Trifunctional compound and application thereof

Abstract

The present invention relates to a trifunctional compound comprising at least one covalent warhead capable of forming a reversible or irreversible covalent linkage with a protein or tissue. The invention also relates to a pharmaceutical composition and a kit comprising the trifunctional compound, and the use of the trifunctional compound for diagnosing or treating diseases.

Description

A 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 drug conjugates (RDCs) are a major form of radiopharmaceuticals, generally binary conjugates of chelators, linkers, and targeting ligands. The ligand portion can be a small molecule, peptide, nucleic acid aptamer, antibody, etc. The linker mainly acts as a spacer to prevent the coupled chelator from significantly reducing the affinity of the targeting molecule and has a certain effect on regulating pharmacokinetic properties. The chelator portion can be coordinated with different diagnostic / therapeutic radionuclides by replacing the chelator according to different diagnostic 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, 211 At, 212Pb, 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, the FDA and EU approval of diagnostic / therapeutic RDCs based on prostate-specific membrane antigen (PSMA) and somatostatin receptor (SSTR) has encouraged more RDC 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 RDCs with a broader spectrum of activity. Furthermore, while some current RDCs achieve a certain level of uptake and retention in tumors, they suffer from significant side effects due to high background levels in non-target organs. There is a continued need for RDCs with high uptake specifically at their target sites.

[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. In addition to PSMA and SSTR mentioned above, tumor-related targets with specific high expression similar to the FAP target also include targets with corresponding RDC reports such as integrin (Intergrin, Front Oncol, 2022, 12, 837952) and chemokine receptor 4 (CXCR4, ChemMedChem 2011, 6, 1789), all of which are targets of the specific trifunctional compounds disclosed in this patent.

[0006] Covalent inhibitors are a class of inhibitors that exert their biological function 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 and 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. Covalent warheads that can be used in vivo and ultimately developed into drugs are generally "latent electrophiles," meaning they are activated and accelerate covalent bond formation only upon binding to specific proteins. This allows for excellent bio-orthogonality, 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, which react only with cysteine ​​residues, these warheads not only possess superior in vivo stability but also, after binding to the target protein, can covalently link to a wider range of nearby lysine, histidine, tyrosine, and serine residues, making them a promising new class of drug covalent warheads.

[0007] The present invention conducts innovative research based on existing technologies. By introducing covalent warheads such as fluorosulfate into the drug, a trifunctional compound is obtained. The uptake and retention of the trifunctional compound at target sites, such as tumors, are greatly improved, while the uptake in non-target organs can still be guaranteed to be always low, thus having significantly reduced toxicity compared to traditional strategies to improve tumor retention.

[0008] Summary of the Invention

[0009] One aspect of the present disclosure provides a trifunctional compound, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein the trifunctional compound has a structure of formula (I), formula (II), formula (III), formula (IV), or formula (V),

[0010] wherein P is a payload, T is a target, C is a covalent warhead, L1 to L8 are independently linker units, and a to h are independently integers of 0-6.

[0011] In another aspect, the present invention provides a pharmaceutical composition comprising the trifunctional compound as described above, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, and a pharmaceutically acceptable carrier.

[0012] In another aspect, the present invention provides a kit comprising the trifunctional compound as described above, a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, or a pharmaceutical composition as described above. In yet another aspect, the present invention provides a method for diagnosing or treating a disease, the method comprising administering to a subject a therapeutically effective amount of the trifunctional compound as described above, a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

[0013] In another aspect, the present invention provides use of the trifunctional compound as described above, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, for preparing a drug for diagnosing or treating a disease.

[0014] In some embodiments, in the trifunctional compound, P is a payload comprising at least one group comprising a radionuclide, or at least one chelating group capable of chelating a radionuclide, or at least one optical dye group;

[0015] T is a target, which contains at least one targeting group that can target a protein or an unknown target cell or specific tissue, wherein the targeting group is a small molecule, a polypeptide, or a nucleic acid aptamer;

[0016] C is a covalent warhead that can form a reversible or irreversible covalent bond with the protein or tissue targeted by T;

[0017] Each of L1, L2, L3, L4, L5, L6, L7 and L8 is independently a linker unit.

[0018] In some embodiments, in the trifunctional compound, C is selected from the group consisting of:

[0019] in:

[0020] Y is selected from O, S and NR 1 the group formed;

[0021] LG is a leaving group that can be replaced by a protein residue, preferably a halogen or OTs, and Ts is p-toluenesulfonyl;

[0022] R and R' are each independently selected from the group consisting of hydrogen, halogen, nitro, cyano, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted amino;

[0023] 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;

[0024] Ar is optionally substituted aryl, or optionally substituted heteroaryl;

[0025] Hal is a halogen, preferably F or Cl;

[0026] p is an integer between 0 and 12.

[0027] In a preferred embodiment, in the trifunctional compound, C is selected from the group consisting of:

[0028] in:

[0029] Ar is an optionally substituted C6-C 10 Aryl, preferably optionally substituted phenyl, or optionally substituted C5-C 12 heteroaryl;

[0030] Hal is F;

[0031] p is an integer between 0 and 6;

[0032] R is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl.

[0033] In some further preferred embodiments, in the trifunctional compound, the C is selected from the group consisting of:

[0034] in:

[0035] Ar is phenyl;

[0036] Hal is F;

[0037] p is an integer between 0 and 4.

[0038] In some embodiments, in the trifunctional compound, the targeting group targets a target selected from the following:

[0039] Fibroblast activation protein-α (FAP-α), prostate-specific membrane antigen (PSMA), polio receptor-associated protein 4 (Nectin-4), programmed death receptor ligand 1 (PD-L1), programmed death receptor 1 (PD-1), human epidermal growth factor receptor 2 (HER2), integrin, gastrin-releasing peptide receptor (GRPR), somatostatin receptor (SSTR), such as SSTR2, folate receptor (FR), such as folate receptor 1 (FOLR1), estrogen receptor (ER), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), chemokine receptor 4 (CXCR4), poly (ADP-ribose) polymerase (PARP), epidermal growth factor receptor (EGFR), fibroblast cytokine receptor (FGFR), disialoganglioside (GD2), vascular endothelial growth factor (VEGF), metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), insulin growth factor 1 (IGF-1), mesothelin (MSLN), tumor endothelial marker (TEM-1), interleukin 12 (IL-12), leukocyte differentiation antigen 13 (CD13), leukocyte differentiation antigen 19 (CD19), leukocyte differentiation antigen 20 (CD20), leukocyte differentiation antigen 22 (CD22), leukocyte differentiation antigen 33 (CD33), leukocyte differentiation antigen 37 (CD37), leukocyte differentiation antigen 38 (CD38), leukocyte differentiation antigen 44 (CD44), death receptor 5 (DR5), peroxidase 1 (Prdx I), large neutral amino acid transporter 1 (LAT1), angiotensin-converting enzyme 2 (ACE2), vesicular monoamine transporter 2 (VMAT2), dopamine transporter (DAT), and Sigma-1 receptor; or certain cell lines whose protein targets are unknown but can be targeted.

[0040] The targeting group optionally comprises one or more warheads or structures that can form reversible or irreversible covalent bonds with targeted proteins or tissues.

[0041] In a preferred embodiment, in the trifunctional compound, the targeting group targets fibroblast activation protein-α, prostate specific membrane antigen (PSMA), integrin, chemokine receptor 4 (CXCR4), folate receptor (FR), such as folate receptor 1 (FOLR1), polio receptor-related protein 4 (Nectin-4), or somatostatin receptor (SSTR), such as SSTR2, which is selected from the following structures:

[0042] The first group targets the structure of fibroblast activation protein-α, which has the general formula (VI), preferably the general formula (VII),

[0043] Wherein A is selected from O, S, NR A , R A Selected from H, C1-C6 alkyl,

[0044] 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;

[0045] Wherein A is selected from O, S, NR A , R A is selected from H, methyl, ethyl, n-propyl, isopropyl;

[0046] The second group: peptidomimetic groups having the general formula (VIII),

[0047] where R B -CH2R B’ , R B’ is aryl, preferably optionally substituted phenyl or naphthyl,

[0048] R c is H or alkyl,

[0049] w and x are independently 1, 2, 3 or 4,

[0050] The peptoid group is preferably of the following structure

[0051] The third group: cyclic peptide groups, which have the general formula (X),

[0052] Among them, each R D Independently selected from H, -CH2R D’ , -CH2COOH, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, and where R D’ is aryl, preferably optionally substituted phenyl or naphthyl, wherein the cyclic peptide is substituted by R D’ and / or R D The terminal -NH2 is connected to the rest of the trifunctional compound, or -CH2R D’ or R D’ May form part of covalent warhead C;

[0053] R E is H or methyl;

[0054] The cyclic peptide group preferably has the formula

[0055] The two wavy lines in each formula represent optional attachment sites;

[0056] The fourth group, the folic acid group, has the general formula (XI),

[0057] The dotted line indicates whether the bond exists or not, and only one of the two dotted bonds exists.

[0058] R FR1 , when present, is H or C1-C4 alkyl,

[0059] R FR2 、R FR3 、R FR4 are each independently selected from H or C1-C4 alkyl,

[0060] Ar G is aryl or heteroaryl, preferably optionally substituted phenyl or pyridyl,

[0061] L FR -CH2-R FR5 -,-(CH2)2-R FR5 -,-(CH2)3-R FR5 -,-(CH2)3-R FR5 -, or -(CH2)4-R FR5 -, R FR5 Selected from carbonyl (-CO-) or NR FR6 , R FR6 is H or C1-C4 alkyl,

[0062] The folic acid group is preferably of the following structure

[0063] The fifth group, a disulfide bond-containing cyclic group, has the general formula (XII),

[0064] Among them, ss1 , Αr ss2 and Al ss3 Each is independently selected from optionally substituted aryl or heteroaryl, preferably optionally substituted C6-C 10 Aryl or C5-C 10 Heteroaryl, preferably Ar ss1 , Αr ss3 is a C6-C 10 Aryl, Al ss2 C5-C 10 heteroaryl,

[0065] s1, s2 and s3 are each independently selected from 1, 2, 3 or 4,

[0066] wherein the disulfide-containing cyclic group is connected to the rest of the trifunctional compound via the site indicated by the wavy line, or -(CH2) s3 Al ss3 or Al ss3 may form part of the covalent warhead C,

[0067] The disulfide bond-containing cyclic group preferably has the structure

[0068] The sixth group, a bicyclic peptide group, preferably a bicyclic peptide group targeting polio receptor-associated protein 4 (Nectin-4),

[0069] More preferably, the bicyclic peptide group is the following structure

[0070] In some embodiments, in the trifunctional compound, the payload comprises at least one group selected from the group consisting of:

[0071] The first group, groups with at least one radionuclide, are selected from:

[0072] 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,

[0073] X is selected from 18 F. 123 I. 124 I. 125 I. 131 I and 211 At or its non-radioactive isotopes,

[0074] q and r are independently integers between 0 and 4;

[0075] The N on the triazole ring at the attachment site shown here may not be present on the payload but rather on the linker;

[0076] The second group, chelating groups that can chelate radionuclides, are selected from:

[0077] The third group, optical dye groups, is selected from:

[0078] In some embodiments, in the trifunctional compound, L1 and L8 are independently selected from the following groups:

[0079] Each of L2, L3, L4, L5, L6 and L7 is independently selected from the following groups:

[0080] single bond,

[0081] L9 is a single bond, -CH2-, -NHCH2-, or

[0082] Cy is selected from

[0083] a to h are independently 0, 1, 2 or 3,

[0084] u, v and o are independently 1, 2, 3, 4 or 5,

[0085] 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,

[0086] * and ** indicate the sites of attachment to two parts of the trifunctional compound, optionally via L2, L3, L4, L5, L6 or L7,

[0087] Provided that in the linking group composed of L1, L2, L3, L4, L5, L6, L7 and L8, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S.

[0088] In a preferred embodiment, the trifunctional compound has the structure of formula (I), at least one L1 is

[0089] L1 preferably has the following structure:

[0090] where R 6 、R 7 , L9, Cy, a, b, o and u have the definitions as described above.

[0091] In some embodiments, the trifunctional compound has the structure of formula (I):

[0092] in:

[0093] L1 is a trivalent linker unit,

[0094] Each L2 and L3 is independently a divalent linking unit;

[0095] a and b are independently integers between 0 and 6;

[0096] The C shown is a covalent warhead selected from the group consisting of:

[0097] in:

[0098] Ar is an optionally substituted C6-C 10 Aryl, preferably optionally substituted phenyl, or optionally substituted C5-C 12 heteroaryl;

[0099] Hal is F or Cl;

[0100] p is an integer between 0 and 6;

[0101] R is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl;

[0102] T shown is a targeting group targeting fibroblast activation protein-α (FAP-α);

[0103] P is a chelating group capable of chelating a radionuclide.

[0104] In a preferred embodiment, the trifunctional compound has the structure of formula (I),

[0105] in:

[0106] The L1 is a trivalent linking group selected from the following:

[0107] Each L2 and L3 is independently selected from the following groups:

[0108] single bond,

[0109] L9 is a single bond, -CH2-, -NHCH2-, or

[0110] Cy is selected from

[0111] a, b and p are independently 0, 1, 2 or 3,

[0112] u is independently 1, 2, 3, 4 or 5,

[0113] R 6 and R 7are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl,

[0114] * is the site for connecting T,

[0115] ** is the site for connecting C,

[0116] Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0117] C is selected from the group consisting of:

[0118] in:

[0119] Ar is phenyl;

[0120] Hal is F;

[0121] p is an integer between 0 and 4;

[0122] T shown is a targeting group targeting fibroblast activation protein-α (FAP-α);

[0123] The P shown is a chelating group capable of chelating radionuclides, which is selected from the following groups:

[0124] In a preferred embodiment, the trifunctional compound has the structure of formula (I),

[0125] in:

[0126] C and P have the meanings as stated above.

[0127] L1 has the following structure:

[0128] a and b are both 1,

[0129] L2 and L3 are independently the following groups:

[0130] single bond,

[0131] wherein u and o are independently 1, 2, 3, 4 or 5,

[0132] Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4,

[0133] * is the site for connecting T,

[0134] ** is the site for connecting C,

[0135] Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0136] T is a compound of the general formula (VI), preferably a compound of the general formula (VII),

[0137] Wherein A is selected from O, S, NR A , R A Selected from H, C1-C6 alkyl,

[0138] 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;

[0139] Wherein A is selected from O, S, NR A , R A Selected from H, methyl, ethyl, n-propyl, isopropyl.

[0140] In some embodiments, the trifunctional compound has the structure of formula (I),

[0141] in:

[0142] The L1 is a trivalent linking group selected from the following:

[0143] Each L2 and L3 is independently selected from the following groups:

[0144] single bond,

[0145] L9 is a single bond, -CH2-, -NHCH2-, or

[0146] Cy is selected from

[0147] a and b are independently 0, 1, 2 or 3,

[0148] u is independently 1, 2, 3, 4 or 5,

[0149] R 6 and R 7are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl,

[0150] * is the site for connecting T,

[0151] ** is the site for connecting C,

[0152] Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0153] C is selected from the group consisting of:

[0154] in:

[0155] Ar is phenyl;

[0156] Hal is F;

[0157] p is an integer between 0 and 4;

[0158] The T shown is a targeting group targeting prostate-specific membrane antigen (PSMA);

[0159] The P shown is a chelating group capable of chelating radionuclides, which is selected from the following groups:

[0160] In a preferred embodiment, the trifunctional compound has the structure of formula (I),

[0161] in:

[0162] C and P have the meanings as stated above.

[0163] L1 has the following structure:

[0164] a, b are both 1,

[0165] L2 and L3 are independently the following groups:

[0166] single bond,

[0167] wherein u and o are independently 1, 2, 3, 4 or 5,

[0168] Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4,

[0169] * is the site for connecting T,

[0170] ** is the site for connecting C,

[0171] Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0172] Said T has the general formula (VIII),

[0173] where R B -CH2R B’ , R B’ is aryl, preferably optionally substituted phenyl or naphthyl,

[0174] R c is H or alkyl,

[0175] w and x are independently 1, 2, 3 or 4,

[0176] The T is preferably the following structure

[0177] In some embodiments, the trifunctional compound has the structure of formula (I),

[0178] in:

[0179] The L1 is a trivalent linking group selected from the following:

[0180] Each L2 and L3 is independently selected from the following groups:

[0181] single bond,

[0182] L9 is a single bond, -CH2-, -NHCH2-, or

[0183] Cy is selected from

[0184] a and b are independently 0, 1, 2 or 3,

[0185] u is independently 1, 2, 3, 4 or 5,

[0186] 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,

[0187] * is the site for connecting C,

[0188] ** is the site for connecting P,

[0189] Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0190] C is selected from the group consisting of:

[0191] in:

[0192] Ar is phenyl;

[0193] Hal is F;

[0194] p is an integer between 0 and 4;

[0195] T is a targeting group targeting a folate receptor (FR), such as folate receptor 1 (FOLR1);

[0196] The P shown is a chelating group capable of chelating radionuclides, which is selected from the following groups:

[0197] In a preferred embodiment, the trifunctional compound has the structure of formula (I),

[0198] in:

[0199] C and P have the meanings as stated above.

[0200] L1 has the following structure:

[0201] a, b are both 1,

[0202] L2 and L3 are independently the following groups:

[0203] single bond,

[0204] where u is 1, 2, 3, 4, or 5,

[0205] Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4,

[0206] * is the site for connecting C,

[0207] ** is the site for connecting P,

[0208] Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0209] Said T has the general formula (XI),

[0210] The dotted line indicates whether the bond exists or not, and only one of the two dotted bonds exists.

[0211] R FR1 , when present, is H or C1-C4 alkyl,

[0212] R FR2 、R FR3 、R FR4 are each independently selected from H or C1-C4 alkyl,

[0213] Ar G is aryl or heteroaryl, preferably optionally substituted phenyl or pyridyl,

[0214] L FR -CH2-R FR5 -,-(CH2)2-R FR5 -,-(CH2)3-R FR5 -,-(CH2)3-R FR5 -, or -(CH2)4-R FR5 -, R FR5 Selected from carbonyl (-CO-) or NR FR6 , R FR6 is H or C1-C4 alkyl,

[0215] The T is preferably the following structure

[0216] In some embodiments, the trifunctional compound has the structure of formula (I),

[0217] in:

[0218] The L1 is a trivalent linking group selected from the following:

[0219] Each L2 and L3 is independently selected from the following groups:

[0220] single bond,

[0221] L9 is a single bond, -CH2-, -NHCH2-, or

[0222] Cy is selected from

[0223] a and b are independently 0, 1, 2 or 3,

[0224] u is independently 1, 2, 3, 4 or 5,

[0225] 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,

[0226] * is the site for connecting T,

[0227] ** is the site for connecting C,

[0228] Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0229] C is selected from the group consisting of:

[0230] in:

[0231] Ar is phenyl;

[0232] Hal is F;

[0233] p is an integer between 0 and 4;

[0234] T shown is a targeting group targeting polio receptor-associated protein 4 (Nectin-4);

[0235] The P shown is a chelating group capable of chelating radionuclides, which is selected from the following groups:

[0236] In a preferred embodiment, the trifunctional compound has the structure of formula (I),

[0237] in:

[0238] C and P have the meanings as stated above.

[0239] L1 has the following structure:

[0240] a, b are both 1,

[0241] L2 and L3 are independently the following groups:

[0242] single bond,

[0243] where u is 1, 2, 3, 4, or 5,

[0244] Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4,

[0245] * is the site for connecting T,

[0246] ** is the site for connecting C,

[0247] Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0248] The T is a bicyclic peptide group, preferably a bicyclic peptide group targeting poliomyelitis receptor-associated protein 4 (Nectin-4),

[0249] More preferably, the bicyclic peptide group is the following structure

[0250] In some embodiments, the trifunctional compound has the structure of formula (II):

[0251] in:

[0252] Each L4 and L5 shown is independently a divalent linking unit;

[0253] c and d are independently integers from 0 to 6;

[0254] The C shown is a covalent warhead selected from the group consisting of:

[0255] in:

[0256] Ar is an optionally substituted C6-C 10 Aryl, preferably optionally substituted phenyl, or optionally substituted C5-C 12 heteroaryl;

[0257] Hal is F or Cl;

[0258] p is an integer between 0 and 6;

[0259] R is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl;

[0260] T shown is a targeting group targeting chemokine receptor 4 (CXCR4);

[0261] P is a chelating group capable of chelating a radionuclide.

[0262] In a preferred embodiment, the trifunctional compound has the structure of formula (II),

[0263] in:

[0264] Each L4 and L5 is independently selected from the following groups:

[0265] single bond,

[0266] c and d are independently 0, 1, 2 or 3,

[0267] u is independently 1, 2, 3, 4 or 5,

[0268] 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,

[0269] Provided that in the linker formed by L4 and L5, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0270] C is selected from the group consisting of:

[0271] in:

[0272] Ar is phenyl;

[0273] Hal is F;

[0274] p is an integer between 0 and 3;

[0275] T shown is a targeting group targeting chemokine receptor 4 (CXCR4);

[0276] The P shown is a chelating group capable of chelating radionuclides, which is selected from the following groups:

[0277] In a preferred embodiment, the trifunctional compound has the structure of formula (II),

[0278] in:

[0279] C and P have the meanings as stated above.

[0280] c and d are both 1,

[0281] L4 and L5 are independently the following groups:

[0282] single bond,

[0283] wherein u and o are independently 1, 2, 3, 4 or 5,

[0284] Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4,

[0285] Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0286] T has the general formula (X),

[0287] Among them, each R D Independently selected from H, -CH2R D’ , -CH2COOH, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, and where R D’ is aryl, preferably optionally substituted phenyl or naphthyl, wherein the cyclic peptide is substituted by R D’ and / or R D The terminal -NH2 is connected to the rest of the trifunctional compound, or -CH2R D’ or R D’ May form part of covalent warhead C;

[0288] R E is H or methyl;

[0289] Said T preferably has the formula

[0290] The two wavy lines in each formula represent optional attachment sites.

[0291] In some embodiments, the trifunctional compound has the structure of formula (II):

[0292] in:

[0293] Each L4 and L5 shown is independently a divalent linking unit;

[0294] c and d are independently integers from 0 to 6;

[0295] The C shown is a covalent warhead selected from the group consisting of:

[0296] in:

[0297] Ar is an optionally substituted C6-C 10 Aryl, preferably optionally substituted phenyl, or optionally substituted C5-C 12 heteroaryl;

[0298] Hal is F or Cl;

[0299] p is an integer between 0 and 6;

[0300] R is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl;

[0301] T is a targeting group targeting somatostatin receptor (SSTR), such as SSTR2;

[0302] P is a chelating group capable of chelating a radionuclide.

[0303] In a preferred embodiment, the trifunctional compound has the structure of formula (II),

[0304] in:

[0305] Each L4 and L5 is independently selected from the following groups:

[0306] single bond,

[0307] c and d are independently 0, 1, 2 or 3,

[0308] u is independently 1, 2, 3, 4 or 5,

[0309] 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,

[0310] Provided that in the linker formed by L4 and L5, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S;

[0311] C is selected from the group consisting of:

[0312] in:

[0313] Ar is phenyl;

[0314] Hal is F;

[0315] p is an integer between 0 and 3;

[0316] T is a targeting group targeting somatostatin receptor (SSTR), such as SSTR2;

[0317] The P shown is a chelating group capable of chelating radionuclides, which is selected from the following groups:

[0318] In a preferred embodiment, the trifunctional compound has the structure of formula (II),

[0319] in:

[0320] C and P have the meanings as stated above.

[0321] c and d are both 1,

[0322] L4 and L5 are independently the following groups:

[0323] single bond,

[0324] wherein u and o are independently 1, 2, 3, 4 or 5,

[0325] Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4,

[0326] Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are O, N or S; and T has the general formula (XII),

[0327] Among them, ss1 , Αr ss2 and Al ss3 Each is independently selected from optionally substituted aryl or heteroaryl, preferably optionally substituted C6-C 10 Aryl or C5-C 10 Heteroaryl, preferably Ar ss1 , Αr ss3 is a C6-C 10 Aryl, Al ss2 C5-C 10 heteroaryl,

[0328] s1, s2 and s3 are each independently selected from 1, 2, 3 or 4,

[0329] wherein the T is connected to the rest of the trifunctional compound via the site indicated by the wavy line, or -(CH2) s3 Al ss3 or Al ss3 may form part of the covalent warhead C,

[0330] Said T preferably has the structure

[0331] In a most preferred embodiment, the trifunctional compound has one of the following structures:

[0332] In some further preferred embodiments, the payload is a chelating group capable of chelating radionuclides, which chelates radionuclides.

[0333] In some embodiments, 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 metals, 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、 197Hg, 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.

[0334] In some embodiments, the present invention also relates to a pharmaceutical composition comprising the trifunctional compound, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, and a pharmaceutically acceptable carrier.

[0335] In some embodiments, the present invention also relates to a kit comprising or consisting of the trifunctional compound as described above, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, or a pharmaceutical composition as described above, and instructions for diagnosing a disease.

[0336] In some embodiments, the present invention also relates to a method for diagnosing or treating a disease, comprising administering to a subject a therapeutically effective amount of the aforementioned trifunctional compound, 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.

[0337] In a preferred embodiment, 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

[0338] Figure 1 shows the general mechanism of action of covalent inhibitors.

[0339] Figure 2 shows 68 Radioactivity purity spectrum of Ga-FAPI-CB-02.

[0340] Figure 3 shows that after protein co-incubation-gel electrophoresis-radioautography-Coomassie blue staining, 177 Covalent attachment efficiency of Lu-FAPI-CB-01 to FAP target protein. From left to right, the incubation time for each lane is 1, 3, 5, 7, 9, 11, and 30 minutes, respectively.

[0341] Figure 4 shows 177 Covalent linkage efficiency of Lu-PSMA-CB-05 and PSMA human recombinant protein.

[0342] Figure 5 shows 177 Covalent linkage efficiency of Lu-FLOR-CB-06 and FOLR1 human recombinant protein.

[0343] FIG6 shows the molecular affinity and binding kinetics curves measured by surface plasmon resonance experiments.

[0344] FIG7 shows the kinetic parameters of FAPI-04 control, hydro-FAPI-CB-02 control (easily obtained by hydrolysis of FAPI-CB-02, structure refer to FIG12a ), FAPI-CB-01 and FAPI-CB-02 measured in single cycle mode.

[0345] FIG8 shows nat Diagram of some modification sites of Lu-FAPI-CB-01 and FAP human recombinant protein.

[0346] Figure 9 shows the IC of FAPI-CB-01 for HT-1080-FAP cells 50 picture.

[0347] FIG10 shows 68 Ga-FAPI-CB-00 and 68 PET / CT contrast imaging of Ga-FAPI-04 in the HT-1080-FAP mouse model.

[0348] FIG11 shows 68 Ga-FAPI-CB-01 and 68 PET / CT contrast imaging of Ga-FAPI-04 in the HT-1080-FAP mouse model.

[0349] FIG12 shows 68 Ga-FAPI-CB-02 and 68 PET / CT contrast imaging of Ga-FAPI-04 in the HT-1080-FAP tumor-bearing mouse model.

[0350] FIG13 shows 86 Y-FAPI-CB-02 and 86 PET / CT contrast imaging of Y-FAPI-04 in the HT-1080-FAP mouse model at 9 hours.

[0351] FIG14 shows the advantages of the compound of the present invention compared to FAPI-04 in the HT-1080-FAP tumor mouse model, specifically showing that the compound of the present invention 86The integrated area under the tumor uptake-time curve (AUC) of Y-labeled FAPI-CB was significantly improved.

[0352] FIG15 shows 68 Uptake of Ga-FAPI-CB-02 in various organs.

[0353] FIG16 shows the advantages of the compound of the present invention compared to FAPI-04 in the HT-1080-FAP tumor mouse model, specifically showing that the present invention 68 The tumor uptake value of Ga-labeled FAPI-CB was significantly increased.

[0354] FIG17 shows the binding selectivity of the precursor molecule to the cognate protein of the target.

[0355] Figure 18 shows the tumor mouse model 177 Lu nuclide therapy experiment.

[0356] Figure 19 shows the tumor mouse model 225 Ac nuclide therapy experiment.

[0357] FIG20 shows the advantages of the compound of the present invention compared to PSMA-617 in a PSMA high-expressing tumor mouse model, specifically showing that the present invention 68 The tumor uptake value of Ga-labeled PSMA-CB was significantly increased.

[0358] FIG21 shows the advantages of the compound of the present invention compared to DOTATATE in a mouse model with high SSTR2 expression tumor, specifically showing that the compound of the present invention 68 The tumor uptake value of Ga-labeled SSTR2-CB was significantly increased.

[0359] FIG22 shows the advantages of the compound of the present invention compared with FOLR1-control in a FOLR1 high-expression tumor mouse model, specifically showing that the present invention 68 The tumor uptake value of Ga-labeled FOLR1-CB was significantly increased.

[0360] FIG23 shows the advantages of the compound of the present invention compared to pentixafor in a CXCR4 high-expressing tumor mouse model, specifically showing that the present invention 68 The tumor uptake value of Ga-labeled CXCR4-CB was significantly increased.

[0361] Figure 24 shows the advantages of the compound of the present invention compared to NCT4-control in a Nectin-4 high-expression tumor mouse model, specifically showing that the present invention 68 The tumor uptake value of Ga-labeled Nectin4-CB was significantly increased. DETAILED DESCRIPTION

[0362] The present invention obtains a trifunctional compound by introducing a fluorosulfate covalent warhead into the 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.

[0363] 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.

[0364] definition

[0365] 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.

[0366] 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.

[0367] 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."

[0368] 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.

[0369] The term "subject" or "patient" as used in this application includes humans and mammals.

[0370] In the context of this application, the term "treatment" may also include prophylaxis, unless specifically stated to the contrary.

[0371] 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.

[0372] 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.

[0373] 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, wherein preferably 3, 4, 5, 6, 7, 8, 9 or 10 atoms form the ring, for example, 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 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-membered 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. Examples of cycloalkyl groups 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 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.

[0374] 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.

[0375] 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. Examples are 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-benzofuranyl, 2-benzofuranyl, benzotriazolyl, benzothiophene, 2-benzothiophene, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoloxazolyl, 2,1-benzoxazolyl, benzothiazolyl, 1,2-benzisothiazolyl, 2,1-benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, quinolinyl, 1,2,3-benzotriazinyl or 1,2,4-benzotriazinyl.

[0376] 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.

[0377] The expression "optionally substituted" means that one, two, three or more hydrogen atoms in the group may be replaced independently of one another by respective substituents. The substituents may be selected from C1-6 alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, halogen, cyano, amino, nitro, -OH, -COOH.

[0378] 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.

[0379] 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.

[0380] 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."

[0381] 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.

[0382] The term "nuclear drug molecule" or "nuclear drug" refers to a molecule or compound that carries or chelates a radionuclide.

[0383] 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.

[0384] The term "PSMA-targeted" structure refers to a fragment derived from a PSMA-targeting molecule, such as a molecular fragment formed by the compounds disclosed in WO2015055318A1 and Journal of Nuclear Medicine, 2015, 56,914.

[0385] 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.

[0386] Suitable pharmaceutically acceptable salts of the compounds of the present disclosure may be acid addition salts of compounds of the present disclosure having a nitrogen atom in a chain or ring with sufficient basicity, such as acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, or acid addition salts formed 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-hydroxyethanesulfonic 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.

[0387] 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.

[0388] 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.

[0389] 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.

[0390] 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.

[0391] 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.

[0392] 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.

[0393] 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.

[0394] "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.

[0395] 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.

[0396] The term "halogen" refers to fluorine, chlorine, bromine, iodine and astatine.

[0397] The term "optionally" means that the situation may or may not occur.

[0398] 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.

[0399] Example

[0400] Reagents and models used

[0401] 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.

[0402] All reagents and compounds synthesized can be purchased through general commercial channels in China (excluding Hong Kong, Macao and Taiwan). Suppliers include WuXi AppTec (China), Shanghai Bid Pharmaceutical Technology Co., Ltd., Sigma-Aldrich (USA), Sichuan Shangfu Technology Co., Ltd., Anage Chemical, and Cytiva (USA).

[0403] Nuclide: 68 GaCl3 is obtained by eluting with 0.6M hydrochloric acid 68 Ge- 68 Ga generator (iThemba LABS, South Africa).86 YCl3 was produced using the 14.6-MeV cyclotron of the China Academy of Atomic Energy. 177 LuCl3 was dissolved in 0.1 M hydrochloric acid and purchased from ITG (Germany).

[0404] Recombinant protein: Human recombinant protein FAP-His was purchased from Jinan Protein Co., Ltd. (China).

[0405] 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).

[0406] 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.

[0407] Instruments used

[0408] Compound preparation and identification: High-performance liquid chromatography (Waters), high-performance liquid chromatography radioactivity detector (Eckert & Ziegler Group), ultra-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), Cytiva surface plasmon resonance (Biacore 8K).

[0409] Cell experiments: Fluorescence confocal microscopy (Nikon A1R-si).

[0410] Animal experiments: Small animal PET / CT (Mediso PET122S).

[0411] Example 1 Synthesis of Compounds

[0412] The compounds FAPI-CB-01 and FAPI-CB-02 of the present invention can be synthesized via the above synthesis route; some FAP-targeting compounds in the patent can also be synthesized via similar synthesis routes.

[0413] Example 1a. Synthesis of FAPI-CB-01

[0414] Starting materials Compound 1 and Int 1 were synthesized by WuXi AppTec (China) from commercially available raw materials using a published route. Compound 1 (2.40 g, 5.08 mmol) was dissolved in dichloromethane (30.0 ml), and N-hydroxysuccinimide (935 mg, 8.13 mmol) and the condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.66 g, 8.63 mmol) were added. The reaction mixture was stirred at 25°C for 1 hour. Raw material 1A (Shanghai Bidex, 750 mg, 4.45 mmol) was added to the stirred solution, followed by N,N-diisopropylethylamine (2.35 g, 18.2 mmol, 3.17 ml). The reaction mixture was stirred at 25°C for 11 hours. The reaction mixture was freed from the solvent under reduced pressure, and the crude product was purified by reverse phase high performance liquid chromatography (water / acetonitrile) to give a red solid compound 2 (1.56 g, yield 58.9%, purity 90.0%). The product was characterized by liquid chromatography-mass spectrometry (LCMS), nuclear magnetic resonance (NMR) and HPLC-MS / MS. 1 H NMR confirmed: LCMS ([M+H] + =587.3, Rt=0.73 min); 1 H NMR 400MHz, DMSO-d6, δppm: 9.10-9.07(m,1H),8.80(d,J=4.4Hz,1H),7.97(d,J=9.6Hz,1H),7.87-7.86(m,1H),7.50(d,J=4.0Hz,1H),7.46 -7.44(m,1H),5.15-5.12(m,1H),4.29-4.10(m,6H),2.96-2.91(m,3h),2.87(s,1H),2.42-2.37(m,4H),1.99-1.95(m,2H),1.39(s,9H).

[0415] Compound 2 (350 mg, 596 μmol) was dissolved in acetonitrile (2.00 ml), p-toluenesulfonic acid (328 mg, 1.91 mmol) was added, and the reaction was stirred at 30°C for 48 hours. The reaction mixture was stripped of solvent under reduced pressure to give a red solid compound 3 (380 mg, 96.7% yield, 90.0% purity, p-toluenesulfonate salt), which was confirmed by LCMS: LCMS ([M+H] + =487.2, Rt=0.50 min).

[0416] Int 1 (purchased from WuXi AppTec, 400 mg, 499 μmol) and compound 3 (390 mg, 592 μmol, p-toluenesulfonate) were dissolved in dimethylformamide (2.00 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 265 mg, 699 μmol) and N,N-diisopropylethylamine (258 mg, 2.00 mmol, 347 μL) were added as condensing agents, and the mixture was stirred at 25°C for 12 hours. The reaction mixture was filtered, and the filtrate was purified by reverse phase HPLC (water / acetonitrile). After lyophilization, a light yellow solid, compound 4 (270 mg, 42.5% yield), was obtained. LCMS ([M+H] + =1269.8, Rt=0.80 min).

[0417] Compound 4 (270 mg, 212 μmol) was dissolved in dichloromethane (2.00 ml), and trifluoroacetic acid (6.6 g, 58.3 mmol, 4.32 ml) and triethylsilane (247 mg, 2.13 mmol, 339 μl) were added. The reaction was carried out under reduced pressure to remove the solvent, and the residue was purified by reverse phase HPLC (water / acetonitrile) to give compound 5 (122.3 mg, 50.8% yield, 95.38% purity, trifluoroacetate) as a light yellow solid. The product was purified by LCMS, HPLC, 1 H NMR confirmed: LCMS ([M+H] + =1001.3,[(M+H+H) / 2] + =501.9, Rt=1.087 min); HPLC: separation column Gemini C18 5 μm 110A 150*4.6 mm, mobile phase A: deionized water containing 0.1% trifluoroacetic acid, mobile phase B: acetonitrile containing 0.075% trifluoroacetic acid, flow rate 1 mL / min, Rt=10.889 min, purity 95.38%; 1H NMR 400MHz, DMSO-d6, δppm: 9.13(br s,1H),8.80(d,J=4.0Hz,1H),8.28(s,1H),8.21(s,1H),7.98(d,J=9.2Hz,1 H),7.87(d,J=2.0Hz,1H),7.51(d,J=4.4Hz,1H),7.45(dd,J=9.2,2.0Hz,1H) ,5.14(d,J=7.6Hz,1H),4.69(s,1H),4.43-4.08(m,6H),4.05-2.53(m,32H), 2.49-2.27(m,6H),2.05-1.90(s,2H),1.78-1.42(m,4H),1.40-1.20(m,2H). Compound Int 2 was prepared as described in the literature (J. Am. Chem. Soc. 2021, 143, 3753). Compound 5 (10.0 mg, 10 μmol) was dissolved in dimethyl sulfoxide (0.2 mL). N,N-diisopropylethylamine (6.5 mg, 50 μmol, 8.8 μL) was added, followed by Int 2 (2.7 mg, 20 μmol). The mixture was reacted at 25°C for 2 hours. After the reaction mixture was filtered, the filtrate was purified by reverse phase HPLC (water / acetonitrile) and lyophilized to obtain the final product, FAPI-CB-01, as a light yellow solid (270 mg, 85.5% yield). The product was confirmed by UPLC-MS (ultra-performance liquid chromatography-mass spectrometry: separation column Acquity UPLC BEH C18 1.7 μm, 2.1×50 mm; 0-0.2 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid); 0.2-3.0 min, linear gradient: 10-95% acetonitrile (containing 0.1% trifluoroacetic acid); 3-4.5 min, linear gradient: 95-10% acetonitrile (containing 0.1% trifluoroacetic acid); 4.5-5.0 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid), flow rate 0.5 mL / min, UPLC-MS ([M+H] + =1203.8,R t =2.7 min, purity 98.5%).

[0418] Example 1b. Synthesis of FAPI-CB-02

[0419] Compound Int 3 was prepared according to the literature (J. Am. Chem. Soc. 2021, 143, 3753). Compound 5 (10.0 mg, 10 μmol) was dissolved in dimethyl sulfoxide (0.2 mL), and N,N-diisopropylethylamine (6.5 mg, 50 μmol, 8.8 μL) was added. Int 3 (2.7 mg, 20 μmol) was then added, and the mixture was reacted at 25°C for 2 hours. After the reaction mixture was filtered, the filtrate was purified by reverse phase HPLC (water / acetonitrile) and lyophilized to obtain the final product, FAPI-CB-02, as a light yellow solid (278 mg, 88.0% yield). The product was confirmed by UPLC-MS (ultra-performance liquid chromatography-mass spectrometry, Waters): separation column: Acquity UPLC BEH C18 1.7 μm, 2.1×50 mm; 0-0.2 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid); 0.2-3.0 min, linear gradient: 10-95% acetonitrile (containing 0.1% trifluoroacetic acid); 3-4.5 min, linear gradient: 95-10% acetonitrile (containing 0.1% trifluoroacetic acid); 4.5-5.0 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid), flow rate 0.5 mL / min, UPLC-MS ([M+H] + =1203.8, R t =2.7 min, purity 98.6%).

[0420] Example 1c. Synthesis of AF488-FAPI-CB-02

[0421] The compound AF488-FAPI-CB-02 of the present invention can be synthesized through the above synthetic route.

[0422] Int 4 (Shanghai Bid, 277 mg, 592 μmol) and compound 3 (390 mg, 592 μmol, p-toluenesulfonate) were dissolved in N,N-dimethylformamide (2.00 ml), and condensing agent 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 265 mg, 699 μmol) and N,N-diisopropylethylamine (258 mg, 2.00 mmol, 347 μL) were added and stirred at 25 ° C for 12 hours. After the reaction mixture was filtered, the filtrate was purified by reverse phase HPLC (water / acetonitrile) and lyophilized to obtain compound 6 as a light yellow solid (221 mg, 40% yield). The product was confirmed by UPLC-MS (ultra-performance liquid chromatography-mass spectrometry, Waters): separation column: Acquity UPLC BEH C18 1.7 μm, 2.1×50 mm; 0-0.2 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid); 0.2-3.0 min, linear gradient: 10-95% acetonitrile (containing 0.1% trifluoroacetic acid); 3-4.5 min, linear gradient: 95-10% acetonitrile (containing 0.1% trifluoroacetic acid); 4.5-5.0 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid), flow rate 0.5 mL / min, UPLC-MS ([M+H] + =937.4, Rt=2.8min, purity 99.2%)

[0423] Compound 6 (20 mg, 21 mmol) was dissolved in dichloromethane (200 μL), and trifluoroacetic acid (611 mg, 5.4 mmol, 400 μL) was added. The reaction solvent was removed under reduced pressure, and the crude residue was purified by reverse phase HPLC (water / acetonitrile) to give compound 7 (12 mg, 67% yield, trifluoroacetic acid salt) as a light yellow solid. UPLC-MS (ultra-performance liquid chromatography-mass spectrometry, Waters) was used for confirmation: separation column: Acquity UPLC BEH C18 1.7 μm, 2.1×50 mm; 0-0.2 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid); 0.2-3.0 min, linear gradient: 10-95% acetonitrile (containing 0.1% trifluoroacetic acid); 3-4.5 min, linear gradient: 95-10% acetonitrile (containing 0.1% trifluoroacetic acid); 4.5-5.0 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid), flow rate: 0.5 mL / min, UPLC-MS ([M+H] + =837.4, Rt=2.4 min, purity 98.6%).

[0424] Compound 7 (12 mg, 14.3 μmol) was dissolved in dimethyl sulfoxide (200 μL), and N,N-diisopropylethylamine (9.3 mg, 71.5 μmol, 12.6 μL) was added followed by Int 3 (6.8 mg, 21.5 μmol). The mixture was reacted at 25°C for 1 hour. The reaction mixture was filtered, and the filtrate was purified by reverse phase HPLC (water / acetonitrile) and lyophilized to give a light yellow compound 8 (6.2 mg, 6.0 μmol, 42% yield). The product was confirmed by UPLC-MS (ultra-performance liquid chromatography-mass spectrometry, Waters): separation column: Acquity UPLC BEH C18 1.7 μm, 2.1×50 mm; 0-0.2 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid); 0.2-3.0 min, linear gradient: 10-95% acetonitrile (containing 0.1% trifluoroacetic acid); 3-4.5 min, linear gradient: 95-10% acetonitrile (containing 0.1% trifluoroacetic acid); 4.5-5.0 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid), flow rate 0.5 mL / min, UPLC-MS ([M+H] + =1039.4, Rt=2.7 min, purity 98.8%).

[0425] Compound 8 (6.2 mg, 6.0 μmol) was dissolved in N,N-dimethylformamide (200 μL), and piperidine (34 mg, 400 μmol, 40 μL) was added. The mixture was reacted at 25°C for 1 hour and purified by reverse phase HPLC (water / acetonitrile). After lyophilization, a light yellow solid compound 9 (3 mg, 3.7 μmol, 62% yield) was obtained. UPLC-MS (ultra-performance liquid chromatography-mass spectrometry, Waters) was used for confirmation: the separation column was Acquity UPLC BEH C18 1.7 μm, 2.1×50 mm; 0-0.2 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid); 0.2-3.0 min, linear gradient: 10-95% acetonitrile (containing 0.1% trifluoroacetic acid); 3-4.5 min, linear gradient: 95-10% acetonitrile (containing 0.1% trifluoroacetic acid); 4.5-5.0 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid), flow rate 0.5 mL / min, UPLC-MS ([M+H] + =817.3, Rt=2.5min, purity 98.6%).

[0426] Compound 9 (3 mg, 3.7 μmol) was dissolved in dimethyl sulfoxide (200 μl), and N,N-diisopropylethylamine (2.4 mg, 18.5 μmol, 3.3 μl) was added followed by Int 5 (2.8 mg, 4.4 μmol). The mixture was reacted at 25°C for 1 hour and purified by reverse phase HPLC (water / acetonitrile). The final product AF488-FAPI-CB-02 (2.0 mg, 40% yield) was obtained as an orange-yellow solid after lyophilization. The product was confirmed by UPLC-MS (ultra-performance liquid chromatography-mass spectrometry, Waters): separation column Acquity UPLC BEH C18 1.7 μm, 2.1×50 mm; 0-0.2 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid); 0.2-3.0 min, linear gradient 10-95% acetonitrile (containing 0.1% trifluoroacetic acid); 3-4.5 min, linear gradient 95-10% acetonitrile (containing 0.1% trifluoroacetic acid); 4.5-5.0 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid), flow rate 0.5 mL / min, UP-LCMS ([M+H] + =1333.3, Rt=3.2 min, purity 99.1%).

[0427] Example 1d. Synthesis of FAPI-CB-00

[0428] Compound FAPI-CB-00 was readily obtained using the method described in Example 1a and confirmed by UPLC-MS (ultra-performance liquid chromatography-mass spectrometry, Waters): separation column: Acquity UPLC BEH C18 1.7 μm, 2.1×50 mm; 0-0.2 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid); 0.2-3.0 min, linear gradient: 10-95% acetonitrile (containing 0.1% trifluoroacetic acid); 3-4.5 min, linear gradient: 95-10% acetonitrile (containing 0.1% trifluoroacetic acid); 4.5-5.0 min, 10% acetonitrile (containing 0.1% trifluoroacetic acid), flow rate: 0.5 mL / min, UPLC-MS ([M+H] + =1214.5, R t =2.67 min, purity 98.7%).

[0429] Example 1e. Synthesis of FOLR1-CB-06

[0430] The synthetic route of compound FOLR1-CB-06 is shown in the figure above. The intermediates are referenced to Synthesis and biological evaluation of 68Ga-labeled Pteroyl-Lys conjugates for folate receptor-targeted tumor imaging were obtained using a novel method. The synthesis steps are as follows: 1) 1.0 g of 2-Cl-Trt resin (0.50 mmol, 1.00 eq) (loading: 0.500 mmol / g) was added to 3 mL of toluene and 2 mL of acetyl chloride, and activated at 60°C without stirring for 3 h. The resin was transferred to a 25 mL solid-phase synthesis tube, washed five times with 10 mL of toluene and five times with 10 mL of ultra-dry dichloromethane, and swollen in 20 mL of ultra-dry dichloromethane for 1 h. After swelling, the resin was washed five times with 10 mL of ultra-dry dichloromethane. Fmoc-Lys(Dde)-OH (0.50 mmol, 266 mg, 1.00 eq) and DIEA (2.00 mmol, 0.35 mL, 4.00 eq) were then added, dissolved in 8 mL of dichloromethane, and reacted on a shaker at room temperature for 12 h. 1 mL of methanol was added to block the unreacted sites, and the mixture was shaken at room temperature for 1 h. After filtration, the mixture was washed five times with 10 mL of DMF.

[0431] 2) Add 15 mL of 20% piperidine / DMF solution and shake at room temperature for 1 h to remove the Fmoc protecting group. Filter and wash five times with 10 mL of DMF. Add Fmoc-Glu-OtBu (1.50 mmol, 637 mg, 3.00 eq), HBTU (1.42 mmol, 540 mg, 2.85 eq), and DIEA (3.00 mmol, 0.50 mL, 6.00 eq), dissolve in 8 mL of DMF, and shake at room temperature for 3 h. Filter and wash five times with 10 mL of DMF.

[0432] 3) Add 15 mL of 20% piperidine / DMF solution and shake at room temperature for 1 h to remove the Fmoc protecting group. Filter and wash five times with 10 mL of DMF. Add pteroic acid (0.65 mmol, 200 mg, 1.30 eq), HATU (0.70 mmol, 266 mg, 1.40 eq), and DIEA (2.00 mmol, 0.37 mL, 4.00 eq), dissolve in 8 mL of DMSO, and shake at room temperature for 3 h. Filter and wash five times with 10 mL of DMF.

[0433] 4) Add 15 mL of 3% hydrazine / DMF solution and shake at room temperature for 1 h to remove the Dye protecting group. Filter and wash five times with 10 mL of DMF. Add DOTA-tri(t-butyl ester) (0.65 mmol, 372 mg, 1.30 eq), HATU (0.7 mmol, 266 mg, 1.40 eq), and DIEA (2.00 mmol, 0.37 mL, 4.00 eq), dissolve in 8 mL of DMF, and shake at room temperature for 3 h. Filter and wash five times with 10 mL of DMF.

[0434] 5) Add 15 mL of 5% TFA / DCM solution and react on a shaker at room temperature for 20 min. Collect the filtrate by filtration and rotary evaporation to obtain a yellow solid, which was separated and purified by HPLC. 4-(aminomethyl)phenol-OSF (1.30 eq), HATU (1.40 eq), and DIEA (4.00 eq) were added and dissolved in 1 mL of DMSO. Stir the mixture at room temperature for 3 h. Separate and purify by HPLC to obtain FR-5.

[0435] 6) Add 3 mL of 95% TFA / DCM solution and stir at room temperature for 2 h. Remove TFA and DCM by rotary evaporation. Dissolve in DMSO and purify by HPLC to obtain FOLR1-CB-06. The product was confirmed by UPLC-MS: [M+H] + =1142.4.

[0436] Example 1f. Synthesis of SSTR2-CB-02

[0437] The synthetic route for compound SSTR2-CB-02 is shown above. Commercially available DOTATATE (14 mg, 10 μmol) was dissolved in H2O (0.5 mL) and DCM (0.5 mL). Triethylamine (14 μmol, 10 equiv.) was added, and the atmosphere was replaced with SO2F2. The reaction was stirred for approximately 6 hours. After removing the DCM by low-pressure rotary evaporation, the mixture was purified by reverse-phase preparative HPLC. The product was confirmed by UPLC-MS: [M+H] + =1516.55.

[0438] Example 1g. Synthesis of Nectin4-CB-02

[0439] The synthetic route of compound Nectine4-CB-02 is shown in the figure above. 68Ga-N188 targeting nectin-4 for PET / CT imaging of advanced urothelial carcinoma and the solid-phase synthesis method described in the previous examples were used to obtain NCT4-int1. Subsequently, NCT4-int2 was readily obtained by solid-phase synthesis, referring to the publication "Discovery of BT8009: A Nectin-4 Targeting Bicycle Toxin Conjugate for the Treatment of Cancer" by Gemma E. Mudd et al. To a DMF solution of NCT4-int1 (9 mg) were added DCC (1.2 equiv.) and pentafluorophenol (1.2 equiv.) to prepare an activated lipid. NCT4-int2 (22 mg) was then added and stirred, and the mixture was purified by reverse-phase preparative HPLC to obtain NCT4-int3. Nectin-CB-02 was obtained by removal of the tert-butyl group in 95% TFA in dichloromethane. The product was confirmed by UPLC-MS: [M+H] + =2887.10.

[0440] Example 1h. Synthesis of CXCR4-CB-03

[0441] The synthetic route for compound CXCR4-CB-03 is shown above. CXCR4-3 was obtained by referring to the "PET Imaging of CXCR4 Receptors in Cancer by a New Optimized Ligand" published by Oliver Demmer et al. and the solid-phase synthesis method described in the previous examples. CXCR4-4 was then obtained by sulfonyl fluorination reaction as described in Example 1f. The tert-butyl group was removed by 95% TFA in dichloromethane, and the mixture was purified by reverse-phase preparative HPLC to yield CXCR4-CB-03. The product was confirmed by UPLC-MS: [M+H] + =1302.55.

[0442] Example 1i. Synthesis of PSMA-CB-05

[0443] The synthetic route for compound PSMA-CB-05 is shown above. PSMA-5 is readily obtained by referring to the method described in "Preclinical Evaluation of a Tailor-Made DOTA-Conjugated PSMA Inhibitor with Optimized Linker Moiety for Imaging and Endoradiotherapy of Prostate Cancer" published by Martina Benesova et al. Subsequently, referring to the condensation reactions described in Examples 1d and 1e, PSMA-CB-05 is readily obtained using commercially available raw materials. The product is then purified by reverse-phase preparative high-pressure liquid chromatography. UPLC-MS analysis confirms the product: [M+H] + =1383.6.

[0444] Example 2 Preparation of Nuclear Drug Molecules from Nuclide-Labeled Precursor Compounds

[0445] 68 Ga, 86 Y. 177 Lu and 225 Radiolabeling of Ac was carried out by incubating the nuclide with 10-50 nmol of the precursor compound (i.e., the compound described in Example 1) at pH 4-4.5 and 90-100°C for 10 minutes. 18 The radiolabeled compound was purified by solid-phase extraction using a Waters column, and its radiochemical purity and stability in saline and human serum were determined by high-performance liquid chromatography (radio-HPLC) equipped with a radioactivity detector. The radio-HPLC conditions were as follows: 0-2 minutes, 10% acetonitrile (containing 0.1% trifluoroacetic acid); 2-10 minutes, linear gradient of 10-60% acetonitrile (containing 0.1% trifluoroacetic acid); 10-12 minutes, linear gradient of 60-90% acetonitrile (containing 0.1% trifluoroacetic acid); 12-15 minutes, linear gradient of 90-10% acetonitrile (containing 0.1% trifluoroacetic acid), flow rate of 1 mL / min, using C 18 A column (4.6 x 150 mm, 5 μm, XBridge, Waters) was used to collect chromatograms.

[0446] 68 The radioactivity purity spectrum of Ga-FAPI-CB-02 is shown in FIG2 , and the purity is 99.2%.

[0447] Example 3 Molecular experiment - target protein co-incubation - gel electrophoresis - autoradiography - Coomassie brilliant blue staining experiment

[0448] The total volume was 20 μL and the concentration was 200 nM. 177Lu-labeled nuclear drug molecules (such as 177 Lu-FAPI-CB-01, 177 Lu-PSMA-CB-05 or 177 Lu-FOLR-CB-06, etc.) are incubated with 4 μM of the target protein at pH 7.4 (serum pH) or pH 6.5 (tumor microenvironment pH) at 37°C for 1 hour. Subsequently, the sample is denatured (e.g., with a strong acid or base) to remove reversible interactions, and then SDS-PAGE is performed to separate the protein and the nuclear drug molecules on the polyacrylamide gel. The gel is then taken for phosphor screen autoradiography (Amersham Typhoon imaging) to obtain radioactive bands of the nuclear drug. Finally, the gel is stained with Coomassie Brilliant Blue, and the target protein appears blue. Comparing the radioactive bands with the Coomassie Brilliant Blue staining results, if the radionuclide-labeled compound forms an irreversible covalent bond with the protein, a radioactive band will be visible at the site of protein staining.

[0449] Figure 3 shows 177 The covalent connection efficiency of Lu-FAPI-CB-01 and FAP human recombinant protein. The light blue image on the left is a Coomassie blue stained gel image, and the dark blue band shows the protein position (due to the strong acid and alkali treatment to destroy the non-covalent binding, the protein is partially denatured, so there are three bands, and the middle band corresponds to the molecular weight position of the remaining undenatured protein); the gray-white image on the right is a radioautograph, which shows that the positions corresponding to the three proteins on the top are all radioactive and gradually deepen over time, while the radioactive drug small molecule band represented by the bottom band gradually becomes lighter over time, indicating that 177 Efficient covalent attachment of Lu-FAPI-CB-01 to FAP protein.

[0450] Similarly, FIG4 shows 177 Covalent linkage efficiency of Lu-PSMA-CB-05 and PSMA human recombinant protein.

[0451] Similarly, FIG5 shows 177 Covalent linkage efficiency of Lu-FLOR-CB-06 and FOLR1 human recombinant protein.

[0452] Example 4 Molecular Experiments - Determination of Molecular Affinity and Binding Kinetics by Surface Plasmon Resonance Experiments

[0453] According to the United States Pharmacopoeia USP43 IMMUNOLOGICAL TEST METHODS—SURFACE PLASMON RESONANCE <1105> , Chinese Pharmacopoeia 2020 Edition Part IV General Chapter 3429 Immunochemistry Non-labeled immunochemical method (IV) Surface plasmon resonance method was used to perform surface plasmon resonance experiments.

[0454] Example 4a Multi-cycle mode measurement

[0455] Measurements were performed using a Cytiva surface plasma chromatograph. Prior to protein injection, a mixture of 400 mM condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 100 mM N-hydroxysuccinimide was used to activate a CM5 sensor chip for 420 seconds at a flow rate of 10 μL / min. Protein was then injected into the Fc2 sample channel at a flow rate of 10 μL / min to achieve protein immobilization levels of approximately 8,000–15,000 RU; no protein was injected into the control group. Subsequently, the unreacted chip was blocked with 1 M ethanolamine hydrochloride-NaOH at a flow rate of 10 μL / min for 420 seconds.

[0456] Subsequently, FAPI-04 (control) or FAPI-CB-01 was diluted with buffer to various concentration gradients. These gradient solutions were injected into the Fc1-Fc2 region of the channel at a flow rate of 30 μL / min. Data were collected during a 60-second association phase and a 90-second dissociation phase. The collected data were analyzed using software to obtain the results shown in Figure 6 and Table 1.

[0457] Table 1 Analysis of surface plasma chromatography results. a represents the binding constant, k d represents the dissociation constant, K D Indicates affinity, R max represents the theoretical maximum binding capacity of the analyte, Chi 2 Indicates the degree of fit, for kinetic fitting: Chi 2 <1 / 10R max .

[0458] The results show that the binding rate of FAPI-CB-01 is significantly increased, the dissociation rate is reduced, and the affinity is increased by about two orders of magnitude compared with the FAPI-04 molecule using this multi-cycle procedure.

[0459] Example 4b Single cycle mode measurement

[0460] Kinetic parameters for the FAPI-04 control, the hydro-FAPI-CB-02 control (easily obtained by hydrolysis of FAPI-CB-02, see Figure 12a for its structure), FAPI-CB-01, and FAPI-CB-02 were measured in single-cycle mode, following the methods described in Willemsen-Seegers, N. et al., "Compound Selectivity and Target Residence Time of Kinase Inhibitors Studied with Surface Plasmon Resonance." Journal of Molecular Biology 429, 574-586 (2017). Kinetic constants for the compounds were determined using single-cycle kinetics. Five consecutive injections were performed, depending on affinity, at compound concentrations of 0.625, 1.25, 2.5, 5, and 10 nM. The association time for each concentration was 100 seconds, and the dissociation time was 1800 seconds. To avoid mass transport limitations, a flow rate of 30 μL / min was used. A blank run was performed under the same conditions before compound injection. The SPR sensorgrams were analyzed using Biacore evaluation software using a dual reference approach. First, the reference channel was subtracted from the channel containing the immobilized protein. Subsequently, the reference curve obtained with the buffer injection was subtracted. The resulting curve was fitted with a 1:1 binding model. Compounds bound according to the induced fit model were fitted with a two-state reaction model. The kinetic constant (k a ,k d ,K D ) The geometric mean method was used. The collected data were analyzed by software to obtain the results shown in Figure 7.

[0461] Example 5 Modification Site

[0462] The total volume was 20 μL, and the concentration was 40 μM natural nat The Lu-labeled precursor compound is incubated with 4 μM FAP human recombinant protein at pH 7.4 (serum pH) and 37°C for 1 hour. Subsequently, protein separation / enzymatic hydrolysis is performed to prepare peptides through a standard tandem mass spectrometry sample preparation process, and the sample is analyzed using a Lumos three-in-one mass spectrometer or other mass spectrometers with HCD-FTMS fragmentation mode. In the secondary mass spectrometry, the parent ion collides with an inert gas, breaking the peptide bond in the peptide chain and forming a series of product ions. These are the N-terminal fragment ions (B series) and the C-terminal fragment ions (Y series). Based on the molecular weight, the site information of the specific peptide modified with the corresponding nuclear drug can be obtained.

[0463] The results are shown in Figure 8. According to the secondary mass spectrometry analysis, natLu-FAPI-CB-01 is covalently linked to FAP at position 450. In the peptide shown in FIG8 , the red-marked (third position) Y represents position 450 tyrosine.

[0464] Example 6 Cellular Experiment to Determine IC 50

[0465] HT-1080-FAP cells were seeded into 24-well plates 24 h in advance, with a density of about 30% per well (estimated by calculation), and 10 5 Countable cells were added to 0.5 mL complete culture medium; the medium was changed to serum-free culture medium; 12 groups of 0-5000 nM concentration gradient FAPI-CB-01 solutions were set up, with n=4 replicates within each group, and PC3-PIP cells were added separately, and 0.75 nM FAPI-CB-01 was added to each well. 68 Ga-FAPI-04 was incubated at 37°C for 1 hour; washed twice with cold PBS, and the radioactivity count was measured after lysis.

[0466] IC of FAPI-CB-01 50 The value was 11.64±0.32 nM, as shown in FIG9 .

[0467] Using a similar method, the affinity of the corresponding targeted PSMA-CB-05, FOLR1-CB-06, SSTR2-CB-02, Nectin4-CB-02, and CXCR4-CB-03 was measured in LNCaP cells, KB cells, AR42J cells, SW-780 cells, or Daudi cells, and all maintained good affinity.

[0468] Example 7 FAP High Expression Tumor Mouse Model Experiment - PET / CT Imaging and Biodistribution

[0469] All PET / CT scans are performed at Mediso PET 122S small animal PET / CT.

[0470] 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 Y-FAPI-04 (control) and 68 Ga / 86 After the Y-FAPI-CB series molecules were injected (Note: the injection of the control and investigational molecules was separated by at least 5 half-lives of the radionuclide to ensure that the radioactive decay of the previous probe was essentially complete and that the metabolism was essentially complete), imaging was performed at designated time points. In some embodiments, mice were anesthetized and then sacrificed by cervical dislocation at certain time points. The radioactivity of various organs was measured by dissection to comprehensively and quantitatively analyze the uptake and retention of nuclear drugs in the tumor.

[0471] Example 7a: 68 PET / CT imaging of Ga-FAPI-CB-00 in HT-1080-FAP tumor-bearing mouse model

[0472] Elute with 5 mL of 0.6 M high-purity hydrochloric acid 68 Ge- 68 Ga generator to obtain Ga-68 hydrochloric acid solution. Take 1 mL of the washed Ga-68 solution, add 100 μL of 3M sodium hydroxide and 130 μL of 3M sodium acetate to adjust the acidity to a final pH of 4.0, add 12 μg of FAPI-CB-00 compound, and heat the reaction mixture to 70°C for 20 minutes. The reaction solution is passed through C 18 The free ions were removed by using a small column, and then C was eluted with ethanol solution. 18 Column. Get the marked 68 Ga-FAPI-CB-00. 68 Ga-FAPI-CB-00 was diluted with physiological saline and 3.7 MBq was injected into each mouse through the tail vein. PET scanning imaging was performed 1 hour later and reconstructed using PET image processing software. The resulting image is shown in Figure 10. 68 Ga-FAPI-CB-00 probe with 68 Ga-FAPI-04 has a significantly higher uptake at the tumor site (because the drug is mainly metabolized by the kidneys, with high uptake in the bladder for a short time and excreted in the urine), but the background is also somewhat increased.

[0473] Example 7b: 68 PET / CT imaging of Ga-FAPI-CB-01 in HT-1080-FAP tumor-bearing mouse model

[0474] Elute with 5 mL of 0.6 M high-purity hydrochloric acid 68 Ge- 68 Ga generator to obtain Ga-68 hydrochloric acid solution. Take 1 mL of the washed Ga-68 solution, add 100 μL of 3M sodium hydroxide and 130 μL of 3M sodium acetate to adjust the acidity to a final pH of 4.0, add 12 μg of FAPI-CB-01 compound, and heat the reaction mixture to 90°C for 10 minutes. The reaction solution is passed through C 18 The free ions were removed by using a small column, and then C was eluted with ethanol solution. 18 Column. Get the marked 68 Ga-FAPI-CB-01. 68Ga-FAPI-CB-01 was diluted with physiological saline and 3.7 MBq was injected into each mouse via the tail vein. PET scanning was performed 1 hour later and reconstructed using PET image processing software. The representative images obtained are shown in Figure 11. 68 Ga-FAPI-CB-01 probe and 68 Compared with Ga-FAPI-04, Ga-FAPI-04 has a significantly higher uptake at the tumor site (because the drug is mainly metabolized by the kidneys, has a high uptake in the bladder for a short time, and is excreted in the urine), and the background increase is relatively limited.

[0475] Example 7c: 68 PET / CT imaging of Ga-FAPI-CB-02 in HT-1080-FAP tumor-bearing mouse model

[0476] Labeling was performed as described in Example 7b. 68 Ga-FAPI-04, 68 Ga-hydro-FAPI-CB-02 (structure shown in Figure 12a) and 68 The labeled drug was diluted with saline and three molecules (approximately 3.7 MBq) were injected into each mouse via the tail vein at intervals of 12 hours. PET scans were performed at 1 hour and reconstructed using PET image processing software. The resulting image is shown in Figure 12b. 68 Ga-FAPI-CB-02 probe showed significantly higher uptake in tumor sites, while the control 68 Ga-FAPI-04, and fluorosulfate hydrolyzed 68 The tumor uptake of Ga-hydro-FAPI-CB-02 was low (quantitative results are shown in Figure 12c), which illustrates the beneficial effect of introducing additional covalent warheads.

[0477] Example 7d: 86 PET / CT imaging and pharmacokinetic data of Y-FAPI-CB-02 in the HT-1080-FAP tumor-bearing mouse model

[0478] The target bombarded by the accelerator was washed with 5 mL of 0.1 M high-purity hydrochloric acid. 86 Sr] SrCO3, to obtain Y-86 hydrochloric acid solution. Take 1mL of the washed Y-86 solution, add 90 microliters of 3M sodium acetate to adjust the acidity to a final pH of 4.0, add 12 micrograms of FAPI-CB-02 precursor, and heat the reaction mixture to 90°C for 10 minutes. Pass the reaction solution through a C18 column to remove free ions, and then elute the C18 column with ethanol solution. Get the labeled 86 Y-FAPI-CB-02. 86Y-FAPI-CB-02 was diluted with physiological saline and injected into each mouse via the tail vein at 29.6 MBq. PET scans were performed at multiple time points over a long period of time and reconstructed using PET image processing software. The contrast images of a representative mouse at 9 hours are shown in Figure 13. 86 Y-FAPI-CB-02 probe with 86 Y-FAPI-04 still has significantly higher uptake and retention in the tumor site compared to the long-term time point.

[0479] Tumor uptake can be semi-quantified using the standard uptake value (SUV) obtained by analyzing the reconstructed PET-CT scan images using the device's built-in software. SUV is the ratio of the radioactivity of the imaging agent taken up locally to the average systemic injected activity: SUV = radioactivity concentration in the lesion (kBq / ml) / (injected dose (MBq) / body weight (kg)). SUV data obtained in this experiment are used to generate a tumor pharmacokinetic curve, defined as the area under the curve (AUC), representing the integrated area of ​​the tumor uptake-time curve. 86 Y-FAPI-CB-02 and 86 Y-FAPI-04 showed a significant improvement of more than 6 times in AUC (n=4, Figure 14), and is expected to greatly enhance the therapeutic efficacy of corresponding therapeutic radionuclide-labeled drugs.

[0480] Example 7e: 177 Biodistribution of Lu-FAPI-CB-02 in HT-1080-FAP tumor-bearing mouse model

[0481] Referring to the marking method in the above embodiment, it is easy to obtain the marked 177 Lu-FAPI-CB-02. Nu / Nu mice (18-20 g) bearing HT1080-FAP xenografts were randomly divided into four groups, with 7-8 mice in each group. The mice were anesthetized under an isoflurane atmosphere (2-3%) and injected with 200 μL of 0.74 MBq of Lu-FAPI-CB-02 via the tail vein. 68 Ga-FAPI-CB-02 formulation. Mice were anesthetized with isoflurane (2-3%) at various time points after injection (60, 120, and 240 minutes) and sacrificed by cervical dislocation. Organs of interest were dissected and removed. The radioactivity and weight of each organ were measured using a gamma counter and balance, and the percentage of radioactivity uptake per unit organ mass (%ID / g) was calculated. The results are shown in Figure 15. 177 The uptake of Lu-FAPI-CB-02 in liver and kidney is relatively high (compared to 177The radioactive substance uptake value shows a trend of gradually decreasing with the extension of time, while the tumor uptake is higher, so the tumor-normal organ uptake ratio gradually increases after a long time.

[0482] Example 8 Tumor Uptake Value

[0483] The same group of 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 almost completely, 18.5 MBq was injected into the tail vein 68 After Ga-FAPI-CB-02, PET-CT was 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)). As shown in Figure 16, compared with FAPI-04 in the most relevant prior art, in the HT-1080-FAP mouse model, the tumor maximum standard uptake value (SUV) of the FAPI-CB-01 and FAPI-CB-02 molecules of the present invention is max ) showed significant increases of approximately 2-fold and 4-fold, respectively (n=4, p<0.005), and also demonstrated significant advantages in PET-CT imaging. Therefore, the radionuclide-labeled FAPI-CB-01 and FAPI-CB-02 of the present invention significantly enhance tumor uptake, which is of great significance for improving clinical tumor detection rates.

[0484] Example 9 Binding selectivity of precursor molecules to homologous proteins of target sites

[0485] Taking the FAP target as an example, its homologous proteins include but are not limited to DPP-4 and PREP. Their structural similarities may lead to a decrease in the homologous protein binding selectivity of the modified precursor molecule, resulting in increased uptake in non-target organs.

[0486] The binding selectivity of homologous proteins was determined by competitive inhibition of enzyme substrates, and the half-maximal inhibitory concentration (IC 50 ), indirectly evaluating its binding / inhibition selectivity.

[0487] With reference to the method described in Extended Structure–Activity Relationship and Pharmacokinetic Investigation of (4-Quinolinoyl)glycyl-2-cyanopyrrolidine Inhibitors of Fibroblast Activation Protein (FAP) published by Jansen et al., Journal of Medicinal Chemistry 57, 3053-3074 (2014), the kinetic characteristics of enzyme activity were determined by measuring the initial velocity of release of p-nitroaniline (pNA) from the substrate (absorbance at 405 nm) using a microplate reader (PerkinElmer, VictorTM X5) at 37°C in a final volume of 200 microliters. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the release of 1 μmol of pNA from the substrate per minute under the detection conditions. All measurements were performed twice to ensure reproducibility. IC 50 The value was defined as the inhibitor concentration that caused a 50% decrease in activity under the assay conditions and was calculated by GraphPad based on the inhibitor concentration and the absorbance of pNA.

[0488] IC of FAP 50 The assay method is as follows: The substrate Z-Gly-Pro-p-nitroanilide (50 μM, Cat# G-58468, HEOWNS) is used to measure FAP activity at pH 7.4. The FAP assay buffer consists of 50 mM Tris pH 7.4, containing 1 M NaCl and 1.0 mg / mL bovine serum albumin. The buffer, substrate, and inhibitor are mixed before adding FAP protein (0.05 μg per well). The mixture is then incubated at 37°C for 6 hours, and pNA release is measured. The inhibitor concentration ranges from 0.01 nM to 200 μM. IC 50 The value is determined as above.

[0489] IC50 Determination of DPP-4: The substrate Z-Gly-Pro-p-nitroanilide (100 μM, Cat# G-58468, HEOWNS) was used to measure DPP-4 activity at pH 8.3. The DPP-4 assay buffer consisted of 50 mM Tris pH 8.3 containing 1 mM EDTA. The buffer, substrate, and inhibitor were mixed before adding DPP-4 protein (0.05 μg per well). The mixture was then incubated at 37°C for 20 minutes, and pNA release was measured. Inhibitor concentrations ranged from 0.01 nM to 200 μM. IC50 values ​​were determined as described above.

[0490] IC50 Determination of PREP: The substrate Gly-Pro-p-nitroanilide (0.1 mM) was used for PREP activity measurement at pH 7.5. The PREP assay buffer consists of 0.1 M K3PO4 pH = 8.3, containing 1 mM EDTA and 10 mM DTT. The buffer, substrate, and inhibitor were mixed, and then PREP protein (0.05 μg per well) was added. The mixture was then incubated at 37°C for 20 minutes to measure the release of pNA. The concentration of the inhibitor ranged from 0.01 nM to 200 μM. IC 50 The value is determined as above.

[0491] IC 50 The results are shown in Figure 17. It can be seen that after covalent modification with FAPI-04, FAPI-CB-01 and FAPI-CB-02 still maintain the original high selectivity of FAPI-04 for FAP protein.

[0492] Example 10 Tumor Mouse Model 177 Lu nuclide therapy experiment

[0493] When the average tumor size reaches about 100 mm 3 At the same time, the same batch of HT-1080-FAP tumor-bearing mice were randomly divided into 5 groups, with n = 7 or 8 mice in each group. Then two rounds of treatment were performed on day 0 and day 4, and the treatment group was injected with the corresponding dose of 177 Lu-labeled FAPI-CB-02 or control FAPI-04 (radioactivity yield>99%, radioactivity purity>99%, specific activity ∼11.2 MBq / nmol) The tumor size and body weight of the mice were monitored every 2 or 3 days.

[0494] As shown in Figure 18, 18a is a schematic diagram of the treatment regimen, 18b is the averaged tumor growth curve, 18c is the Kaplan-Meier curve (survival curve) 50 days after treatment, and 18d is the detailed tumor growth curve of each mouse in each treatment group. It can be seen that compared with the saline control group or the FAPI-04 control group, the same or higher doses ( 177 Lu activity) has a significantly better tumor inhibitory effect and may also have a better therapeutic effect when using a lower dose.

[0495] Example 11 Tumor Mouse Model 225 Ac nuclide therapy experiment

[0496] When the average tumor size reaches about 100 mm 3At the same time, the same batch of HT-1080-FAP tumor-bearing mice were randomly divided into 5 groups, with n = 7 or 8 mice in each group. Then two rounds of treatment were performed on day 0 and day 4, and the treatment group was injected with the corresponding dose of 225 Ac-labeled FAPI-CB-02 or control FAPI-04 (radioactivity purity>99%, specific activity ∼10 kBq / nmol) The tumor size and body weight of the mice were monitored every 2 or 3 days.

[0497] As shown in Figure 19, 19a is a schematic diagram of the treatment regimen, 19b is an averaged tumor growth curve, and 19c is a detailed tumor growth curve for each mouse in each treatment group. It can be seen that compared with the saline control group or the FAPI-04 control group, the same or higher dose ( 225 Ac activity) has a significantly better tumor inhibitory effect and may also have a better therapeutic effect when using a lower dose.

[0498] Example 12 PSMA High Expression Tumor Mouse Model Experiment - PET / CT Imaging

[0499] All PET / CT scans are performed at Mediso PET 122S small animal PET / CT.

[0500] A certain number (n=4) of mice bearing LNCaP tumors (the construction method refers to the method in Preclinical Evaluation of a Tailor-Made DOTA-Conjugated PSMA Inhibitor with Optimized Linker Moiety for Imaging and Endoradiotherapy of Prostate Cancer published by Martina Benesova et al.) were intravenously injected with 7.4-18.5 MBq of 68 Ga-PSMA-617 (control, labeling precursor is commercially available, structure is uniquely disclosed) and 68 After Ga-PSMA-CB-05 molecule (Note: The injection of control and investigation molecules should be separated by at least 12 hours, and the injection time should be kept until the previous molecule 68 Ga decay is almost complete and metabolism is basically completed), imaging is performed at the 2-hour time point, and the tumor SUV is obtained by software max The bar chart of the obtained results is shown in Figure 20. 68 Ga-PSMA-CB-05 68 Ga-PSMA-617 had significantly higher drug tumor uptake (p<0.05).

[0501] Example 13 SSTR2 High Expression Tumor Mouse Model Experiment - PET / CT Imaging

[0502] All PET / CT scans are performed at Mediso PET 122S small animal PET / CT.

[0503] A certain number (n=4) of AR42J tumor mice (the construction method refers to the combination radionuclide therapy using Marion de Jong et al.) 177 Lu-and 90 Y-labeled somatostatin analogs (methods described in the literature), 7.4–18.5 MBq of 68 Ga-DOTATATE (control, labeling precursor is commercially available, structure is publicly available) and 68 After Ga-SSTR2-CB-02 molecule (Note: The injection of control and investigation molecules should be separated by at least 12 hours, and the injection of the previous molecule should be stopped at 12 hours). 68 Ga decay is almost complete and metabolism is basically completed), imaging is performed at the 2-hour time point, and the tumor SUV is obtained by software max The bar chart of the obtained results is shown in Figure 21. 68 Ga-SSTR2-CB-02 68 Ga-DOTATATE had significantly higher drug tumor uptake (p<0.05).

[0504] Example 14 FOLR1 High Expression Tumor Mouse Model Experiment - PET / CT Imaging

[0505] All PET / CT scans are performed at Mediso PET 122S small animal PET / CT.

[0506] [Corrected 04.07.2023 according to Rule 91] A certain number (n=4) of KB tumor mice (construction method refers to Synthesis and biological evaluation of Xuran Zhang et al. 68 Ga-labeled Pteroyl-Lys conjugates for folate receptor-targeted tumor imaging) were injected intravenously with 7.4-18.5 MBq of 68Ga-FOLR1-control (control, the structure is shown in Figure 22a, refer to the Synthesis and biological evaluation of Xuran Zhang et al. 68 Ga-labeled Pteroyl-Lys conjugates for folate receptor-targeted tumor imaging) and 68 After the injection of Ga-FOLR1-CB-06 molecules (Note: The injection of control and observation molecules should be separated by at least 12 hours, and the injection of the previous molecule should be stopped after the injection of Ga-FOLR1-CB-06 molecules). 68 Ga decay is almost complete and metabolism is basically completed), imaging is performed at the 2-hour time point, and the tumor SUV is obtained by software max The bar chart of the obtained results is shown in Figure 22. 68 Ga-FOLR1-CB-06 68 Ga-FOLR1 had significantly higher drug tumor uptake (p<0.05).

[0507] Example 15 CXCR4 High Expression Tumor Mouse Model Experiment - PET / CT Imaging

[0508] All PET / CT scans are performed at Mediso PET 122S small animal PET / CT.

[0509] A certain number (n=4) of Daudi tumor-bearing mice (the construction method refers to the method in Oliver Demmer et al., PET Imaging of CXCR4 Receptors in Cancer by a New Optimized Ligand) were taken and injected intravenously with 7.4-18.5MBq of 68 Ga-pentixafor (control, labeled precursor commercially available, structure specifically disclosed) and 68 After the injection of Ga-CXCR4-CB-03 molecules (Note: The injection of control and investigation molecules should be separated by at least 12 hours, 68 Ga decay is almost complete and metabolism is basically completed), imaging is performed at the 2-hour time point, and the tumor SUV is obtained by software max The bar chart of the obtained results is shown in Figure 23. 68 Ga-CXCR4-CB-03 68 Ga-pentixafor had significantly higher drug tumor uptake (p<0.05).

[0510] Example 16 Nectin-4 High Expression Tumor Mouse Model Experiment - PET / CT Imaging

[0511] All PET / CT scans are performed at Mediso PET 122S small animal PET / CT.

[0512] A certain number (n=4) of SW-780 tumor mice were selected (the construction method was based on the Discovery of BT8009: A Nectin-4 Targeting Bicycle Toxin Conjugate for the Treatment of Cancer published by Gemma E. Mudd et al. and the First-in-human study of the radioligand 68 Ga-N188 targeting nectin-4 for PET / CT imaging of advanced urothelial carcinoma) were injected intravenously with 7.4–18.5 MBq of 68 Ga-NCT4-control (control, structure shown in Figure 24a, refer to the First-in-human study of the radioligand published by Xiaojiang Duan et al. 68 Ga-N188 targeting nectin-4 for PET / CT imaging of advanced urothelial carcinoma (synthesis method is readily available) or 68 After Ga-Nectin4-CB-02 molecule (Note: The injection of control and investigation molecules should be separated by at least 12 hours, 68 Ga decay is almost complete and metabolism is basically completed), imaging is performed at the 2-hour time point, and the tumor SUV is obtained by software max The bar chart of the obtained results is shown in Figure 24b. 68 Ga-Nectin4-CB-02 compares 68 Ga-NCT4-control had significantly higher drug tumor uptake (p<0.05).

[0513] 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.

[0514] 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, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein the trifunctional compound has a structure of formula (I), formula (II), formula (III), formula (IV), or formula (V), wherein P is a payload comprising at least one group comprising a radionuclide, at least one chelating group capable of chelating a radionuclide, or at least one optical dye group; T is a target, which comprises at least one targeting group that can target a protein or tissue, wherein the targeting group is a small molecule, a polypeptide, or a nucleic acid aptamer; C is a covalent warhead that can form a reversible or irreversible covalent bond with the protein or tissue targeted by T; Each of L1, L2, L3, L4, L5, L6, L7 and L8 is independently a linker unit; a, b, c, d, e, f, g, and h are independently integers between 0 and 6.

2. The trifunctional compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein C is selected from the group consisting of: in: Y is selected from O, S and NR 1 the group formed; LG is a leaving group that can be replaced by a protein residue, preferably a halogen or OTs, and Ts is p-toluenesulfonyl; R and R' are each independently selected from the group consisting of hydrogen, halogen, nitro, cyano, 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; Ar is optionally substituted aryl, or optionally substituted heteroaryl; Hal is a halogen, preferably F or Cl; p is an integer between 0 and 12.

3. The trifunctional compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein C is selected from the group consisting of: in: Ar is an optionally substituted C6-C 10 Aryl, preferably optionally substituted phenyl, or optionally substituted C5-C 12 heteroaryl; Hal is F; p is an integer between 0 and 6; R is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl.

4. The trifunctional compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein C is selected from the group consisting of: in: Ar is phenyl; Hal is F; p is an integer between 0 and 4.

5. The trifunctional compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the targeting group targets a target selected from the group consisting of: Fibroblast activation protein-α (FAP-α), prostate-specific membrane antigen (PSMA), polio receptor-associated protein 4 (Nectin-4), programmed death receptor ligand 1 (PD-L1), programmed death receptor 1 (PD-1), human epidermal growth factor receptor 2 (HER2), integrin, gastrin-releasing peptide receptor (GRPR), somatostatin receptor (SSTR), such as SSTR2, folate receptor (FR), such as folate receptor 1 (FOLR1), estrogen receptor (ER), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), chemokine receptor 4 (CXCR4), poly (ADP-ribose) polymerase (PARP), epidermal growth factor receptor (EGFR), fibroblast cytokine receptor (FGFR), disialoganglioside (GD2), vascular endothelial growth factor (VEGF), metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), insulin growth factor 1 (IGF-1), mesothelin (MSLN), tumor endothelial marker (TEM-1), interleukin 12 (IL-12), leukocyte differentiation antigen 13 (CD13), leukocyte differentiation antigen 19 (CD19), leukocyte differentiation antigen 20 (CD20), leukocyte differentiation antigen 22 (CD22), leukocyte differentiation antigen 33 (CD33), leukocyte differentiation antigen 37 (CD37), leukocyte differentiation antigen 38 (CD38), leukocyte differentiation antigen 44 (CD44), death receptor 5 (DR5), peroxidase 1 (Prdx I), large neutral amino acid transporter 1 (LAT1), angiotensin-converting enzyme 2 (ACE2), vesicular monoamine transporter 2 (VMAT2), dopamine transporter (DAT), and Sigma-1 receptor, The targeting group optionally comprises one or more warheads or structures that can form reversible or irreversible covalent bonds with targeted proteins or tissues.

6. The trifunctional compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the targeting group targets fibroblast activation protein-α (FAP-α), prostate-specific membrane antigen (PSMA), integrin, chemokine receptor 4 (CXCR4), folate receptor (FR), such as folate receptor 1 (FOLR1), polio receptor-related protein 4 (Nectin-4), or somatostatin receptor (SSTR), such as SSTR2, which is selected from the following structures: The first group targets the structure of fibroblast activation protein-α, which has the general formula (VI), preferably the general formula (VII), Wherein A is selected from O, S, NR A , R A Selected from H, C1-C6 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, 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; Wherein A is selected from O, S, NR A , R A is selected from H, methyl, ethyl, n-propyl, isopropyl; The second group: peptidomimetic groups having the general formula (VIII), where R B -CH2R B’ , R B’ is aryl, preferably optionally substituted phenyl or naphthyl, R c is H or alkyl, w and x are independently 1, 2, 3 or 4, The peptoid group is preferably of the following structure The third group: cyclic peptide groups, which have the general formula (X), Among them, each R D Independently selected from H, -CH2R D’ , -CH2COOH, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, and where R D’ is aryl, preferably optionally substituted phenyl or naphthyl, wherein the cyclic peptide is substituted by R D’ and / or R D The terminal -NH2 is connected to the rest of the trifunctional compound, or -CH2R D’ or R D’ May form part of covalent warhead C; R E is H or methyl; The cyclic peptide group preferably has the formula The two wavy lines in each formula represent optional attachment sites; The fourth group, the folic acid group, has the general formula (XI), The dotted line indicates whether the bond exists or not, and only one of the two dotted bonds exists. R FR1 , when present, is H or C1-C4 alkyl, R FR2 、R FR3 、R FR4 are each independently selected from H or C1-C4 alkyl, Ar G is aryl or heteroaryl, preferably optionally substituted phenyl or pyridyl, L FR -CH2-R FR5 -,-(CH2)2-R FR5 -,-(CH2)3-R FR5 -,-(CH2)3-R FR5 -, or -(CH2)4-R FR5 -, R FR5 Selected from carbonyl (-CO-) or NR FR6 , R FR6 is H or C1-C4 alkyl, The folic acid group is preferably of the following structure The fifth group, a disulfide bond-containing cyclic group, has the general formula (XII), Among them, ss1 , Αr ss2 and Al ss3 Each is independently selected from optionally substituted aryl or heteroaryl, preferably optionally substituted C6-C 10 Aryl or C5-C 10 Heteroaryl, preferably Ar ss1 , Αr ss3 is a C6-C 10 Aryl, Al ss2 C5-C 10 heteroaryl, s1, s2 and s3 are each independently selected from 1, 2, 3 or 4, wherein the disulfide-containing cyclic group is connected to the rest of the trifunctional compound via the site indicated by the wavy line, or -(CH2) s3 Al ss3 or Al ss3 may form part of the covalent warhead C, The disulfide bond-containing cyclic group preferably has the structure The sixth group, a bicyclic peptide group, preferably a bicyclic peptide group targeting polio receptor-associated protein 4 (Nectin-4), More preferably, the bicyclic peptide group is the following structure 7. The trifunctional compound, pharmaceutically acceptable salt, stereoisomer or solvate thereof according to any one of claims 1 to 6, wherein the payload comprises at least one group selected from the group consisting of 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 but rather on the linker; The second group, chelating groups that can chelate radionuclides, are selected from: The third group, optical dye groups, is selected from:

8. The trifunctional compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein L1 and L8 are independently selected from the following groups: Each of L2, L3, L4, L5, L6 and L7 is independently selected from the following groups: single bond, L9 is a single bond, -CH2-, -NHCH2-, or Cy is selected from a to h are independently 0, 1, 2 or 3, u, v and o are independently 1, 2, 3, 4 or 5, 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, * and ** indicate the sites of attachment to two parts of the trifunctional compound, optionally via L2, L3, L4, L5, L6 or L7, Provided that in the linking group composed of L1, L2, L3, L4, L5, L6, L7 and L8, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S.

9. The trifunctional compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (I), at least one L1 is L1 preferably has the following structure: where R 6 、R 7 , L9, Cy, a, b, o and u have the definitions as described in claim 8.

10. The trifunctional compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (I): General formula (I) in: L1 is a trivalent linker unit, Each L2 and L3 is independently a divalent linking unit; a and b are independently integers between 0 and 6; The C shown is a covalent warhead selected from the group consisting of: in: Ar is an optionally substituted C6-C 10 Aryl, preferably optionally substituted phenyl, or optionally substituted C5-C 12 heteroaryl; Hal is F or Cl; p is an integer between 0 and 6; R is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl; T shown is a targeting group targeting fibroblast activation protein-α (FAP-α); P is a chelating group capable of chelating a radionuclide.

11. The trifunctional compound according to claim 10, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (I), in: The L1 is a trivalent linking group selected from the following: Each L2 and L3 is independently selected from the following groups: single bond, L9 is a single bond, -CH2-, -NHCH2-, or Cy is selected from a, b and p are independently 0, 1, 2 or 3, u is independently 1, 2, 3, 4 or 5, 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, Provided that in the linker formed by L1, L2 and L3, 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: Ar is phenyl; Hal is F; p is 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 radionuclides, which is selected from the following groups:

12. The trifunctional compound according to claim 10 or 11, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (I), in: C and P have the meanings as defined in claim 11. L1 has the following structure: a and b are both 1, L2 and L3 are independently the following groups: single bond, wherein u and o are independently 1, 2, 3, 4 or 5, Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4, * is the site for connecting T, ** is the site for connecting C, Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S; T is a compound of the general formula (VI), preferably a compound of the general formula (VII), Wherein A is selected from O, S, NR A , R A Selected from H, C1-C6 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, 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; Wherein A is selected from O, S, NR A , R A Selected from H, methyl, ethyl, n-propyl, isopropyl.

13. The trifunctional compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (I), in: The L1 is a trivalent linking group selected from the following: Each L2 and L3 is independently selected from the following groups: single bond, L9 is a single bond, -CH2-, -NHCH2-, or Cy is selected from a and b are independently 0, 1, 2 or 3, u is independently 1, 2, 3, 4 or 5, 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, Provided that in the linker formed by L1, L2 and L3, 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: Ar is phenyl; Hal is F; p is an integer between 0 and 4; The T shown is a targeting group targeting prostate-specific membrane antigen (PSMA); The P shown is a chelating group capable of chelating radionuclides, which is selected from the following groups:

14. The trifunctional compound according to claim 13, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (I), in: C and P have the meanings as defined in claim 13. L1 has the following structure: a, b are both 1, L2 and L3 are independently the following groups: single bond, wherein u and o are independently 1, 2, 3, 4 or 5, Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4, * is the site for connecting T, ** is the site for connecting C, Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S; Said T has the general formula (VIII), where R B -CH2R B’ , R B’ is aryl, preferably optionally substituted phenyl or naphthyl, R c is H or alkyl, w and x are independently 1, 2, 3 or 4, The T is preferably the following structure 15. The trifunctional compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (I), in: The L1 is a trivalent linking group selected from the following: Each L2 and L3 is independently selected from the following groups: single bond, L9 is a single bond, -CH2-, -NHCH2-, or Cy is selected from a and b are independently 0, 1, 2 or 3, u is independently 1, 2, 3, 4 or 5, 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 C, ** is the site for connecting P, Provided that in the linker formed by L1, L2 and L3, 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: Ar is phenyl; Hal is F; p is an integer between 0 and 4; T is a targeting group targeting a folate receptor (FR), such as folate receptor 1 (FOLR1); The P shown is a chelating group capable of chelating radionuclides, which is selected from the following groups:

16. The trifunctional compound according to claim 15, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (I), in: C and P have the meanings as defined in claim 15. L1 has the following structure: a, b are both 1, L2 and L3 are independently the following groups: single bond, where u is 1, 2, 3, 4, or 5, Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4, * is the site for connecting C, ** is the site for connecting P, Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S; Said T has the general formula (XI), The dotted line indicates whether the bond exists or not, and only one of the two dotted bonds exists. R FR1 , when present, is H or C1-C4 alkyl, R FR2 、R FR3 、R FR4 are each independently selected from H or C1-C4 alkyl, Ar G is aryl or heteroaryl, preferably optionally substituted phenyl or pyridyl, L FR -CH2-R FR5 -,-(CH2)2-R FR5 -,-(CH2)3-R FR5 -,-(CH2)3-R FR5 -, or -(CH2)4-R FR5 -, R FR5 Selected from carbonyl (-CO-) or NR FR6 , R FR6 is H or C1-C4 alkyl, The T is preferably the following structure 17. The trifunctional compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (I), in: The L1 is a trivalent linking group selected from the following: Each L2 and L3 is independently selected from the following groups: single bond, L9 is a single bond, -CH2-, -NHCH2-, or Cy is selected from a and b are independently 0, 1, 2 or 3, u is independently 1, 2, 3, 4 or 5, R 6 and R 7 independently selected from hydrogen, optionally substituted alkyl, optionally substituted The group consisting of cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl, * is the site for connecting T, ** is the site for connecting C, Provided that in the linker formed by L1, L2 and L3, 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: Ar is phenyl; Hal is F; p is an integer between 0 and 4; T shown is a targeting group targeting polio receptor-associated protein 4 (Nectin-4); The P shown is a chelating group capable of chelating radionuclides, which is selected from the following groups:

18. The trifunctional compound according to claim 17, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (I), in: C and P have the meanings as defined in claim 17. L1 has the following structure: a, b are both 1, L2 and L3 are independently the following groups: single bond, where u is 1, 2, 3, 4, or 5, Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4, * is the site for connecting T, ** is the site for connecting C, Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S; The T is a bicyclic peptide group, preferably a bicyclic peptide group targeting poliomyelitis receptor-associated protein 4 (Nectin-4), More preferably, the bicyclic peptide group is the following structure 19. The trifunctional compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (II): in: Each L4 and L5 shown is independently a divalent linking unit; c and d are independently integers from 0 to 6; The C shown is a covalent warhead selected from the group consisting of: in: Ar is an optionally substituted C6-C 10 Aryl, preferably optionally substituted phenyl, or optionally substituted C5-C 12 heteroaryl; Hal is F or Cl; p is an integer between 0 and 6; R is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl; T shown is a targeting group targeting chemokine receptor 4 (CXCR4); P is a chelating group capable of chelating a radionuclide.

20. The trifunctional compound according to claim 19, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has a structure of formula (II): in: Each L4 and L5 is independently selected from the following groups: single bond, c and d are independently 0, 1, 2 or 3, u is independently 1, 2, 3, 4 or 5, 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, Provided that in the linker formed by L4 and L5, 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: Ar is phenyl; Hal is F; p is an integer between 0 and 3; T shown is a targeting group targeting chemokine receptor 4 (CXCR4); The P shown is a chelating group capable of chelating radionuclides, which is selected from the following groups:

21. The trifunctional compound according to claim 19 or 20, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (II), in: C and P have the meanings as defined in claim 20. c and d are both 1, L4 and L5 are independently the following groups: single bond, wherein u and o are independently 1, 2, 3, 4 or 5, Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4, Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are selected from N, O and S; T has the general formula (X), Among them, each R D Independently selected from H, -CH2R D’ , -CH2COOH, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, and where R D’ is aryl, preferably optionally substituted phenyl or naphthyl, wherein the cyclic peptide is substituted by R D’ and / or R D The terminal -NH2 is connected to the rest of the trifunctional compound, or -CH2R D’ or R D’ May form part of covalent warhead C; R E is H or methyl; Said T preferably has the formula The two wavy lines in each formula represent optional attachment sites.

22. The trifunctional compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has the structure of general formula (II): in: Each L4 and L5 shown is independently a divalent linking unit; c and d are independently integers from 0 to 6; The C shown is a covalent warhead selected from the group consisting of: in: Ar is an optionally substituted C6-C 10 Aryl, preferably optionally substituted phenyl, or optionally substituted C5-C 12 heteroaryl; Hal is F or Cl; p is an integer between 0 and 6; R is selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl; T is a targeting group targeting somatostatin receptor (SSTR), such as SSTR2; P 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 the structure of general formula (II), in: Each L4 and L5 is independently selected from the following groups: single bond, c and d are independently 0, 1, 2 or 3, u is independently 1, 2, 3, 4 or 5, R 6 and R 7 are independently selected from hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted A group consisting of heteroaryl groups, Provided that in the linker formed by L4 and L5, 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: Ar is phenyl; Hal is F; p is an integer between 0 and 3; T is a targeting group targeting somatostatin receptor (SSTR), such as SSTR2; The P shown is a chelating group capable of chelating radionuclides, 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 the structure of general formula (II), in: C and P have the meanings as defined in claim 23. c and d are both 1, L4 and L5 are independently the following groups: single bond, wherein u and o are independently 1, 2, 3, 4 or 5, Each R 6 and R 7 are independently selected from hydrogen, optionally substituted C1-C4, Provided that in the linker formed by L1, L2 and L3, the heteroatoms are not directly connected by covalent bonds, and the heteroatoms are O, N or S; and T has the general formula (XII), Among them, ss1 , Αr ss2 and Al ss3 Each is independently selected from optionally substituted aryl or heteroaryl, preferably optionally substituted C6-C 10 Aryl or C5-C 10 Heteroaryl, preferably Ar ss1 , Αr ss3 is a C6-C 10 Aryl, Al ss2 C5-C 10 heteroaryl, s1, s2 and s3 are each independently selected from 1, 2, 3 or 4, wherein the T is connected to the rest of the trifunctional compound via the site indicated by the wavy line, or -(CH2) s3 Al ss3 or Al ss3 may form part of the covalent warhead C, Said T preferably has the structure 25. The trifunctional compound according to any one of claims 1 to 24, 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 7 to 25, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the payload is a chelating group capable of chelating a radionuclide, which chelates a radionuclide.

27. The trifunctional compound according to any one of claims 7 to 26, or a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein 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 metals, 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 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 use in diagnosing a disease.

30. A method for diagnosing or treating a disease, the method 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, glioma, and bone metastasis.

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