum).

[0486] Generally, Al 18 F radiolabeling was performed by adding commercially available, accelerator-prepared 18 The F anion aqueous solution was concentrated to 10 μL using a nitrogen blowdown instrument and 2 μL of 6 mM Al 3+ 0.2M sodium acetate solution (pH = 4.0, 12 nmol), 30 μL acetonitrile, 20 μL 0.2M sodium acetate buffer (pH = 4.0), heated at 100 ° C for 5 min to prepare [Al 18 F] 2+; Then, 20 nmol of the corresponding FAPI-CB series precursor molecules were added and incubated at 100°C for 15 minutes. 18 The protein was purified by a Waters column, and its radiochemical purity was determined by high performance liquid chromatography (radio-HPLC) equipped with a radioactivity detector. The stability in phosphate buffer (0.01 M, pH 7.4) and human serum was also tested at 37°C (acetonitrile precipitation was required to remove proteins in human serum).

[0487] Radio-HPLC characterization conditions: separation column, C 18 5μm 4.6×150mm; eluent A: H2O (0.1% trifluoroacetic acid); eluent B: acetonitrile (0.1% trifluoroacetic acid); 0-2 minutes, 90% phase A; 2-10 minutes, 90% to 40% phase A, linear mode 6; 10-12 minutes, 40% phase A; 12-13 minutes, 40% to 90% phase A, linear mode 6; 13-15 minutes, 90% phase A; retention time: product 7.4 minutes, purity about 98.6%; hydrolysis by-product 6.1 minutes, accounting for about 1.4%.

[0488] 68 Ga, 177 Lu, 86 Y and 225 Ac-labeled FAPI-CB-22, FAPI-CB-28, FAPI-CB-30, FAPI-CB-31, FAPI-CB-44, FAPI-CB-45, FAPI-CB-47, FAPI-CB-50, FAPI-CB-53, FAPI-CB-59, FAPI-CB-77, FAPI-CB-86, FAPI-CB-93, FAPI-CB-94; Al 18 F-labeled FAPI-CB-36 and FAPI-CB-95 can be easily obtained by similar methods as above and disclosed prior art.

[0489] 68 The radioactive HPLC spectrum of Ga-FAPI-CB-30 is shown in Figure 2. The left figure is the quality control chart after labeling, the middle figure is the stability monitoring chart after serum co-incubation for 1 hour, and the right figure is the stability monitoring chart after serum co-incubation for 2 hours.

[0490] 68 Ga-FAPI-CB-31, 68 Ga-FAPI-CB-50、Al 18 The radioactive HPLC spectra of F-FAPI-CB-36 are shown in the left, middle, and right panels of FIG3 , respectively.

[0491] Example 3 Molecular experiment - target protein co-incubation - acid-base treatment - gel electrophoresis - autoradiography - Coomassie brilliant blue staining experiment

[0492] The total volume was 20 μL and the precursor concentration was 200 nM. 177 Lu-FAPI-CB-30 is incubated with 4 μM FAP human recombinant protein at pH 7.4 (serum pH) or pH 6.5 (tumor microenvironment pH) at 37°C for 1 hour. The sample is then denatured with a strong acid or base to eliminate reversible interactions. SDS-PAGE is then performed on a polyacrylamide gel to separate the protein and the nuclear drug molecules. The gel is then subjected to phosphorautoradiography (Amersham Typhoon imaging) to visualize radioactive bands of the nuclear drug. Finally, the gel is stained with Coomassie Brilliant Blue, which displays the target protein in blue. Comparing the radioactive bands with the Coomassie Brilliant Blue staining indicates that irreversible covalent binding of the nuclear drug to the protein will be observed in the protein-stained region above the gel.

[0493] Figure 4 shows the protein co-incubation - gel electrophoresis - radioautography - Coomassie blue staining after the obtained 177 Covalent linkage efficiency of Lu-FAPI-CB-30 to FAP human recombinant protein. The left panel shows a control incubated at pH 7.4 for 1 hour, while the right panel shows a sample treated with strong acid after 1 hour of incubation, demonstrating continued covalent linkage.

[0494] Example 4 Molecular Experiment - Long-term Co-incubation of Target Protein - Gel Electrophoresis - Autoradiography - Coomassie Brilliant Blue Staining Experiment

[0495] The precursor molecule concentration is 150nM 177 Lu-FAPI-CB-30 was incubated with 3 μM FAP human recombinant protein at pH 7.4 and 37°C. Subsequently, equal volumes of samples were taken at different time points, mixed with protein loading buffer, and placed in a -80°C freezer. The samples were uniformly denatured (95°C, 5 minutes) and subjected to SDS-PAGE to separate the protein and the nuclear drug molecules on a polyacrylamide gel. The gel was then subjected to phosphorscreen autoradiography (Amersham Typhoon imaging) to obtain radioactive bands of the nuclear drug. Finally, the gel was stained with Coomassie Brilliant Blue, resulting in the target protein appearing blue. Comparing the radioactive bands with the Coomassie Brilliant Blue staining results indicates that if the nuclear drug and protein are irreversibly covalently bound, radioactive bands will be visible in the protein-stained area above the gel.

[0496] Figure 5 shows the protein co-incubation - gel electrophoresis - radioautography - Coomassie blue staining after the obtained 177The covalent bonding ratio of Lu-FAPI-CB-30 molecules to human FAP recombinant protein. The left image is an autoradiogram, and the right image shows the grayscale values ​​of the bands quantified using image quantification tools such as ImageJ, corresponding to the covalent bonding ratio.

[0497] Example 5 Molecular Experiment - Long-term Co-incubation of Target Protein - Gel Electrophoresis - Autoradiography - Coomassie Brilliant Blue Staining Experiment

[0498] The precursor molecule concentration is 150nM 177 The Lu-FAPI-CB series of molecules were incubated with 3 μM of human recombinant FAP protein at pH 7.4 and 37°C. Subsequently, equal volumes of samples were taken at specific time points, mixed with protein loading buffer, and placed in a -80°C freezer. The samples were uniformly denatured (95°C, 5 minutes) and subjected to SDS-PAGE to separate the protein and the nuclear drug molecules on a polyacrylamide gel. The gel was then subjected to phosphor-screen autoradiography (Amersham Typhoon imaging) to visualize radioactive bands of the nuclear drug. Finally, the gel was stained with Coomassie Brilliant Blue, which displayed the target protein in blue. Comparing the radioactive bands with the Coomassie Brilliant Blue staining revealed that irreversible covalent binding of the nuclear drug to the protein would result in radioactive bands visible in the protein-stained region above the gel.

[0499] Figure 6 shows the protein co-incubation - gel electrophoresis - radioautography - Coomassie blue staining after the obtained 177 The covalent bonding ratio of Lu-FAPI-CB series molecules to FAP human recombinant protein. The lanes in the figure from left to right are 177 Lu-labeled FAPI-CB-22, FAPI-CB-28, FAPI-CB-31, FAPI-CB-44, FAPI-CB-45, FAPI-CB-47, FAPI-CB-50.

[0500] Example 6 Animal Experiment - PET / CT Imaging and Biodistribution

[0501] All PET / CT scans are performed at Mediso PET 122S small animal PET / CT is performed.

[0502] A certain number of HT-1080-FAP tumor mice were injected with 7.4-18.5 MBq of the drug into the tail vein at the designated time points. 68 Ga / 86 After the addition of Y-FAPI-04 (control) and radionuclide-labeled -FAPI-CB series molecules, imaging was performed at designated time points. At certain time points, the mice were anesthetized and killed by cervical dislocation. The organs were dissected and the radioactivity was measured to comprehensively and quantitatively analyze the uptake and retention of nuclear drugs in the tumor.

[0503] Example 7a: 68 Ga-FAPI-CB-30 and 68 Ga-FAPI-CB-02 or 68 PET / CT contrast imaging of Ga-FAPI-04 in HT-1080-FAP tumor-bearing mouse model

[0504] Elute with 5 mL of 0.6 M high-purity hydrochloric acid 68 Ge- 68 Ga generator (iThemba LABS, South Africa) was used to obtain Ga-68 hydrochloric acid solution. 1 mL of the washed Ga-68 solution was added with 100 μL of 3M sodium hydroxide and 130 μL of 3M sodium acetate to adjust the acidity to a final pH of 4.0. 12 μg of FAPI-CB-02 precursor (structure shown in FIG7a, using essentially the same steps as in Example 1a, replacing the corresponding reactants, to synthesize FAPI-CB-02) was added, and the reaction mixture was heated to 90°C for 10 minutes. The reaction solution was passed through C 18 Column (Waters), free ions were removed, and then C was eluted with ethanol solution. 18 Column. Get the marked 68 Ga-FAPI-CB-02. 68 Ga-FAPI-CB-02 was diluted with saline and injected into each mouse at approximately 3.7 MBq (approximately 200 μl). PET scans were performed 90 minutes after injection and reconstructed using PET image processing software. Furthermore, PET imaging was performed on the same mouse 24 hours after labeling the control FAPI-CB-30 using the same procedure. The resulting images are shown in Figure 7b. 68 Ga-FAPI-CB-30 probe in tumor site 68 Ga-FAPI-CB-02 uptake was comparable, but blood pool background was lower.

[0505] Comparison of the same process 68 Ga-FAPI-CB-30 and 68 Ga-FAPI-04, as shown in Figure 7c 68 Ga-FAPI-CB-30 tumor uptake was enhanced without a significant increase in background.

[0506] Example 7b: 68 Ga-FAPI-CB-31 and 68 PET / CT contrast imaging of Ga-FAPI-04 in HT-1080-FAP tumor-bearing mouse model

[0507] Elute with 5 mL of 0.6 M high-purity hydrochloric acid68 Ge- 68 Ga generator (iThemba LABS, South Africa) was used to obtain Ga-68 hydrochloric acid solution. 1 mL of the washed Ga-68 solution was added with 100 μL of 3M sodium hydroxide and 130 μL of 3M sodium acetate to adjust the acidity to a final pH of 4.0. 9 μg of FAPI-04 precursor was added and the reaction mixture was heated to 90°C for 10 minutes. The reaction solution was passed through a C 18 Column (Waters), free ions were removed, and then C was eluted with ethanol solution. 18 Column. Get the marked 68 Ga-FAPI-04. 68 Ga-FAPI-04 was diluted with saline and injected into each mouse at approximately 3.7 MBq (approximately 200 μL). PET scans were performed 90 minutes after injection and reconstructed using PET image processing software. Furthermore, PET imaging of the control FAPI-CB-31 was performed on the same mouse 24 hours apart using the same procedure. The resulting images are shown in Figure 8. 68 Ga-FAPI-CB-31 probe is more sensitive to the tumor 68 Ga-FAPI-04 uptake was enhanced with a similar blood pool background (the structure of FAPI-04 is shown in FIG8a ).

[0508] Example 7c: Al 18 F-FAPI-CB-36 and 68 PET / CT contrast imaging of Ga-FAPI-04 in HT-1080-FAP tumor-bearing mouse model

[0509] According to the method in Example 2, the marked 68 Ga-FAPI-04 was diluted with physiological saline and injected into each mouse at a volume of about 3.7 MBq (about 200 μl). PET scanning imaging was performed 90 minutes after injection and reconstructed using PET image processing software. 18 F labeling process to obtain control Al 18 F-FAPI-CB-36 was used to perform PET imaging on the same mouse every 24 hours, and the tumor uptake of SUV was obtained. max As shown in Figure 9, it can be seen that Al 18 The F-FAPI-CB-36 probe is more sensitive to the tumor site 68 Ga-FAPI-04 uptake significantly increased the SUV of mouse tumors max The average value can reach 2.08, while the mice in the same group 68 Ga-FAPI-04 SUV max The average value is only 0.78.

[0510] Example 7d: 68 Ga-FAPI-CB-50 and 68 PET / CT contrast imaging of Ga-FAPI-04 in HT-1080-FAP tumor-bearing mouse model

[0511] Elute with 5 mL of 0.6 M high-purity hydrochloric acid 68 Ge- 68 Ga generator (iThemba LABS, South Africa) was used to obtain Ga-68 hydrochloric acid solution. 1 mL of the washed Ga-68 solution was added with 100 μL of 3M sodium hydroxide and 130 μL of 3M sodium acetate to adjust the acidity to a final pH of 4.0. 9 μg of FAPI-04 precursor was added and the reaction mixture was heated to 90°C for 10 minutes. The reaction solution was passed through a C 18 Column (Waters), free ions were removed, and then C was eluted with ethanol solution. 18 Column. Get the marked 68 Ga-FAPI-04. 68 Ga-FAPI-04 was diluted with saline and injected into each mouse at approximately 3.7 MBq (approximately 200 μl). PET scans were performed 90 minutes after injection and reconstructed using PET image processing software. Furthermore, PET imaging of the control FAPI-CB-50 was performed on the same mouse at 24-hour intervals using the same process. The images obtained 2 hours after injection are shown in Figure 10a. 68 Ga-FAPI-CB-50 probe is more sensitive to the tumor 68 Ga-FAPI-04 uptake was improved, and the blood pool background was reduced to a certain extent; as shown in Figure 10b, the SUV of the tumor and blood pool were examined. max ratio, 68 Ga-FAPI-CB-50 has a better clearance rate.

[0512] Example 7e: 68 Ga-FAP-2286 and 68 PET / CT contrast imaging of Ga-FAPI-CB-45 in HT-1080-FAP tumor-bearing mouse model

[0513] According to the method in Example 2, the marked 68Ga-FAP-2286 (precursor structure shown in Figure 11A, commercially available or easily obtained by referring to patent WO 2021 / 005125A1 applied by 3B PHARMACEUTICALS GMBH) was diluted with saline and injected into each mouse with approximately 3.7 MBq (volume of approximately 200 μl). PET scan imaging was performed 90 minutes after injection and reconstructed using PET image processing software. In addition, the control was labeled using the same process. 68 Ga-FAPI-CB-45 was used to perform PET imaging on the same mouse every 24 hours. The resulting images are shown in Figure 11B. 68 Ga-FAPI-CB-45 probe is more sensitive to tumor 68 Ga-FAP-2286 uptake was enhanced without a significant increase in background.

[0514] Example 7f: 68 Ga-FAPI-CB-53 and 68 PET / CT contrast imaging of Ga-FAPI-04 in HT-1080-FAP tumor-bearing mouse model

[0515] Elute with 5 mL of 0.6 M high-purity hydrochloric acid 68 Ge- 68 Ga generator (iThemba LABS, South Africa) was used to obtain Ga-68 hydrochloric acid solution. 1 mL of the washed Ga-68 solution was added with 100 μL of 3M sodium hydroxide and 130 μL of 3M sodium acetate to adjust the acidity to a final pH of 4.0. 9 μg of FAPI-04 precursor was added and the reaction mixture was heated to 90°C for 10 minutes. The reaction solution was passed through a C 18 Column (Waters), free ions were removed, and then C was eluted with ethanol solution. 18 Column. Get the marked 68 Ga-FAPI-04. 68 Ga-FAPI-04 was diluted with saline and injected into each mouse at approximately 3.7 MBq (approximately 200 μL). PET scans were performed 1 hour after injection and reconstructed using PET image processing software. Furthermore, the same procedure was used to label the control FAPI-CB-53. PET images were performed on the same mouse 24 hours apart. The images obtained 1 hour after injection are shown in Figure 12. 68 Ga-FAPI-CB-53 probe is more sensitive to the tumor site 68 Ga-FAPI-04 uptake was enhanced, and the blood pool background was maintained at a reduced level.

[0516] Example 7g: 68 Ga-FAPI-CB-59 and 68PET / CT contrast imaging of Ga-FAPI-04 in HT-1080-FAP tumor-bearing mouse model

[0517] Using a similar process as in Example 2, we obtain 68 Ga-FAPI-CB-59 was used for PET imaging in HT-1080-FAP mice. The images obtained 1 hour and 3 hours after injection are shown in Figure 13. 68 The Ga-FAPI-CB-59 probe had a high uptake at the tumor site, and the tumor uptake at 3 hours was almost unchanged compared to 1 hour, demonstrating good tumor retention.

[0518] Example 7h: Al 18 F-FAPI-CB-77 and Al 18 PET / CT contrast imaging of F-FAPI-74 in HT-1080-FAP tumor-bearing mouse model

[0519] According to the method in Example 2, the labeled Al 18 F-FAPI-74 (structure shown in FIG14a) was diluted with physiological saline and injected into each mouse with about 7.4 MBq (volume about 200 μl). PET scanning imaging was performed 2 hours after injection and reconstructed using PET image processing software. In addition, Al 18 F labeling process to obtain control Al 18 F-FAPI-CB-77 was used to perform PET imaging on the same mouse at 48-hour intervals. The obtained imaging images are shown in FIG14 b. It can be seen that although Al 18 The F-FAPI-CB-77 probe is more sensitive to the tumor site than Al 18 The uptake of F-FAPI-74 was reduced to a certain extent, and the background uptake in the gallbladder and intestine was significantly reduced, indicating that it may still have certain advantages in the exploration of digestive system tumors.

[0520] Example 7i: 68 Ga-FAPI-CB-86 and 68 PET / CT contrast imaging of Ga-FAPI-04 in HT-1080-FAP tumor-bearing mouse model

[0521] Elute with 5 mL of 0.6 M high-purity hydrochloric acid 68 Ge- 68 Ga generator (iThemba LABS, South Africa) was used to obtain Ga-68 hydrochloric acid solution. 1 mL of the washed Ga-68 solution was added with 100 μL of 3M sodium hydroxide and 130 μL of 3M sodium acetate to adjust the acidity to a final pH of 4.0. 9 μg of FAPI-04 precursor was added and the reaction mixture was heated to 90°C for 10 minutes. The reaction solution was passed through a C18 Column (Waters), free ions were removed, and then C was eluted with ethanol solution. 18 Column. Get the marked 68 Ga-FAPI-04. 68 Ga-FAPI-04 was diluted with saline and injected into each mouse at approximately 3.7 MBq (approximately 200 μl). PET scans were performed 90 minutes after injection and reconstructed using PET image processing software. Furthermore, PET imaging of the control FAPI-CB-50 was performed on the same mouse using the same procedure at 24-hour intervals. The images obtained 2 hours after injection are shown in Figure 15a. 68 Ga-FAPI-CB-50 probe is more sensitive to the tumor 68 Ga-FAPI-04 uptake was improved, and the blood pool background was reduced to a certain extent; as shown in Figure 15b, the SUV of the tumor and blood pool were examined. max ratio, 68 Ga-FAPI-CB-50 has a better clearance rate.

[0522] Example 7j: 68 Ga-FAPI-CB-93 and 68 PET / CT contrast imaging of Ga-FAPI-04 in HT-1080-FAP tumor-bearing mouse model

[0523] Elute with 5 mL of 0.6 M high-purity hydrochloric acid 68 Ge- 68 Ga generator (iThemba LABS, South Africa) was used to obtain Ga-68 hydrochloric acid solution. 1 mL of the washed Ga-68 solution was added with 100 μL of 3M sodium hydroxide and 130 μL of 3M sodium acetate to adjust the acidity to a final pH of 4.0. 9 μg of FAPI-04 precursor was added and the reaction mixture was heated to 90°C for 10 minutes. The reaction solution was passed through a C 18 Column (Waters), free ions were removed, and then C was eluted with ethanol solution. 18 Column. Get the marked 68 Ga-FAPI-04. 68 Ga-FAPI-04 was diluted with saline and injected into each mouse at approximately 3.7 MBq (approximately 200 μl). PET scan imaging was performed 90 minutes after injection and reconstructed using PET image processing software. Furthermore, PET imaging of the control FAPI-CB-50 was performed on the same mouse at 24-hour intervals using the same process. The image obtained 2 hours after injection is shown in Figure 16a. 68 Ga-FAPI-CB-50 probe is more sensitive to the tumor 68Ga-FAPI-04 uptake was improved, and the blood pool background was reduced to a certain extent; as shown in Figure 16b, the SUV of the tumor and blood pool were examined. max ratio, 68 Ga-FAPI-CB-50 has a better clearance rate.

[0524] Example 7k: 68 Ga-FAPI-CB-94 and 68 PET / CT contrast imaging of Ga-FAPI-04 in HT-1080-FAP tumor-bearing mouse model

[0525] The labeled 68 Ga-FAPI-04 was diluted with saline and injected into each mouse at approximately 3.7 MBq (approximately 200 μL). PET scans were performed 1 hour after injection and reconstructed using PET image processing software. Furthermore, PET images of the control FAPI-CB-94 were performed on the same mouse 24 hours after injection using the same procedure. The images obtained 1 hour after injection are shown in Figure 17. 68 Ga-FAPI-CB-94 is more active in the tumor 68 Ga-FAPI-04 uptake was enhanced without a significant increase in blood pool background.

[0526] Example 71: A1 18 F-FAPI-CB-95 and Al 18 PET / CT contrast imaging of F-FAPI-74 in HT-1080-FAP tumor-bearing mouse model

[0527] According to the method in Example 2, the labeled Al 18 F-FAPI-74 (structure shown in FIG18a) was diluted with physiological saline and injected into each mouse with about 7.4 MBq (volume about 200 μl). PET scanning imaging was performed 2 hours after injection and reconstructed using PET image processing software. In addition, Al 18 F labeling process to obtain control Al 18 F-FAPI-CB-95 was used to perform PET imaging on the same mouse at 48-hour intervals. The resulting images are shown in Figure 18b. 18 The F-FAPI-CB-95 probe is more sensitive to the tumor site than the Al 18 The uptake of F-FAPI-74 was improved to a certain extent, and the background uptake in the gallbladder and intestine was significantly reduced.

[0528] Example 8 Tumor Uptake Value

[0529] The same mice bearing HT-1080-FAP tumors (4 mice in each group) were injected with 18.5 MBq of 68 PET-CT scan was performed on the first day after Ga-FAPI-04, and 24 hours after 68 After Ga decayed completely, 18.5 MBq of the same specific activity was injected into the tail vein. 68 After Ga-FAPI-CB, PET-CT is used to scan again to obtain image data. The tumor uptake value can be semi-quantified by analyzing the reconstructed image after PET-CT scanning using the device's own software to obtain the standard uptake value (SUV). SUV refers to the ratio of the radioactivity of the imaging agent taken up by local tissue to the average injected activity of the whole body: SUV = radioactivity concentration of the lesion (kBq / ml) / (injection dose (MBq) / body weight (kg)). Compared to FAPI-04 in the most relevant prior art, in the HT-1080-FAP mouse model, the maximum standard uptake value (SUV) of the tumor of the FAPI-CB molecule in certain embodiments of the present invention is max ), mean standard uptake value (SUV mean ) or the percentage of injected dose (ID% / g) at the 1-hour time point showed a significant increase of more than 3 times (n=4, p<0.0001, see Figure 19), and there was also a significant improvement in the ratio of tumor to blood pool standard uptake values ​​(see Figure 20), and it also had obvious advantages in PET-CT representative images; in addition, in some embodiments, there was no significant difference in blood pool uptake of FAPI-CB molecules compared with FAPI-04 at the 1-hour time point, but tumor uptake was higher (see Figure 21).

[0530] In certain embodiments, as 68 Ga-FAPI-CB-47 and 68 PET-CT results of Ga-FAPI-04, Figure 22a shows the tumor uptake value 68 Ga-FAPI-CB-47 showed a significant increase, and the tumor uptake increased over time. Figure 22b shows 68 Ga-FAPI-CB-47 has a fast clearance rate in the blood pool, with blood pool uptake reduced by approximately two-thirds in 2 hours compared to 0.5 hours, and the cumulative toxicity is controllable.

[0531] Therefore, the tumor uptake value of the radionuclide-labeled FAPI-CB compound of the present invention is significantly improved, and the background uptake is controllable or reduced, which is of great significance for improving the clinical tumor detection rate or tumor treatment.

[0532] Example 9 Cancer patients 68 Ga-FAPI-CB-50 PET / CT imaging

[0533] Elute the GMP-compliant clinical product with 3 mL of 0.05 M high-purity hydrochloric acid. 68 Ge- 68 Ga generator to obtain Ga-68 hydrochloric acid solution. Take the eluted Ga-68 solution with pH 4.0, add 300 μg FAPI-CB-50 precursor, and heat the reaction mixture to 90°C for 15 minutes. 18 Column (Waters), free ions were removed, and then C was eluted with 80% ethanol solution. 18 Column. Get the marked 68 Ga-FAPI-CB-50. 68 Ga-FAPI-CB-50 was diluted with sterile membrane-filtered physiological saline and injected into the forearm vein of a cancer patient at approximately 188 MBq. PET / CT scans were performed 60 and 180 minutes after injection, and the images were reconstructed using PET-CT image processing software. The resulting images are shown in Figure 23a. 68 The Ga-FAPI-CB-50 probe had a higher uptake in the tumor site, with the highest standardized uptake value (SUV max ) can reach about 45, and the lesions basically do not decrease over time, while the non-target organ uptake value gradually decreases.

[0534] The patient underwent 18 F-FDG imaging, the resulting image is shown in Figure 23b. 68 Compared with it, Ga-FAPI-CB-50 clearly shows better tumor imaging effect.

[0535] Example 10 Cancer patients 177 Lu-FAPI-CB-30 SPECT / CT imaging

[0536] According to the method in Example 2, clinical carrier-free 177 Lu labeling 177 Lu-FAPI-CB-30 (radioactive yield>99%, radioactive purity>99%, molar activity~37MBq / nmol). 177 Lu-FAPI-CB-30 was diluted with sterile membrane-filtered physiological saline and injected into the forearm vein of a cancer patient at approximately 1.11 GBq. SPECT / CT scans were performed 24, 48, and 120 hours after injection. The images were reconstructed using SPECT / CT image processing software, and the resulting images are shown in Figure 24. 177Lu-FAPI-CB-30 has a higher uptake at the tumor site (the dotted circle indicates peritoneal metastasis), and the uptake of the lesions decreases slowly over time, while the uptake value of non-target organs decreases faster, providing a high target-to-target ratio, suggesting a wider therapeutic window for this candidate molecule.

[0537] Example 11 Tumor model mice 177 Lu-FAPI-CB-86 SPECT / CT imaging

[0538] According to the method in Example 2, the marked 177 Lu-FAPI-CB-86 was diluted in saline and injected into the tail vein of HT-1080-FAP tumor-bearing mice at a dose of approximately 25.9 MBq (molar activity ~29.6 MBq / nmol, in a volume of approximately 200 μL). PET scans were performed 90 minutes after injection and reconstructed using SPECT / CT image processing software. The results are shown in Figure 25 , with Figure 25a showing a representative SPECT / CT image of the mouse and Figure 25b showing the tumor SUV versus time curve. These results demonstrate that the radionuclide molecule remains in FAP-overexpressing tumors for a prolonged period and exhibits a significant therapeutic effect (tumor reduction).

[0539] Example 12 Tumor Model Mice 177 Treatment with Lu-FAPI-CB-30

[0540] When the average tumor size reaches about 100 mm 3 At day 0, the same batch of HT-1080-FAP tumor-bearing mice were randomly divided into three groups: negative control group (injected with saline, number of mice n = 8), low-dose group (injected with 22.2 MBq of 177 Lu-FAPI-CB-30, number of mice n = 6, high dose group (injection of 44.4 MBq) 177 Lu-FAPI-CB-30, number of mice n=6). Only one round of treatment was performed on day 0. 177 Lu-labeled FAPI-CB-30 was prepared as described in Example 2 (radioactivity yield>99%, radioactivity purity>99%, molar activity ∼29.6 MBq / nmol). Tumor size and body weight of mice were monitored every 2 or 3 days.

[0541] As shown in Figure 26, Figure 26a is a schematic diagram of the treatment plan, Figure 26b is the averaged tumor growth curve, Figure 26c is the tumor growth curve of each group of individual mice, and Figure 26d is the averaged normalized body weight curve. It can be seen that compared with the saline control group, 177Lu-FAPI-CB-30 has a significant tumor inhibitory effect and is dose-dependent, and its toxicity is controllable as shown by changes in mouse body weight.

[0542] Example 13 68 Ga-FAPI-CB-30 and 68 PET / CT contrast imaging of Ga-FAPI-04 in a mouse model of renal fibrosis

[0543] A group of mice with left renal fibrosis were prepared by unilateral ureteral ligation to induce left renal obstruction in mice (FAP was highly expressed in the fibrotic area). 68 Ga-FAPI-04 was diluted with physiological saline and injected into each mouse model at 11.2 MBq (volume approximately 200 μl, number of mice n = 4). PET scan imaging was performed 1 hour after injection and reconstructed using PET image processing software. In addition, the same process was used to label the control. 68 Ga-FAPI-CB-30 was used for PET imaging in another 4 mouse models. The representative images (coronal plane) obtained 1 hour after injection are shown in FIG27a. 68 Ga-FAPI-CB-30 uptake was higher in the left kidney with more severe renal fibrosis, while 68 Ga-FAPI-04 is just the opposite. Figure 27b shows the quantitative results of kidney SUV values. This example illustrates 68 Advantages of Ga-FAPI-CB-30 in monitoring and staging renal fibrosis.

[0544] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The methods described herein can be performed in any order that is logically possible, except for the specific order disclosed.

[0545] The representative examples are intended to help illustrate the present invention and are not intended to, and should not be construed as, limiting the scope of the present invention. Indeed, various modifications of the present invention and many other embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art, including the examples and the scientific and patent literature references cited herein. The examples contain important additional information, illustrations, and guidance that can be employed in the practice of the present invention in its various embodiments and equivalents.

Claims

1. A trifunctional compound, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound comprises at least one target T, at least one payload P, It is characterized in that In addition to the covalent warhead that may be carried by the target T itself, the trifunctional compound includes at least one covalent warhead C as follows: The payload P is any optical functional group, radiolabeled functional group, radiolabeled functional group, or small molecule cytotoxic drug functional group that can be used for optical imaging, positron emission tomography imaging, single photon emission computed tomography imaging, chemotherapy or radiotherapy; The target T comprises a targeting group that can target a protein or tissue, and the targeting group is a small molecule; The covalent warhead C can form a reversible or irreversible covalent connection with the protein or tissue targeted by the target T; Wherein the covalent warhead C is selected from the group consisting of: in: Y is selected from O, S and NR 1 The group composed of; LG is a leaving group, which can be replaced by a protein residue, preferably a halogen or OTs, and Ts is p-toluenesulfonyl; R, R' and R" are independently selected from the group consisting of hydrogen, halogen, nitro, cyano, optionally substituted thiol, optionally substituted seleno, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted amino; R 1 is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl; Het is an optionally substituted heterocyclic group or heteroaryl group, wherein when the heteroaryl group contains a N atom, the N atom may be present in a free form or in the form of an onium salt; Hal is a halogen, preferably F or Cl; Each p is independently an integer between 0 and 12; The at least one targeting agent T, the at least one payload P, and the at least one covalent warhead C are connected via a linker moiety.

2. The trifunctional compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the covalent warhead C is selected from the group consisting of: in: Het is an optionally substituted C 4 -C 12 Heterocyclic group, or optionally substituted C 5 -C 12 Heteroaryl, wherein when the heteroaryl contains a N atom, the N atom may be present in a free form or in the form of an onium salt; Hal is F; Each p is independently an integer between 0 and 6; R 1 is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl.

3. The trifunctional compound according to claim 1, its pharmaceutically acceptable salt, stereoisomer or solvate, wherein C is selected from the group consisting of: in: Het is C 4 , C 5 or C 6 Heterocyclic group, or C 5 or C 6 Heteroaryl, wherein the heterocyclic group or heteroaryl group contains one or two heteroatoms selected from N, O or S, wherein when the heteroaryl group contains a N atom, the N atom may exist in a free form or in the form of an onium salt, preferably in a free form, and more preferably Het is azetidinyl, pyridyl, pyrrolyl or N-oxypyridyl; Hal is F; Each p is independently an integer between 0 and 4.

4. A trifunctional compound, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound comprises at least one target T, at least one payload P, It is characterized in that In addition to the covalent warhead that may be carried by the target T itself, the trifunctional compound includes at least one covalent warhead C as follows: The payload P is any optical functional group, radiolabeled functional group, radiolabeled functional group, or small molecule cytotoxic drug functional group that can be used for optical imaging, positron emission tomography imaging, single photon emission computed tomography imaging, chemotherapy or radiotherapy; The target T comprises a targeting group that can target a protein or tissue, and the targeting group is a small molecule; The covalent warhead C can form a reversible or irreversible covalent connection with the protein or tissue targeted by the target T; Wherein the covalent warhead C is selected from the group consisting of: in: Y is selected from O, S and NR 1 The group composed of; LG is a leaving group, which can be replaced by a protein residue, preferably a halogen or OTs, and Ts is p-toluenesulfonyl; R, R' and R" are independently selected from the group consisting of hydrogen, halogen, nitro, cyano, optionally substituted thiol, optionally substituted seleno, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted amino; R 1 is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl; Het is an optionally substituted heterocyclic group or heteroaryl group, wherein when the heteroaryl group contains a N atom, the N atom may be present in a free form or in the form of an onium salt; Hal is a leaving group, preferably halogen, OH or NH 2 , more preferably F or Cl; Each p is independently an integer between 0 and 12; The at least one targeting agent T, the at least one payload P, and the at least one covalent warhead C are connected via a linker moiety.

5. The trifunctional compound according to claim 4, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the covalent warhead C is selected from the group consisting of: in: Het is an optionally substituted C 4 -C 12 Heterocyclic group, or optionally substituted C 5 -C 12 Heteroaryl, wherein when the heteroaryl contains a N atom, the N atom may be present in a free form or in the form of an onium salt; Hal is F; Each p is independently an integer between 0 and 6; R 1 is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl.

6. The trifunctional compound according to claim 4, its pharmaceutically acceptable salt, stereoisomer or solvate, wherein C is selected from the group consisting of: in: Het is C 4 , C 5 or C 6 Heterocyclic group, or C 5 or C 6 Heteroaryl, wherein the heterocyclic group or heteroaryl group contains one or two heteroatoms selected from N, O or S, wherein when the heteroaryl group contains a N atom, the N atom may exist in a free form or in the form of an onium salt, preferably in a free form, and more preferably Het is azetidinyl, Pyridyl, pyrrolyl or N-oxide pyridyl; Hal is F; Each p is independently an integer between 0 and 4.

7. A trifunctional compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound comprises one to three, for example one or two, payloads P, and one to three, for example one or two, targeting agents T, and in addition to the covalent warhead that may be carried by the targeting agent T itself, the trifunctional compound comprises one to three, for example one or two, covalent warheads C, and the linker moieties each independently comprise 0 to 6 linker units, and the linker units can be divalent linkers, such as single bonds, or trivalent linkers.

8. A trifunctional compound, a pharmaceutically acceptable salt, stereoisomer or solvate thereof according to any one of claims 1 to 7, wherein the trifunctional compound has a structure of formula (I), formula (II) or formula (III), in: The effective loads P and P' are each independently the same or different groups containing radionuclides, or chelating groups that can chelate radionuclides, optical dye groups, or small molecule cytotoxic drug groups; The target substances T and T' are each independently the same or different targeting groups that can target proteins or tissues, and the targeting groups are small molecules; In the general formula (I), general formula (II) and general formula (III), in addition to the covalent warhead that may be carried by the target T itself, there are one, two or three identical or different covalent warheads C, each of which is independently connected to the target T by the same or different linker moieties L. 4 Connect to P, T, L shown in the general formula 1 , T' or P'; Each L 1 , L 2 and L 3 is a linker moiety, each of which independently comprises 0 to 6 A connection base unit.

9. A trifunctional compound, a pharmaceutically acceptable salt, stereoisomer or solvate thereof according to any one of claims 1 to 8, wherein the trifunctional compound has a structure of formula (Ia), formula (Ib), formula (Ic), formula (IIa) or formula (IIIa), in: Each L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 , L 11 , L 12 , L 13 , L 14 , L 15 , L 16 and L 17 are independently linker moieties, each of which independently comprises 0 to 6 linker units; a, b, c, d, e and f are each independently 0 or 1, and when it is 0, it means that the group in the bracket marked with the number does not exist, wherein the sum of a, b and c is at least 1, and the sum of c, d and e is at least 1; C, C' and C" each independently have the meaning as defined in claims 1 to 6.

10. The trifunctional compound according to any one of claims 1 to 9, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the targeting group targets fibroblast activation protein-α (FAP-α).

11. A trifunctional compound, a pharmaceutically acceptable salt, a stereoisomer or a solvate thereof according to any one of claims 1 to 10, wherein the targeting group has the general formula (IV), preferably has the general formula (IVa), more preferably has the general formula (IVc), the general formula (IVd), the general formula (IVe) or the general formula (IVf), Wherein in the general formula (IV), A is selected from O, S, NR A , R A Selected from H, C 1 -C 6 alkyl, There are multiple R on the tetrahydropyrrole ring shown f Group, each R f The groups are each independently selected from H, F, Cl, -CN, -B(OH) 2 , C 1 -C 6 Alkyl, α-chloroketo, optionally two R f The groups may be linked to each other to form a cycloalkyl group, preferably C 3 -C 7 Cycloalkyl, B 1 and B 2 independently selected from O or S, R f1 and R f2 Independently selected from H, D and C 1 -C 4 alkyl, Ar is a C containing a N atom 6 -C 10 heteroaryl; Wherein in the general formula (IVa), A is selected from O, S, NR A , R A Selected from H, C 1 -C 6 alkyl, There are multiple R on the tetrahydropyrrole ring shown f Group, each R f The groups are each independently selected from H, F, Cl, -CN, C 1 -C 6 Alkyl, α-chloroketo, optionally two R f The groups may be linked to each other to form a cycloalkyl group, preferably C 3 -C 7 Cycloalkyl; Wherein in the general formula (IVb), (IVc), (IVd) or (IVe), A is selected from O, S, NR A , R A Selected from H, methyl, ethyl, n-propyl, isopropyl.

12. A trifunctional compound, a pharmaceutically acceptable salt, a stereoisomer or a solvate thereof according to any one of claims 1 to 9, wherein the targeting group has the general formula (V), The peptide sequences are drawn from left to right in the direction of N-terminus to C-terminus. Xaa1 is the residue of an amino acid of the general formula (VI), in: R 1a Yes-NH-R 1a’ , R 1a’ is a protecting group or an amino acid, preferably the protecting group is R 1a” -C(O)- or R 1a” -S(O 2 )-, and R 1a” It is C 1 -C 6 Alkyl, wherein optionally, 1 -CH 2 -group is replaced by -S- or -O-, R 1b is H or methyl, g is 0 or 1, The carbonyl group of Xaa1 is covalently linked to the nitrogen of Xaa2. and the sulfur atom of Xaa1 is covalently linked to Yc as a thioether; Xaa2 and Xaa3 are independently selected from the residues of amino acids of formula (VII) or (VIII), in, h is 0, 1, or 2, i is 1 or 2, j is 1, 2, or 3, The amino acid of the general formula (VII) may be represented by a group consisting of methyl, OH, NH 2 and one or two substituents in F; Xaa4 is the residue of an amino acid of the general formula (IX), in, R 4a Selected from H, OH, COOH and CONH 2 , q is 1, 2 or 3, wherein optionally, the 1, 2 or 3 CH 2 One or two hydrogen atoms of the group are each and independently replaced by a methyl group or an ethyl group, R 4b is H or methyl; Xaa5 is the residue of an amino acid of the general formula (X), in: R 5a is OH or NH 2 , m is 1, 2 or 3, R 5b is H or methyl; Xaa6 is an amino acid selected from aromatic La-amino acids, preferably the residue of an amino acid of the general formula (XI), in, R 6a and R 6b are each independently selected from H, methyl, ethyl, propyl and isopropyl, preferably H, R 6c represents 0 to 3 substituents, each of which is independently selected from F, Cl, Br, NO 2 NH 2 , CN, CHF 3 , OH, OR 6d and C 1 -C 4 alkyl, R 6d is selected from methyl, ethyl, propyl and isopropyl, l is 0 or 1, preferably 0, The CR shown 6a R 6b (CH 2 ) l Ph 6c The moiety may constitute part of the covalent warhead C in formula (I), (II) or (III); Xaa7 is an aminothiol or amino acid residue of the general formula (XII), in, R 7a It is H, -COOH, -CONH 2 or CH 2 OH, n is 1 or 2; Yc is a structure of the general formula (XIII), It connects the S atom of Xaa1 and the S atom of Xaa7 while forming two thioether bonds, thereby forming a cyclic structure of the general formula (XIV), Wherein, the substitution pattern of the aromatic group in the general formula (XIII) is ortho, para or meta, g is 0 or 1, n is 1 or 2, Y 1 is CH or N, Y 2 Yes CR L1 , R L1 is a linker unit connected to other parts in general formula (I); The targeting group preferably has formula (Va), 13. The trifunctional compound, pharmaceutically acceptable salt, stereoisomer or solvate thereof according to any one of claims 1 to 12, wherein the payload P comprises at least one group selected from the following group, The first group, groups with at least one radionuclide, are selected from: Where R 2 , R 3 , R 4 and R 5 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, X is selected from 18 F. 123 I. 124 I. 125 I. 131 I and 211 At or its non-radioactive isotopes, q and r are independently integers between 0 and 4; The N on the triazole ring at the attachment site shown here may not be present on the payload P but rather on the linker unit; The second group, chelating groups that can chelate radionuclides, are selected from: The third group, optical dye groups, are selected from: The fourth group, small molecule cytotoxic drug group, is selected from:

14. The trifunctional compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein L 1 , when it is a trivalent linking group, that is, when the covalent warhead C is connected, or L 4 Independently selected from the following groups: L 1 To L 17 The divalent linking group units contained in are independently selected from the following groups: single bonds, The methylene group in the divalent linking unit listed may be replaced by one or more -COR L2 or -COOR L2 substituted by a group, wherein each R L2 Independently selected from H, C 1 -C 6 Alkyl, C 6 -C 10 Aryl, C 6 -C 10 Aryl substituted C 1 -C 6 alkyl; L 18 is a single bond, -CH 2 -、-NHCH 2 -,or Cy is selected from u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, v and o are independently 1, 2, 3, 4 or 5, Each R 6 and R 7 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, * is the site for connecting the target T, ** is the site for connecting the covalent warhead C, The condition is that L 1 To L 17 In the above-mentioned formula, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S.

15. The trifunctional compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein L 1 , when it is a trivalent linking group, that is, when the covalent warhead C is connected, or L 4 Independently selected from the following groups: L 1 To L 17 The divalent linking group units contained in are independently selected from the following groups: single bonds, The methylene group in the divalent linking unit listed may be replaced by one or more -COR L2 or -COOR L2 substituted by a group, wherein each R L2 Independently selected from H, C 1 -C 6 Alkyl, C 6 -C 10 Aryl, C 6 -C 10 Aryl substituted C 1 -C 6 alkyl; L 18 is a single bond, -CH 2 -、-NHCH 2 -,or Cy is selected from u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, u12, v and o are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, Each R 6 and R 7 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, * is the site for connecting the target T, ** is the site for connecting the covalent warhead C, The condition is that L 1 To L 17 In the above-mentioned formula, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S.

16. A trifunctional compound according to any one of claims 1 to 11 and 13 to 15, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has a structure of general formula (Ia), L 4 for L 4 Preferably, it has the following structure: Where R 6 , R 7 , L 18 , Cy, o and u have the definitions as set forth in claim 14 or 15.

17. The trifunctional compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has a structure of general formula (Ia) or general formula (Ib): in: L 4 is a trivalent linking group portion; Each shown L 5 , L 6 , L 7 and L 8 are independently divalent linker moieties, each of which independently comprises 0 to 6, preferably 0 to 3 linker units; The C shown is a covalent warhead selected from the group consisting of: Het is an optionally substituted C 4 -C 12 Heterocyclic group, or optionally substituted C 5 -C 12 Heteroaryl, wherein when the heteroaryl contains a N atom, the N atom may be present in a free form or in the form of an onium salt; Hal is F or Cl; Y is selected from O, S and NR 1 The group composed of; Each p is independently an integer between 0 and 6; Each R 1 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl; each R is independently selected from the group consisting of hydrogen, halogen, nitro, cyano, optionally substituted thiol, optionally substituted seleno, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted amino; T shown is a targeting group targeting fibroblast activation protein-α (FAP-α); P shown is a chelating group capable of chelating a radionuclide.

18. The trifunctional compound according to claim 17, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has a structure of general formula (Ia) or general formula (Ib), in: The L 4 is a trivalent linking group selected from the following: Each of the L 5 , L 6 , L 7 and L 8 are independently divalent linker moieties, each of which independently comprises 0 to 3, preferably 0 or 1 linker units, wherein the linker units are independently selected from the following groups: single bond, The methylene group in the divalent linking unit listed may be replaced by one or more -COR L2 or -COOR L2 substituted by a group, wherein each R L2 Independently selected from H, C 1 -C 6 Alkyl, C 6 -C 10 Aryl, C 6 -C 10 Aryl substituted C 1 -C 6 alkyl; L 18 is a single bond, -CH 2 -、-NHCH 2 -,or Cy is selected from u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, v and o are independently 1, 2, 3, 4 or 5, Each R 6 and R 7 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, * is the site for connecting T, ** is the site for connecting C, The condition is that L 5 , L 6 , L 7 and L 8 In the above, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S; C is selected from the group consisting of: in: Het is C 4 , C 5 or C 6 Heterocyclic group, or C 5 or C 6 Heteroaryl, the heterocyclic group or heteroaryl group contains one or two heteroatoms selected from N, O or S. When the heteroaryl group contains a N atom, the N atom may exist in a free form or in the form of an onium salt, preferably in a free form; preferably Het is C 4 , C 5 or C 6 Heterocyclic group, or C 5 or C 6 Heteroaryl, wherein the heterocyclic or heteroaryl group contains one heteroatom of N, O or S, preferably N; more preferably Het is azetidinyl, pyridyl, pyrrolyl, or N-oxypyridyl; Hal is F; Each p is independently an integer between 0 and 4; T shown is a targeting group targeting fibroblast activation protein-α (FAP-α); The P shown is a chelating group capable of chelating a radionuclide, which is selected from the following groups:

19. The trifunctional compound according to claim 17 or 18, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has a structure of general formula (Ia), in: C and P have the meanings as defined in claim 18. L 4 It has the following structure: Each of the L 5 and L 6 Independently, the following groups: single bond, wherein u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, v and o are independently 1, 2, 3, 4 or 5, Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C 1 -C 4 , * is the site for connecting T, ** is the site for connecting C, The condition is that L 5 and L 6 In the above, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S; T is the general formula (IV), preferably has the general formula (IVa), more preferably has the general formula (IVc), the general formula (IVd), the general formula (IVe) or the general formula (IVf), Wherein in the general formula (IV), A is selected from O, S, NR A , R A Selected from H, C 1 -C 6 alkyl, There are multiple R on the tetrahydropyrrole ring shown f Group, each R f The groups are each independently selected from H, F, Cl, -CN, -B(OH) 2 , C 1 -C 6 Alkyl, α-chloroketo, optionally two R f The groups may be linked to each other to form a cycloalkyl group, preferably C 3 -C 7 Cycloalkyl, B 1 and B 2 independently selected from O or S, R f1 and R f2 Independently selected from H, D or C 1 -C 4 alkyl, Ar is a C containing a N atom 6 -C 10 heteroaryl; Wherein in the general formula (IVa), A is selected from O, S, NR A , R A Selected from H, C 1 -C 6 alkyl, There are multiple R on the tetrahydropyrrole ring shown f Group, each R f The groups are each independently selected from H, F, Cl, -CN, C 1 -C 6 Alkyl, optionally two R on adjacent carbons f The groups may be linked to each other to form a cycloalkyl group, preferably C 3 -C 7 Cycloalkyl; Wherein in the general formula (IVb), (IVc), (IVd) or (IVe), A is selected from O, S, NR A , R A Selected from H, methyl, ethyl, n-propyl, isopropyl.

20. The trifunctional compound according to claim 17 or 18, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has a structure of general formula (Ib), in: In the general formula (Ib), C and P have the meanings as defined in claim 18. Each of the L 7 and L 8 Independently, the following groups: single bond, wherein u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, v and o are independently 1, 2, 3, 4 or 5, Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C 1 -C 4 , The condition is that L 7 and L 8 In the above, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S; T has the general formula (V), The peptide sequences are drawn from left to right in the direction of N-terminus to C-terminus. Xaa1 is the residue of an amino acid of the general formula (VI), in: R 1a Yes-NH-R 1a’ , R 1a’ is a protecting group or an amino acid, preferably the protecting group is R 1a” -C(O)- or R 1a” -S(O 2 )-, and R 1a” It is C 1 -C 6 Alkyl, wherein optionally, 1 -CH 2 -group is replaced by -S- or -O-, R 1b is H or methyl, g is 0 or 1, The carbonyl group of Xaa1 is covalently linked to the nitrogen of Xaa2. and the sulfur atom of Xaa1 is covalently linked to Yc as a thioether; Xaa2 and Xaa3 are independently selected from the residues of amino acids of formula (VII) or (VIII), in, h is 0, 1, or 2, i is 1 or 2, j is 1, 2, or 3, And the amino acid of the general formula (VII) can be represented by Selected from methyl, OH, NH 2 and one or two substituents in F; Xaa4 is the residue of an amino acid of the general formula (IX), in, R 4a Selected from H, OH, COOH and CONH 2 , q is 1, 2 or 3, wherein optionally, the 1, 2 or 3 CH 2 One or two hydrogen atoms of the group are each and independently replaced by a methyl group or an ethyl group, R 4b is H or methyl; Xaa5 is the residue of an amino acid of the general formula (X), in: R 5a is OH or NH 2 , m is 1, 2 or 3, R 5b is H or methyl; Xaa6 is an amino acid selected from aromatic La-amino acids, preferably the residue of an amino acid of the general formula (XI), in, R 6a and R 6b are each independently selected from H, methyl, ethyl, propyl and isopropyl, preferably H, R 6c represents 0 to 3 substituents, each of which is independently selected from F, Cl, Br, NO 2 NH 2 , CN, CHF 3 , OH, OR 6d and C 1 -C 4 alkyl, R 6d is selected from methyl, ethyl, propyl and isopropyl, l is 0 or 1, preferably 0, The CR shown 6a R 6b (CH 2 ) l Ph 6c The moiety may constitute a part of C in the general formula (Ib); Xaa7 is an aminothiol or amino acid residue of the general formula (XII), in, R 7a It is H, -COOH, -CONH 2 or CH 2 OH, n is 1 or 2; Yc is a structure of the general formula (XIII), It connects the S atom of Xaa1 and the S atom of Xaa7 while forming two thioether bonds, thereby forming a cyclic structure of the general formula (XIV), Wherein, the substitution pattern of the aromatic group in the general formula (XIII) is ortho, para or meta, g is 0 or 1, n is 1 or 2, Y 1 is CH or N, Y 2 Yes CR L1 , R L1 It is a linker unit connected to other parts in the general formula (Ib).

21. The trifunctional compound according to claim 17 or 18, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has a structure of general formula (Ib), in: L 7 , L 8 , C, and P have the meanings as set forth in claim 20, T has the general formula (V), The peptide sequences are drawn from left to right in the direction of N-terminus to C-terminus. Xaa1 is the residue of an amino acid of the general formula (VI), in: R 1a Yes-NH-R 1a’ , R 1a’ is a protecting group or an amino acid, preferably the protecting group is R 1a” -C(O)- or R 1a” -S(O 2 )-, and R 1a” It is C 1 -C 6 Alkyl, wherein optionally, 1 -CH 2 -group is replaced by -S- or -O-, R 1b is H or methyl, g is 0 or 1, The carbonyl group of Xaa1 is covalently linked to the nitrogen of Xaa2. and the sulfur atom of Xaa1 is covalently linked to Yc as a thioether; Xaa2 and Xaa3 are independently selected from the residues of amino acids of formula (VII) or (VIII), in, h is 0, 1, or 2, i is 1 or 2, j is 1, 2, or 3, The amino acid of the general formula (VII) may be represented by a group consisting of methyl, OH, NH 2 and one or two substituents in F; Xaa4 is the residue of an amino acid of the general formula (IX), in, R 4a Selected from H, OH, COOH and CONH 2 , q is 1, 2 or 3, wherein optionally, the 1, 2 or 3 CH 2 One or two hydrogen atoms of the group are each and independently replaced by a methyl group or an ethyl group, R 4b is H or methyl; Xaa5 is the residue of an amino acid of the general formula (X), in: R 5a is OH or NH 2 , m is 1, 2 or 3, R 5b is H or methyl; Xaa6 is the residue of an amino acid of the general formula (XI), in, R 6a and R 6b are each independently selected from H, methyl, ethyl, propyl and isopropyl, preferably H, R 6c represents 0 to 1 substituents, each of which is independently selected from F, Cl, NO 2 NH 2 , CN, CHF 3 , OH and C 1 -C 4 alkyl, R 6d is selected from methyl, ethyl, propyl and isopropyl, l is 0 or 1, preferably 0, The CR shown 6a R 6b (CH 2 ) l Ph 6c The moiety may constitute a part of C in the general formula (Ib); Xaa7 is an aminothiol or amino acid residue of the general formula (XII), in, R 7a is H or -COOH, n is 1 or 2; Yc is a structure of the general formula (XIII), It connects the S atom of Xaa1 and the S atom of Xaa7 while forming two thioether bonds, thereby forming a cyclic structure of the general formula (XIV), Wherein, the substitution pattern of the aromatic group in the general formula (XIII) is ortho, para or meta, g is 0 or 1, n is 1 or 2, Y 1 is CH or N, Y 2 Yes CR L1 , R L1 is a linker unit connected to other parts in general formula (Ib); Said T preferably has the formula (Va), 22. The trifunctional compound according to claim 4, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has a structure of general formula (Ia) or general formula (Ib): in: L 4 is a trivalent linking group portion; Each shown L 5 , L 6 , L 7 and L 8 are independently divalent linker moieties, each of which independently comprises 0 to 6, preferably 0 to 3 linker units; The C shown is a covalent warhead selected from the group consisting of: Het is an optionally substituted C 4 -C 12 Heterocyclic group, or optionally substituted C 5 -C 12 Heteroaryl, wherein when the heteroaryl contains a N atom, the N atom may be present in a free form or in the form of an onium salt; Hal is F or Cl; Y is selected from O, S and NR 1 The group composed of; Each p is independently an integer between 0 and 6; Each R 1 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl; each R is independently selected from the group consisting of hydrogen, halogen, nitro, cyano, optionally substituted thiol, optionally substituted seleno, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted amino; T shown is a targeting group targeting fibroblast activation protein-α (FAP-α); P shown is a chelating group capable of chelating a radionuclide.

23. The trifunctional compound according to claim 22, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has a structure of general formula (Ia) or general formula (Ib), in: The L 4 is a trivalent linking group selected from the following: Each of the L 5 , L 6 , L 7 and L 8 are independently divalent linker moieties, each of which independently comprises 0 to 3, preferably 0 or 1 linker units, wherein the linker units are independently selected from the following groups: single bond, The methylene group in the divalent linking unit listed may be replaced by one or more -COR L2 or -COOR L2 substituted by a group, wherein each R L2 Independently selected from H, C 1 -C 6 Alkyl, C 6 -C 10 Aryl, C 6 -C 10 Aryl substituted C 1 -C 6 alkyl; L 18 is a single bond, -CH 2 -、-NHCH 2 -,or Cy is selected from u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, u12, v and o are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, Each R 6 and R 7 are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, * is the site for connecting T, ** is the site for connecting C, The condition is that L 5 , L 6 , L 7 and L 8 In the above, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S; C is selected from the group consisting of: in: Het is C 4 , C 5 or C 6 Heterocyclic group, or C 5 or C 6 Heteroaryl, the heterocyclic group or heteroaryl group contains one or two heteroatoms selected from N, O or S. When the heteroaryl group contains a N atom, the N atom may exist in a free form or in the form of an onium salt, preferably in a free form; preferably Het is C 4 , C 5 or C 6 Heterocyclic group, or C 5 or C 6 Heteroaryl, wherein the heterocyclic or heteroaryl group contains one heteroatom of N, O or S, preferably N; more preferably Het is azetidinyl, pyridyl, pyrrolyl, or N-oxypyridyl; Hal is F; Each p is independently an integer between 0 and 4; T shown is a targeting group targeting fibroblast activation protein-α (FAP-α); The P shown is a chelating group capable of chelating a radionuclide, which is selected from the following groups:

24. The trifunctional compound according to claim 22 or 23, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has a structure of general formula (Ia), in: C and P have the meanings as defined in claim 23. L 4 It has the following structure: Each of the L 5 and L 6 Independently, the following groups: single bond, wherein u, u1, u2, u3, u4, u5, u6, u7, u8, u9, u10, u11, u12, v and o are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C 1 -C 4 , * is the site for connecting T, ** is the site for connecting C, The condition is that L 5 and L 6 In the above, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S; T is the general formula (IV), preferably has the general formula (IVa), more preferably has the general formula (IVc), the general formula (IVd), the general formula (IVe) or the general formula (IVf), Wherein in the general formula (IV), A is selected from O, S, NR A , R A Selected from H, C 1 -C 6 alkyl, There are multiple R on the tetrahydropyrrole ring shown f Group, each R f The groups are each independently selected from H, F, Cl, -CN, -B(OH) 2 , C 1 -C 6 Alkyl, α-chloroketo, optionally two R f The groups may be linked to each other to form a cycloalkyl group, preferably C 3 -C 7 Cycloalkyl, B 1 and B 2 independently selected from O or S, R f1 and R f2 Independently selected from H, D or C 1 -C 4 alkyl, Ar is a C containing a N atom 6 -C 10 heteroaryl; Wherein in the general formula (IVa), A is selected from O, S, NR A , R A Selected from H, C 1 -C 6 alkyl, There are multiple R on the tetrahydropyrrole ring shown f Group, each R f The groups are each independently selected from H, F, Cl, -CN, C 1 -C 6 Alkyl, optionally two R on adjacent carbons f The groups may be linked to each other to form a cycloalkyl group, preferably C 3 -C 7 Cycloalkyl; Wherein in the general formula (IVb), (IVc), (IVd) or (IVe), A is selected from O, S, NR A , R A Selected from H, methyl, ethyl, n-propyl, isopropyl.

25. The trifunctional compound according to claim 1 or 4, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, having one of the following structures:

26. The trifunctional compound according to any one of claims 13 to 25, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the effective load P is a chelating group capable of chelating a radionuclide, which chelates a radionuclide.

27. The trifunctional compound according to any one of claims 13 to 26, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the radionuclide carried or chelated by the trifunctional compound is a positron nuclide, or a beta emitter, or an alpha emitter, or an Auger electron emitting isotope, or an X-ray emitting isotope, or a fluorescent isotope, or a stable metal / non-metal element coordinated with a radionuclide, preferably 11 C. 13 N. 15 O. 18 F and its coordination elements, 47 Sc, 51 Cr, 67 Ga, 68 Ga, 86 Y. 90 Y. 64 Cu, 67 Cu, 72 As, 72 Se, 89 Zr, 97 Such as 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 8. 123 I. 124 I. 131 I. 142 Pr, 151 Eu, 153 Eu, 169 Eu, 159 Gd, 161 Tb, 177 Lu, 198 Au, 199 Ag, 201 Tl, 211 At 203 Pb, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 99m Tc, 111 In, 149 Pm, 153 Sm, 165 Dy, 169 2. 186 Re, 188 Re, 197 Hg, 227 Th, 67 Ga, 68 Ga, 86 Y. 90 Yes, 55 Yes 139 Yes 140 Yes 149 Tb 152 Tb 155 Tb 166 Cough, 175 Yes, 226 Th 223 Out 230 U.

28. A pharmaceutical composition comprising the trifunctional compound according to any one of claims 1 to 27, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, and a pharmaceutically acceptable carrier.

29. A kit comprising or consisting of the trifunctional compound according to any one of claims 1 to 27, a pharmaceutically acceptable salt, stereoisomer or solvate thereof or the pharmaceutical composition according to claim 28, and instructions for diagnosing a disease.

30. A method for diagnosing or treating a disease, preferably a disease involving FAP, comprising administering to a subject a therapeutically effective amount of a trifunctional compound according to any one of claims 1 to 27, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the disease is preferably a central nervous system disease, a metabolic disease, preferably a cardiovascular metabolic disease, or cancer.

31. The method of claim 30, wherein the cancer is selected from the group consisting of prostate cancer, breast cancer, pancreatic cancer, liver cancer, lung cancer, gastric cancer, kidney cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck cancer, thymic cancer, cervical cancer, endometrial cancer, neuroendocrine tumors, thyroid cancer, intestinal cancer, brain glioma and bone metastasis.

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