Bicyclic peptide radionuclide ligand and chelate

Abstract

Disclosed are a bicyclic peptide radionuclide ligand and a chelate, specifically relating to formula (V).

Description

Bicyclic peptide nuclide ligands and chelates

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. CN202411734852.3, filed on November 29, 2024, entitled "Bicyclic Peptide Nucleotide Ligands and Chelates", and Chinese Patent Application No. CN202510416237.6, filed on April 3, 2025, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to bicyclic peptide nuclide ligands and chelates; specifically, to bicyclic peptide nuclide ligands of formula (V) and their chelates. Background Technology

[0004] Radionuclide drug conjugates (RDCs) are structurally composed of a targeting component (ligand), a linker, a chelator, and a radionuclide. They selectively bind to their target, causing the molecule to accumulate at the lesion site. The radionuclide releases gamma rays, positrons, alpha particles, or beta particles during decay, enabling imaging diagnosis and precise radiotherapy. Currently, two radionuclide conjugate therapies are on the market: (lutetium Lu 177 dotatate) and (lutetium Lu 177 vipivotide tetraxetan) is used to treat somatostatin receptor-positive gastrointestinal and pancreatic neuroendocrine tumors and PSMA-positive metastatic castration-resistant prostate cancer.

[0005] Nectin-4 (also known as PVRL4) is an adhesion molecule located in epithelial cells, which supports homogeneous and heterogeneous cell adhesion through the interaction of its extracellular portion. Studies have found that Nectin-4 protein is overexpressed in various human tumors and is associated with poor prognosis in some tumors (such as pancreatic cancer, breast cancer, and gastric cancer). Enfortumab Vedotin, an antibody-drug conjugate targeting this protein, was approved by the FDA in 2019 for the treatment of metastatic urothelial carcinoma, and several other indications are currently in clinical trials. Summary of the Invention

[0006] On the one hand, the present invention provides a bicyclic peptide nuclide ligand of formula (V),

[0007] in,

[0008] RM is a chelating ligand, independently selected from DOTA, DO3A, DOTAGA, Bn-DOTA, Bn-Oxo-DO3A, Nota, NODAGA, Bn-NOTA, NOPO, DOTAM, Bn-TCMC, Bn-CHX-A”-DTPA, CB-TE2A, CB-DO2A, 2B3M-DTPA, Bn-PCTA, TETA, mDOTA, and nDOTA;

[0009] G is selected independently.

[0010] L is selected independently The left end of L* may be connected to RM or not connected to any segment including RM, and the carbonyl end on the right side** forms an amide bond with the nitrogen end of formula (V);

[0011] L1 is independently selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -OCH2-, -OCH2CH2-, -OCH2CH2CH2-, -CH2OCH2CH2-, -CH2CH2OCH2CH2-, -CONHCH2-, -CONHCH2CH2-, -CH2CONHCH2-, -CH2CH2CONHCH2-, and L1 is connected in L in order from left to right;

[0012] L2 is independently selected from

[0013] The # terminal connects to RM, the ## terminal connects to NH, and terminals without the # terminal can exist independently.

[0014] Xi, Xii, and Xiii are each independently selected from Cys, hCys, βCys, or Pen;

[0015] A3 is selected independently.

[0016] A4 is selected independently.

[0017] A5 is selected independently.

[0018] A6 is selected independently.

[0019] M is independently selected from --NH2, R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from H and -C. 1-6 Alkyl, -C 1-6 Alkylene-R 21 -C 5-12 cycloalkyl, -C 1-6 alkylene-heteroatom-C 1-3 Alkyl, -C 1-6 alkylene-amino, -C 1-6 alkylene-naphthalene ring, -CO-R 21 -C 1-6 Alkyl-benzene ring;

[0020] R 21 Selected from H, NH2, SCH3, hydroxyl, phenyl, p-hydroxyphenyl, imidazole, indole, -COOH, -CONH2,

[0021] n, m, u, p, q, v, w are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0022] This invention provides bicyclic peptide ligands represented by formulas (I) and (II).

[0023] in,

[0024] RM is a chelating ligand, independently selected from DOTA, DO3A, DOTAGA, Bn-DOTA, Bn-Oxo-DO3A, Nota, NODAGA, Bn-NOTA, NOPO, DOTAM, Bn-TCMC, Bn-CHX-A”-DTPA, CB-TE2A, CB-DO2A, 2B3M-DTPA, Bn-PCTA, TETA, mDOTA, and nDOTA;

[0025] L1 is independently selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -OCH2-, -OCH2CH2-, -OCH2CH2CH2-, -CH2OCH2CH2-, -CH2CH2OCH2CH2-, -CONHCH2-, -CONHCH2CH2-, -CH2CONHCH2-, -CH2CH2CONHCH2-, and L1 is connected in L in order from left to right;

[0026] L2 is independently selected from The # terminal connects to RM, the ## terminal connects to NH, and terminals without the # terminal can exist independently.

[0027] Xi, Xii, and Xiii are each independently selected from Cys, hCys, βCys, or Pen;

[0028] M is independently selected from --NH2,

[0029] R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from H and -C. 1-6 Alkyl, -C 1-6 Alkylene-R 21 -C 5-12 cycloalkyl, -C 1-6 alkylene-heteroatom-C 1-3 Alkyl, -C 1-6 alkylene-amino, -C 1-6 alkylene-naphthalene ring, -CO-R 21 -C 1-6 Alkyl-benzene ring;

[0030] R 21 Independently selected from H, NH2, SCH3, hydroxyl, phenyl, p-hydroxyphenyl, imidazole, indole, -COOH, -CONH2,

[0031] n, m, u, p, q, v, w are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0032] The present invention also provides a bicyclic peptide ligand of formula (Ⅳ),

[0033] in,

[0034] RM is a chelating ligand, independently selected from DOTA, DO3A, DOTAGA, Bn-DOTA, Bn-Oxo-DO3A, Nota, NODAGA, Bn-NOTA, NOPO, DOTAM, Bn-TCMC, Bn-CHX-A”-DTPA, CB-TE2A, CB-DO2A, 2B3M-DTPA, Bn-PCTA or TETA;

[0035] L1 is independently selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -OCH2-, -OCH2CH2-, -OCH2CH2CH2-, -CH2OCH2CH2-, -CH2CH2OCH2CH2-, -CONHCH2-, -CONHCH2CH2-, -CH2CONHCH2-, -CH2CH2CONHCH2-, and L1 is connected in L in order from left to right;

[0036] L2 is independently selected from The # terminal connects to the RM, and the ## terminal connects to the NH.

[0037] Xi, Xii, and Xiii are each independently selected from Cys, hCys, βCys, or Pen;

[0038] M is independently selected from --NH2,

[0039] R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from H and -C. 1-6 Alkyl, -C 1-6 Alkylene-R 21 -C 5-12 cycloalkyl, -C 1-6 alkylene-heteroatom-C 1-3 Alkyl, -C 1-6 alkylene-amino, -C 1-6 alkylene-naphthalene ring, -CO-R 21 ;

[0040] R 21 Independently selected from H, NH2, SCH3, hydroxyl, phenyl, p-hydroxyphenyl, imidazole, indole, -COOH, -CONH2,

[0041] n, m, u, p, and q are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively.

[0042] This invention also provides a bicyclic peptide ligand of formula (Ⅱ-a),

[0043] A3 is selected from

[0044] L1 is independently selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -OCH2-, -OCH2CH2-, -OCH2CH2CH2-, -CH2OCH2CH2-, -CH2CH2OCH2CH2-, -CONHCH2--, -CONHCH2CH2-, -CH2CONHCH2-, -CH2CH2CONHCH2-, and L1 is connected in L in order from left to right.

[0045] L2 is independently selected from The # terminal connects to RM, the ## terminal connects to NH, and terminals without the # terminal can exist independently.

[0046] Xi, Xii, and Xiii are each independently selected from Cys, hCys, βCys, or Pen;

[0047] M is independently selected from NH2.

[0048] R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from H and -C. 1-6 Alkyl, -C 1-6 Alkylene-R 21 -C 5-12 cycloalkyl, -C 1-6 alkylene-heteroatom-C 1-3 Alkyl, -C 1-6 alkylene-amino, -C 1-6 alkylene-naphthalene ring, -CO-R 21 -C 1-6 Alkyl-benzene ring;

[0049] R 21 Independently selected from H, NH2, SCH3, hydroxyl, phenyl, p-hydroxyphenyl, imidazole, indole, -COOH, -CONH2,

[0050] n, m, u, p, q, v, w are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0051] In some embodiments of the present invention, the above n is independently selected from 0 or 1, and other variables are as defined in the present invention.

[0052] In some embodiments of the present invention, R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -(CH2)2CH(CH3)2, -CH(CH3)CH2CH3, -CH2CH(CH3)CH2CH3, -COOH, -CH2COOH, -(CH2)2COOH, -(CH2)3COOH, and -CH 2CONH2, -(CH2)2CONH2, -(CH2)3CONH2, -CH2NH2, -(CH2)2NH2, -(CH2)3NH2, -(CH2)4NH2, -(CH2)5NH2, -( CH2)2SCH3, -(CH2)3SCH3, -CH2Ph, -(CH2)2Ph, -CH2Ph(p-OH), -(CH2)2OH, -CH(CH3)OH, -CH2CH(CH3)OH, Other variables are as defined in this invention.

[0053] In some embodiments of the present invention, the above-mentioned structural segments In the context, when u is selected from 1, the segment is independently selected from... Other variables are as defined in this invention.

[0054] In some embodiments of the present invention, the above-mentioned structural segments Independently selected

[0055] Other variables are as defined in this invention.

[0056] In some embodiments of the present invention, the L2 mentioned above is independently selected from...

[0057] The # terminal is connected to RM, the ## terminal is connected to NH, and the terminal without the # terminal can exist independently. Other variables are as defined in this invention.

[0058] In some embodiments of this invention, the aforementioned RM is DOTA:

[0059] In some embodiments of the present invention, the aforementioned RM is DO3A:

[0060] In some embodiments of the present invention, the aforementioned RM is DOTAGA:

[0061] In some embodiments of the present invention, the aforementioned RM is Bn-DOTA:

[0062] In some embodiments of the present invention, the aforementioned RM is Bn-Oxo-DO3A:

[0063] In some embodiments of the present invention, the aforementioned RM is NOTA:

[0064] In some embodiments of the present invention, the aforementioned RM is NODAGA:

[0065] In some embodiments of the present invention, the aforementioned RM is Bn-NOTA:

[0066] In some embodiments of the present invention, the aforementioned RM is NOPO:

[0067] In some embodiments of the present invention, the aforementioned RM is DOTAM:

[0068] In some embodiments of the present invention, the aforementioned RM is Bn-TCMC:

[0069] In some embodiments of the present invention, the aforementioned RM is Bn-CHX-A”-DTPA:

[0070] In some embodiments of the present invention, the aforementioned RM is CB-TE2A:

[0071] In some embodiments of the present invention, the aforementioned RM is CB-DO2A:

[0072] In some embodiments of the present invention, the aforementioned RM is 2B3M-DTPA:

[0073] In some embodiments of the present invention, the aforementioned RM is Bn-PCTA:

[0074] In some embodiments of the present invention, the aforementioned RM is TETA:

[0075] In some embodiments of the present invention, the aforementioned RM is mDOTA:

[0076] In some embodiments of the present invention, the aforementioned RM is nDOTA:

[0077] In some embodiments of the present invention, the aforementioned bicyclic peptide nuclide ligand is selected from:

[0078] The independent u is selected from 0 or 1, and L1, L2, R1, R2, R3, R4, R5, R6, R7, R8, R9, A3, A4, A5, A6, and M are as defined in this invention. Preferably, A3 in Ve is selected from...

[0079] On the other hand, the present invention also provides a bicyclic peptide nuclide chelate, wherein the chelate is formed by chelating the bicyclic peptide nuclide ligand of the present invention with a radionuclide, wherein the radionuclide is selected from... 18 F, 51 Cr 67 Ga、 68 Ga、 111 In、 99m Tc, 186 Re、 188 Re、 139 La、 140 La、 175 Yb、 153 Sm、 166 Ho、 86 Y、 88 Y、 90 Y、 149 Pm, 165 Dy、 169 Er、 177 Lu、 47 Sc、 142 Pr, 159 Gd, 212 Bi、 213 Bi、 72 As、 72 Se、 97 Ru、 109 Pd, 105 Rh、 101m Rh、 119 Sb, 128 Ba、 123 I, 125 I, 124 I, 131 I, 197 Hg, 211 At、 151 Eu、 153 Eu、 169 Eu、 201 Tl、 203 Pb, 212 Pb, 64 Cu、67 Cu、 198 Au、 225 Ac、 227 Th、 199 Ag、 169 Yb、 62 Zn, 65 Zn, 89 Zr and 95 Zr.

[0080] In some embodiments of the present invention, the radionuclides are selected from... 225 Ac、 211 At、 212 Bi、 213 Bi、 11 C 64 Cu、 67 Cu、 18 F, 68 Ga、 123 I, 124 I, 125 I, 131 I, 111 In、 177 Lu、 212 Pb, 186 Re、 188 Re、 153 Sm、 86 Y、 90 Y and 89 Zr.

[0081] In some embodiments of the present invention, the radionuclides are selected from... 68 Ga and 177 Lu.

[0082] The radionuclides described in this invention include diagnostic radionuclides and therapeutic radionuclides.

[0083] In some embodiments of the present invention, the aforementioned radionuclide is a diagnostic radionuclide selected from... 68 Ga、 64 Cu、 18 F, 86 Y、 90 Y、 89 Zr、 111 In、 99m Tc, 11 C 123 I, 125 I and 124 At least one of I.

[0084] In some embodiments of the present invention, the aforementioned radionuclide is a diagnostic radionuclide selected from... 177 Lu、125 I, 131 I, 211 At、 111 In、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.

[0085] In some embodiments of the present invention, the aforementioned radionuclide is a therapeutic radionuclide, selected from... 68 Ga and 177 At least one of Lu.

[0086] In some embodiments of the present invention, the chelation of the bicyclic peptide nuclide ligand with the radionuclide is the chelation of DOTA with the radionuclide: M represents a radioactive nuclide.

[0087] In some embodiments of the present invention, the chelation of the bicyclic peptide nuclide ligand with the radionuclide is the chelation of Bn-DOTA with the radionuclide:

[0088] In some embodiments of the present invention, the chelation of the bicyclic peptide nuclide ligand with the radionuclide is the chelation of Bn-Oxo-DO3A with the radionuclide:

[0089] In some embodiments of the present invention, the chelation of the bicyclic peptide nuclide ligand with the radionuclide is the chelation of TETA with the radionuclide:

[0090] In some embodiments of the present invention, the chelation of the bicyclic peptide nuclide ligand with the radionuclide is the chelation of NOA with the radionuclide:

[0091] In some embodiments of the present invention, the chelation of the bicyclic peptide nuclide ligand with the radionuclide is the chelation of Bn-NOTA with the radionuclide:

[0092] In some embodiments of the present invention, the chelation of the bicyclic peptide nuclide ligand with the radionuclide is the chelation of 2B3M-DTPA with the radionuclide:

[0093] In some embodiments of the present invention, the chelation of the bicyclic peptide nuclide ligand with the radionuclide is the chelation of Bn-PCTA with the radionuclide:

[0094] Some solutions of this invention are derived from any combination of the solutions mentioned above.

[0095] In some embodiments of the present invention, a pharmaceutical composition is also provided comprising the bicyclic peptide nuclide chelate as described in any of the above claims, and a pharmaceutically acceptable carrier.

[0096] In some embodiments of the present invention, a kit is also provided, comprising or consisting of a bicyclic peptide nuclide chelate as described in any of the above claims or a pharmaceutical composition as described above, and instructions for use in diagnosing or treating diseases.

[0097] The present invention also provides the following bicyclic peptide ligands, Attached Figure Description

[0098] Figure 1 shows BCY8234 and 177 Lu-BR001 177 Schematic diagram of competitive binding of Lu-BR002 to PC3-Nectin4 cells;

[0099] Figure 2 shows BCY8234 and 177 Lu-BR001 177 Schematic diagram of competitive binding of Lu-BR002 in MDA-MB-468 cells;

[0100] Figure 3 is 177 Schematic diagram showing the uptake of Lu-labeled compounds in Nectin-4 positive PC3-Nectin4 cells;

[0101] Figure 4 is 177 Schematic diagram of uptake of Lu-labeled compounds in Nectin-4 negative PC3 cells;

[0102] Figure 5 is 177 A schematic diagram illustrating the biodistribution of Lu-labeled compounds in PC3-Nectin4 tumor-bearing mice;

[0103] Figure 6 is 177 Another schematic diagram illustrating the biodistribution of Lu-labeled compounds in PC3-Nectin4 tumor-bearing mice;

[0104] Figure 7 is 177 Another schematic diagram illustrating the biodistribution of Lu-labeled compounds in PC3-Nectin4 tumor-bearing mice;

[0105] Figure 8 is 177 Another schematic diagram showing the uptake of Lu-labeled compounds in Nectin-4 positive PC3-Nectin4 cells;

[0106] Figure 9 is 177 Another schematic diagram showing the uptake of Lu-labeled compounds in Nectin-4 positive PC3-Nectin4 cells;

[0107] Figure 10 is 177 Another schematic diagram illustrating the biodistribution of Lu-labeled compounds in PC3-Nectin4 tumor-bearing mice;

[0108] Figure 11 is 177 Another schematic diagram illustrating the biodistribution of Lu-labeled compounds in PC3-Nectin4 tumor-bearing mice;

[0109] Figure 12 is 68 Schematic diagram of Ga-BR005 uptake in Nectin-4 positive PC3-Nectin4 cells;

[0110] Figure 13 is 177 SPECT imaging results of Lu-BR005 in PC3-Nectin4 tumor-bearing mice;

[0111] Figure 14 is 177 Schematic diagram of SPECT image distribution of Lu-BR005 in PC3-Nectin4 tumor-bearing mice;

[0112] Figure 15 is 212 Schematic diagram of body weight changes in PC3-Nectin4 tumor-bearing mice in a pharmacodynamic assay of Pb-BR005;

[0113] Figure 16 is 212 A schematic diagram of tumor volume in PC3-Nectin4 tumor-bearing mice in a pharmacodynamic assay using Pb-BR005.

[0114] Definitions and Explanations

[0115] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0116] The term “pharmaceutically acceptable” as used herein refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0117] The term "pharmaceutically acceptable salt" refers to a salt of the compounds of this invention, prepared by reacting a compound having specific substituents discovered in this invention with a relatively non-toxic acid or base. When the compounds of this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting a neutral form of such compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. When the compounds of this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting a neutral form of such compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Exemplary acid addition salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, hydrogen sulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannins, pantothenates, hydrogen tartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucuronides, glycosides, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and dihydroxynaphthyl acid (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthylcarboxylic acid)). Exemplary base addition salts include, but are not limited to, ammonium salts; alkali metal salts, such as potassium and sodium salts; alkaline earth metal salts, such as calcium and magnesium salts; and salts with organic bases, such as dicyclohexylamine, N-methyl-D-glucosamine, morpholine, thiomorpholine, piperidine, pyrrolidine, mono-, di-, or tri-lower alkylamines (e.g., ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl-, or dimethyl-propylamine), or mono-, di-, or tri-hydroxylower alkylamines (e.g., mono-, di-, or triethanolamine). Pharmaceutically acceptable salts may involve another molecule, such as an acetate ion, a succinate ion, or other counterions. The counterion can be any organic or inorganic part that stabilizes the charge on the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. The case where multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Therefore, pharmaceutically acceptable salts may have one or more charged atoms and / or one or more counterions. It should also be understood that non-pharmaceutically acceptable salts also fall within the scope of this disclosure, as these may be used as intermediates in the preparation of pharmaceutically acceptable salts, or may be useful during storage or transport.

[0118] As used herein, the term "treatment" includes the relief of symptoms associated with a specific disorder or condition. For example, as used herein, the term "treatment of cancer" includes the relief of symptoms associated with cancer. In one embodiment, the term "treatment of cancer" refers to a reduction in the size of a cancerous tumor. In one embodiment, the term "treatment of cancer" refers to a reduction in the invasiveness of cancer. In one embodiment, the term "treatment of cancer" refers to an increase in progression-free survival. As used herein, the term "progression-free survival" refers to the length of time during and after cancer treatment where a patient lives with the disease, i.e., cancer, without disease recurrence or symptom increase.

[0119] The term "prevention" includes preventing specific barriers or conditions. For example, as used herein, the term "cancer prevention" refers to preventing the onset or persistence of cancer-related symptoms. In one implementation, the term "cancer prevention" refers to slowing or stopping the progression of cancer. In another implementation, the term "cancer prevention" refers to slowing or preventing metastasis.

[0120] The term "therapeutic effective amount" refers to an amount of a conjugate containing a therapeutic agent that is sufficient to alleviate or prevent one or more symptoms of the treated disorder or condition to a certain extent. The result may be a reduction and / or alleviation of the signs, symptoms, or cause of the disease or condition, or any other desired change in the biological system. In one embodiment, the term "therapeutic effective amount" refers to an amount of the conjugate sufficient to result in a reduction in the size of a cancerous tumor. In one embodiment, the term "therapeutic effective amount" refers to an amount of the conjugate sufficient to result in an increase in progression-free survival. As used herein, the term "effective amount" refers to an amount in which the conjugate effectively achieves the desired pharmacological effect or therapeutic improvement without excessive adverse side effects, or achieves the desired pharmacological effect or therapeutic improvement with a reduced profile of side effects. Therapeutic effective amounts can be determined, for example, through routine experiments including, but not limited to, dose-escalation clinical trials. The term "therapeutic effective amount" includes, for example, a preventative effective amount. In one embodiment, a preventative effective amount is an amount sufficient to prevent metastasis. It should be understood that the "effective amount" or "therapeutic effective amount" can vary among subjects due to variations in the metabolism of the compound and any of the subject's age, weight, general condition, the condition being treated, the severity of the condition being treated, and the prescribing physician's judgment. Those skilled in the art can determine the appropriate "effective amount" in any individual case using routine experiments.

[0121] Those skilled in the art of organic and / or medicinal chemistry will understand that many organic compounds can form complexes with solvents in which they react, precipitate, or crystallize. These complexes are called “solvents.” For example, a complex with water is called a “hydrate.” The terms “pharmaceutically acceptable solvate” or “solvent” refer to the association of one or more solvent molecules with a compound of this disclosure. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0122] Unless otherwise stated, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereo isomers, enantiomers, optical isomers, diastereomers and tautomers.

[0123] The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

[0124] Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

[0125] Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" arise because the single bonds of double bonds or cyclic carbon atoms cannot rotate freely.

[0126] Unless otherwise stated, the term "diastereomer" refers to a stereoisomer of a molecule having two or more chiral centers and being in a non-mirror relationship with each other.

[0127] Unless otherwise stated, "(+)" indicates right-handed rotation, "(-)" indicates left-handed rotation, and "(±)" indicates racemic rotation.

[0128] Unless otherwise specified, This indicates that the compound is a mixture of cis (Z-type) and trans (E-type) forms.

[0129] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration of the center of a solid is represented by a straight solid line key. and straight dashed key The relative configuration of the center of a solid is indicated by a wavy line. Indicates wedge-shaped solid line key or wedge-shaped dashed key Or use wavy lines Indicates a straight solid line key and straight dashed key

[0130] In this invention, unless otherwise stated, the terms "tautomer" or "tautomer form" refer to isomers with different functional groups in dynamic equilibrium at room temperature, capable of rapidly interconverting into each other. If tautomerization is possible (e.g., in solution), chemical equilibrium of the tautomer can be achieved. For example, proton tautomers (also called prototropic tautomers) involve interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers involve interconversions involving the rearrangement of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between the two tautomers, pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

[0131] Unless otherwise stated, the terms "rich in one isomer," "isomer enrichment," "rich in one enantiomer," or "enantiomer enrichment" mean that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0132] Unless otherwise stated, the terms "isomer excess" or "enantiomer excess" refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, then the isomer or enantiomer excess (ee value) is 80%.

[0133] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the desired enantiomer in pure form. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0134] The compounds of this invention may contain atomic isotopes in non-natural proportions on one or more atoms constituting the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium. 3 H), Iodine-125 125 I) or C-14 14 C). For example, deuterium can be used to replace hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than that between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have advantages such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds of this invention, regardless of radioactivity, are included within the scope of this invention.

[0135] The terms “optional” or “optionally” refer to events or conditions that may occur but are not required to occur as described below, and the description includes both cases where said events or conditions occur and cases where said events or conditions do not occur.

[0136] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which can include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the resulting compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. The term "optionally substituted" means that it may or may not be substituted. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis of chemical feasibility. The phrase "when each of a certain group is optionally substituted" means that any of that group in the aforementioned structure is optionally substituted.

[0137] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 0-2 Rs, the group can optionally be substituted by at most two Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0138] When the number of a linking group is 0, such as -(CRR)0-, it indicates that the linking group is a single bond.

[0139] When the number of a substituent is 0, it means that the substituent does not exist. For example, -A-(R)0 means that the structure is actually -A.

[0140] When a substituent is vacant, it means that the substituent does not exist. For example, if X is vacant in AX, it means that the structure is actually A.

[0141] When one of the variables is selected as a single bond, it means that the two groups it connects to are directly connected. For example, when L in ALZ represents a single bond, it means that the structure is actually AZ.

[0142] When a substituent can be cross-bonded to two or more atoms on a ring, this substituent can bond with any atom on that ring, for example, structural units. This indicates that the substituent R can be substituted at any position on the cyclohexyl or cyclohexadiene. When the listed substituents do not specify which atom they are attached to the substituted group, such substituents can be bonded to any of their atoms. For example, a pyridyl group as a substituent can be attached to the substituted group through any carbon atom on the pyridine ring.

[0143] When the listed linking groups do not specify their linking direction, the linking direction is arbitrary, for example, The linker group L is -MW-. In this case, -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form a ring. Alternatively, rings A and B can be connected in the opposite direction to the left-to-right reading order to form a ring. The combination of linking groups, substituents, and / or their variants is permitted only if such a combination produces a stable compound.

[0144] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of that group can be connected to other groups by chemical bonds. When the chemical bond connection is non-directional and the connectable site contains H atoms, the number of H atoms at that site will decrease accordingly with the number of chemical bonds connected, resulting in a group with a corresponding valence. The chemical bonds connecting the site to other groups can be straight solid line bonds. Straight dashed key or wavy line For example, a straight solid line bond in -OCH3 indicates that the oxygen atom in that group is connected to other groups; The straight dashed bond in the diagram indicates that the group is connected to other groups through both ends of the nitrogen atom in the group; The wavy lines in the diagram indicate that the phenyl group is connected to other groups through the carbon atoms at positions 1 and 2.

[0145] Unless otherwise specified, the term "C" 1-6 "alkyl" is used to denote a straight-chain or branched saturated hydrocarbon group consisting of 1 to 6 carbon atoms. The C 1-6 Alkyl groups include C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-4 C6 and C5 alkyl groups, etc.; C 1-6 Examples of alkyl groups include, but are not limited to, methyl, ethyl, isopropyl, isobutyl, etc.

[0146] Unless otherwise stated, polypeptides or cyclic peptides in this invention are written from left to right in order from nitrogen terminus to carbon terminus.

[0147] The following abbreviations are used in this invention: eq represents equivalent amount; SPPS represents solid-phase polypeptide synthesis; TFA represents trifluoroacetic acid; Boc2O represents di-tert-butyl dicarbonate; DIEA represents diisopropylethylamine; DMF represents N,N-dimethylformamide; DCM represents dichloromethane; Cs2CO3 represents cesium carbonate; ACN represents acetonitrile; HFIP represents hexafluoroisopropanol; HATU represents 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; HBTU represents benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate; HOAt represents N-hydroxy-7-azabenzotriazole; HOBt represents 1-hydroxybenzotriazole; EDC represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Oxyma represents ethyl 2-oxime cyanoacetate; DIC represents N,N'-diisopropylcarbodiimide; Pd(PPh3)4 represents tetratetraphenylphosphine palladium; 3-MPr represents 3-mercaptopropionic acid; Fmoc represents 9-fluorenylmethoxycarbonyl; Alloc represents allyloxycarbonyl; OAll represents carboxylic acid allyl ester; tBu represents tert-butyl; OtBu represents tert-butoxy; Boc represents tert-butoxycarbonyl; Trt represents triphenylmethyl; Mtt represents methyltriphenylmethyl; Pbf represents 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; NHS represents N-hydroxysuccinimide; TIS represents triisopropylsilane; PhSiH3 represents phenylsilane; DMBA represents N,N-dimethylbenzylamine; DTT represents DL-1,4-dithiothreitol; Cit represents L-citrulline; Val represents L-valine; Glutaryl represents β-Ala represents Sar represents Sar10 represents Cys represents L-cysteine; hCys represents βCys represents Pen represents N-methyl-Dap represents 1Nal represents 1-naphthylalanine; hArg represents L-homogeneous arginine; Hyp represents L-hydroxyproline; Trp represents L-tryptophan; Pro represents L-proline; Thr represents L-threonine; Ser represents L-serine; Asp represents L-aspartic acid; dAsp represents D-aspartic acid; Asn represents L-asparagine; Lys represents L-lysine; Met represents L-methionine; DOTA represents... DO3A represents DOTAGA represents Bn-DOTA represents Bn-Oxo-DO3A represents NOTA represents NODAGA represents Bn-NOTA represents NOPO represents DOTAM represents Bn-TCMC Representative Bn-CHX-A”-DTPA represents CB-TE2A represents CB-DO2A represents 2B3M-DTPA represents Bn-PCTA represents TETA represents TATA stands for DOTA active ester refers to mDOTA represents nDOTA represents PBS represents phosphate buffered saline solution; DMSO represents dimethyl sulfoxide; P20 represents polyoxyethylene sorbitol; DDTA represents sodium diethyldithiocarbamate trihydrate (CAS: 20624-25-3); EDT represents 1,2-ethylenedithiol (CAS: 540-63-6); TCEP represents tris(2-carboxyethyl)phosphine; Fmoc-hCit-OH represents...

[0148] Compounds are named in accordance with conventional naming principles in the field, and commercially available compounds are named according to the supplier's catalog.

[0149] The present invention will be described in detail below with reference to embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope thereof.

[0150] Example 1: BR001

[0151] 1.1 Synthesis of peptide sequence P1

[0152] 1.1.1 Resin swelling

[0153] Weigh 1.52 g of Rink Amide MBHAResin (substitution degree S = 0.33 mmol / g) and add it to the reaction column. Then add DMF (20 mL) to the column and purge with nitrogen for 30 min, purging until no liquid flows out. Add 20% piperidine / DMF (20 mL) to the column and purge with nitrogen for 15 min, purging until no liquid flows out. Wash five times with DMF (20 mL each time), 1 min each time, purging until no liquid flows out. Ninhydrin is detected; the resin turns blue.

[0154] 1.1.2 Coupling of amino acids

[0155] Repeat steps a and b in the order shown in Table 1 until the reaction is complete.

[0156] a. Weigh the amino acid raw material and add it to the above resin. Add DMF (15-30 mL) to the reaction column, replenish with organic base, purge with nitrogen, and add condensing agent after the amino acid dissolves. Adjust the nitrogen gas to ensure uniform aeration of the resin.

[0157] b. React at 25℃ for 20 min, and the resin is colorless and transparent when tested for ninhydrin.

[0158] c. Remove the reaction solution, wash with DMF 5 times, 1 minute each time, and drain the waste until no liquid flows out.

[0159] d. Add 20% piperidine / DMF (20-30 mL) to the reaction column, purge with nitrogen for 20 min, and vent until no liquid flows out. Ninhydrin is detected; the resin turns blue.

[0160] Table 1 Material Feeding Table

[0161] 1.1.3 Coupling of Boc anhydrides

[0162] Add 20 mL of the pre-prepared end-capping solution (Boc₂O:DIEA:DMF = 10:5:85 by volume) to the reaction column, and adjust the nitrogen atmosphere to ensure uniform aeration of the resin. React at 25°C for 10 min. The resin should be colorless and transparent according to the tetrachlorobenzoquinone assay. Remove the reaction solution, wash the column five times with DMF (10 mL each time), 1 min each time, and drain the waste until no more liquid flows out.

[0163] 1.1.4 De-Alloc and OAll

[0164] Add PhSiH3 (20.0 eq) and DCM (30 mL) to the reaction column, purge with nitrogen, then add Pd(PPh3)4 (0.20 eq), purge with nitrogen for 20 min, repeat the reaction twice, and remove waste until no liquid flows out. Wash with DMF 5 times (30 mL each time), 1 min each time, and remove waste until no liquid flows out.

[0165] 1.1.5 Amide ring closure

[0166] Add 2.00 eq of DIEA and 30 mL of DMF to the reaction column. Purge with nitrogen until dissolved, then add 0.95 eq of HATU. Adjust the nitrogen level to ensure uniform aeration of the resin. React at 25°C for 0.5 h, washing 5 times (30 mL each time) with DMF for 1 min each time, draining the waste until no liquid flows out.

[0167] 1.1.6 Crude peptide cutting, drying and purification

[0168] The dried peptide resin from step 1.1.5 was added to the prepared cleavage solution (H2O:TIS:3-MPr:TFA = 2.5%:2.5%:2.5%:92.5%, volume ratio), shaken on a shaker for 2.5 h, filtered, and the filtrate was added to 10 volumes of ice-cold isopropyl ether, centrifuged, and washed 5 times with isopropyl ether. The crude peptide was obtained by vacuum drying for 2 h. The crude peptide was dissolved in 15% ACN / H2O, loaded onto a reverse-phase column (A:H2O; B:ACN), and passed through the column to obtain the highly pure peptide sequence P1.

[0169] 1.2 TATA Loop Closure

[0170] Peptide sequence P1 was dissolved at a concentration of 1 mmol / L in 30% ACN / H2O, and TATA (1.10 eq) was added. The pH was adjusted to 8–9 with Cs2CO3, and the reaction was allowed to proceed for 2 h. 1 mL of the liquid was added dropwise with a thiol detection reagent for MS detection. After detection, the pH was adjusted to 7 with 1 M HCl, and the crude peptide was obtained by lyophilization. The crude peptide was dissolved in 35% ACN / H2O and purified using a reverse-phase column (A: H2O; B: ACN) to obtain compound P2.

[0171] 1.3 Synthesis of the trifluoroacetate of compound BR001

[0172] Compound P2 was dissolved in 10.00 mL of DMF, and DOTA active ester (Cas: 170908-81-3, 2.00 eq) was added repeatedly until the reaction was complete. The final product was obtained by reverse-phase column chromatography purification, yielding the trifluoroacetate of compound BR001 (purity 96.15%, HRMS m / z: 985.42 (M+3H)). + ) / 3).

[0173] Example 2: BR002

[0174] Following the synthetic method of compound BR001, the 16th amino acid precursor in Table 1 was replaced with Fmoc-Lys(OAll)-OH (Cas: 281655-70-7) by replacing Fmoc-Lys(OAll)-OH; the 18th amino acid precursor was replaced with Fmoc-Asp(OAll)-OH by replacing Fmoc-Glu(OAll)-OH, thus obtaining the trifluoroacetate of compound BR002 (purity 92.84%, HRMS m / z: 994.76 (M+3H)). + ) / 3).

[0175] Example 5: BR005

[0176] 5.1 Synthesis of peptide sequence P5

[0177] 5.1.1 Resin swelling

[0178] Weigh 9.8 g of MBHA Resin (substitution degree S = 0.51 mmol / g) and add it to the reaction flask. Add DCM (70 mL) to the reaction flask and stir and wash twice, 30 min each time. Drain the flask until no liquid flows out. Add DCM (70 mL) to the reaction flask again and wash once, 2 min each time. Drain the flask. Add 5% DIEA / DMF (70 mL / time) to the reaction flask and stir and wash three times, 2 min each time. Drain the flask and remove waste until no liquid flows out. Add DCM and stir and wash four times (70 mL / time), 2 min each time. Drain the flask until no liquid flows out. Ninhydrin detection: resin turns blue.

[0179] 5.1.1.1 Lever arm Fmoc-Nle-OH condensation

[0180] a. Add the pre-treated Fmoc-Nle-OH / DIC / HOBt (Fmoc-Nle-OH 2.5 eq, DIC 2.5 eq, HOBt 2.5 eq; dissolve the amino acids and HOBt 0.5 h in advance (DMF:DCM = 2:1), and activate with DIC for at least 1 h) to the reaction flask containing the resin. Stir the reaction mixture.

[0181] b. React at a reaction temperature of 25℃ for 180-480 min. Detect ninhydrin; the resin is colorless and transparent or pale yellow.

[0182] 5.1.1.2 Lever arm Fmoc-Rink Linker compression

[0183] a. Add DMF (100-120 mL) to the reaction flask, wash the resin 6 times, 2 min each time, and drain.

[0184] b. Add 20% piperidine / DMF (100-120 mL) to the reaction flask, stir twice (5 min the first time, 30 min the second time), drain until no liquid flows out.

[0185] c. Add DMF and wash 6 times, 2 minutes each time, drain until no liquid flows out. Ninhydrin test: resin turns blue.

[0186] d. Add the processed amino acid Fmoc-Rink Linker / DIC / HOBt (Fmoc-Rink Linker 2.5 eq, DIC 2.5 eq, HOBt 2.5 eq) (dissolve the amino acid and HOBt 0.5 h in advance (DMF:DCM = 2:1), and activate with DIC for at least 1 h) to the reaction flask containing the resin. Stir the reaction mixture.

[0187] e. React at a reaction temperature of 10℃ for 180-480 min. Detect ninhydrin; the resin is colorless and transparent or pale yellow.

[0188] 5.1.2 Coupling of amino acids

[0189] Repeat steps a, e, in the order shown in Table 2 until the reaction is complete. a. Add DMF (100-120 mL) to the reaction flask, wash the resin 6 times, 2 min each time, drain.

[0190] b. Add 20% piperidine / DMF (100-120 mL) to the reaction flask, stir twice (5 min the first time, 30 min the second time), drain until no liquid flows out.

[0191] c. Add DMF and wash 6 times, 2 minutes each time, drain until no liquid flows out. Ninhydrin test: resin turns blue.

[0192] d. Add the treated amino acids (dissolve the amino acids and HOAt (DMF:DCM = 2:1) 0.5 h in advance, and activate with DIC for at least 1 h) to the reaction flask containing the resin. Stir the reaction mixture.

[0193] e. React at a reaction temperature of 10℃ for 180-480 min. Detect ninhydrin; the resin is colorless and transparent or pale yellow.

[0194] Table 2 Material Feeding Table

[0195] 5.1.3 De-Alloc and OAll

[0196] Pd(PPh3)4 (0.54 eq) and DMBA (20 eq) were dissolved in DMF / DCM (1:1) and added to a reaction flask containing resin. The mixture was stirred for 4-6 hours, then drained until no liquid flowed out. The resin was then washed with 0.5% DDTC / DMF, DMF, 5% DIEA / DMF, and DCM, respectively.

[0197] 5.1.4 Amide ring closure

[0198] Dissolve HOAt / DIC (5.0 eq / 5.0 eq) in DMF and add it to a reaction flask containing peptide resin. Stir and react at 0-10℃ for 10-20 h. Then, stir and react once more (2-4 h) with HBTU / HOAt / DIEA (1.9 eq / 2.0 eq / 4.0 eq) at 5-10℃. Wash 13 times (70-120 ml / time) with DMF and DCM alternately, 2 min each time, and drain the waste until no liquid flows out.

[0199] 5.1.5 Crude peptide cutting and drying

[0200] The dried peptide resin was added to the prepared cleavage solution (TFA:H2O:EDT:TIS:ArOH:ArSCH3 = 81.5:5:2.5:1:5:5, DTT and NH4I need to be added), shaken on a shaker for 4 hours, filtered, the filtrate was concentrated, and 12-15 times the volume of frozen methyl tert-butyl ether was added to precipitate the peptide. The precipitate was washed 5-9 times with frozen methyl tert-butyl ether. The crude peptide sequence P5 was obtained by vacuum drying for 1-2 hours.

[0201] 5.1.6 Peptide Sequence P5 Purification

[0202] The crude peptide sequence P5 powder was dissolved in 50% acetonitrile water, and nitrogen gas was passed through while stirring. The solution was filtered through an organic membrane. Purification was performed using a C18 silica gel matrix (UniHybrid 10-200C18) and a trifluoroacetic acid system. The organic phase was eluted in a gradient from 20% to 40%. The fractions were collected and lyophilized to obtain the trifluoroacetate of peptide sequence P5. MS: 2793.18.

[0203] 5.2 TATA Loop Closure:

[0204] Peptide sequence P5 was dissolved in 50% ACN / H2O at a concentration of 0.5 mmol / L. TATA (5 eq) was dissolved in 50% ACN / H2O. A buffer solution (50 mmol / L ammonium bicarbonate + 5 mmol / L ethylenediaminetetraacetic acid) was prepared according to the above buffer preparation method and cooled to 0±3℃. The pH was measured to be between 7.9 and 8.5. If the pH was higher than 8.5, it was adjusted to between 7.9 and 8.5 with 10% TFA / H2O. If the pH was higher than 9.0 when peptide sequence P5 and TATA solution were added, it was adjusted to between 7.9 and 9.0 with 10% TFA / H2O (pH value was monitored). After monitoring, the peptide sequence P5 solution and dissolved TATA were added dropwise simultaneously under N2 protection. The reaction temperature was controlled at 0±3℃. After 1 h of reaction, the temperature was raised to 10±3℃. The reaction was monitored by HPLC. After the reaction was completed, the crude product solution of compound BR005 was obtained.

[0205] 5.3 Preparation of the trifluoroacetate of compound BR005

[0206] The crude solution was then purified by high-performance liquid chromatography (HPLC) using a C18 silica gel matrix (UniHybrid 10-200C18) and a 0.1% trifluoroacetic acid system. The organic phase was purified by gradient elution from 23% to 33%, followed by lyophilization to obtain the trifluoroacetate salt of compound BR005. Product purity: 99.23%, MS: 10¹⁵.0 (M+3H). + ) / 3.

[0207] Purification conditions

[0208] Example 6: BR006

[0209] Referring to the synthesis method of compound BR005, the 19th amino acid raw material in Table 2 was replaced with trans-4-(N-fluorene-9-yl)methoxy)carbonyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid (CAS: 167690-53-1) to obtain the trifluoroacetate of compound BR006 (product purity: 97.72%, MS: 1008.2 (M+3H)). + ) / 3).

[0210] Example 7: BR007

[0211] Following the synthetic method of compound BR005, the 19th amino acid starting material in Table 2 was replaced with Fmoc-1Nal-OH instead of 2-(((9H-fluorene-9-yl)methoxy)carbonyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid, thus yielding the trifluoroacetate of compound BR007 (product purity: 99.04%, MS: 1027.6 (M+3H)). + ) / 3).

[0212] Example 8: BR008

[0213] Following the synthetic method of compound BR005, the 17th amino acid starting material in Table 2 was replaced with Fmoc-D-Phe-OH; the 19th amino acid starting material was replaced with 2-(((9H-fluorene-9-yl)methoxy)carbonyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid, thus obtaining the trifluoroacetate of compound BR008 (product purity: 96.31%, MS: 1010.9 (M+3H)). + ) / 3).

[0214] Example 9: BR009

[0215] 9.1 Synthesis of peptide sequence P9

[0216] 9.1.1 Resin swelling

[0217] Weigh 19.6 g of MBHAResin (substitution degree S = 0.51 mmol / g) and add it to the reaction flask. Add DCM (100-200 mL) to the reaction flask and stir and wash twice, 30 min each time. Drain the flask until no liquid flows out. Add DCM (100-200 mL) to the reaction flask again and wash once, 2 min each time. Drain the flask. Add 5% DIEA / DMF (100-200 mL) to the reaction flask and stir and wash three times, 2 min each time. Drain the flask until no liquid flows out. Add DCM and stir and wash four times (100-200 mL each time, 2 min each time). Drain the flask until no liquid flows out. Ninhydrin is detected; the resin turns blue.

[0218] 9.1.1.1 Lever arm Fmoc-Nle-OH condensation

[0219] a. Add the prepared Fmoc-Nle-OH / DIC / HOBt (Fmoc-Nle-OH 2.5eq, DIC 2.5eq, HOBt 2.5eq, (dissolve amino acids and HOBt 0.5h in advance (DMF:DCM=2:1), and activate with DIC for at least 1h) to a reaction flask containing resin and stir to react.

[0220] b. React at a reaction temperature of 25℃ for 180-480 min. Detect ninhydrin; the resin is colorless and transparent or pale yellow.

[0221] 9.1.1.2 Lever arm Fmoc-Rink Linker compression

[0222] a. Add DMF (200-250 mL) to the reaction flask, wash the resin 6 times, 2 min each time, and drain.

[0223] b. Add 20% piperidine / DMF (200-250 mL) to the reaction column flask, stir twice (5 min the first time, 30 min the second time), drain until no liquid flows out.

[0224] c. Add DMF and wash 6 times, 2 minutes each time, drain until no liquid flows out. Ninhydrin test: resin turns blue.

[0225] d. Add the processed amino acid Fmoc-Rink Linker / DIC / HOBt (Fmoc-Rink Linker 2.5 eq, DIC 2.5 eq, HOBt 2.5 eq (dissolve the amino acid and HOBt 0.5 h in advance (DMF:DCM = 2:1), and activate with DIC for at least 1 h) to the reaction flask containing the resin. Stir the reaction mixture.

[0226] e. React at a reaction temperature of 10℃ for 180-480 min. Detect ninhydrin; the resin is colorless and transparent or pale yellow.

[0227] 9.1.2 Coupling of amino acids

[0228] Repeat step ae in the order shown in Table 3 until the reaction is complete.

[0229] a. Add DMF (100-120 mL) to the reaction flask, wash the resin 6 times, 2 min each time, and drain.

[0230] b. Add 20% piperidine / DMF (100-120 mL) to the reaction column flask, stir twice (5 min the first time and 30 min the second time), drain until no liquid flows out.

[0231] c. Add DMF and wash 6 times, 2 minutes each time, drain until no liquid flows out. Ninhydrin test: resin turns blue.

[0232] d. Add the treated amino acids (dissolve the amino acids and HOAt (DMF:DCM = 2:1) 0.5 h in advance, and activate with DIC for at least 1 h) to the reaction flask containing the resin. Stir the reaction mixture.

[0233] e. React at a reaction temperature of 10℃ for 180-480 min. Detect ninhydrin; the resin is colorless and transparent or pale yellow.

[0234] Table 3 Material Feeding Table

[0235] 9.1.3 Remove Mtt

[0236] Prepare a 50% HFIP / DCM solution and add it to the reaction flask containing the resin. Wash three times (250-400 mL / time), 30 min each time, draining the solution until no liquid flows out. Wash the resin four times with DCM (250-400 mL / time), 2 min each time, draining the solution. Then wash the resin four times with DMF (150-280 mL / time), 2 min each time, draining the solution. Dissolve tert-butyl isocyanate in DMF and add it to the reaction flask containing the resin, stirring. Then add Na2CO3 to the reaction flask and continue the reaction. After the reaction is complete, wash the resin with DCM / H2O, DMF, DCM, and MeOH, then transfer the resin to dry.

[0237] 9.1.4 Crude peptide cutting and drying

[0238] The dried peptide resin from step 9.1.3 was added to the prepared cleavage solution (TFA:H2O:EDT:TIS:ArOH:ArSCH3 = 81.5:5:2.5:1:5:5, DTT and NH4I need to be added), and shaken on a shaker for 4 hours. The solution was filtered, the filtrate was concentrated, and 12-15 times its volume of frozen methyl tert-butyl ether was added to precipitate the peptide. The precipitate was washed 5-9 times with frozen methyl tert-butyl ether. The crude P9 peptide was obtained by vacuum drying for 1-2 hours, with a purity of 46.11%.

[0239] 9.1.5 Peptide Sequence P9 Purification

[0240] The crude linear peptide was dissolved in 30% ACN / H2O and 2 mmol / L TCEP + 0.2% TFA / H2O solution. After complete dissolution, the solution was heated in a water bath at 32°C for 2 hours. The solution was then filtered through filter paper and a filter membrane. The filtered product was labeled for further purification and then purified by high performance liquid chromatography using C18 silica gel matrix packing material and a 2 mmol / L TCEP + 0.2% TFA aqueous solution system. The organic phase was increased from 29% to 39% for gradient elution purification. Finally, the product was lyophilized to obtain a high-purity P9 linear peptide (product purity 89.3%, molecular weight 2461.86).

[0241] 9.2 The thiol groups of the 3, 7, and 17 side chains of the linear peptide form a ring with TATA.

[0242] Peptide sequence P9 was dissolved in 50% ACN / H2O at a concentration of 0.5 mmol / L to prepare TATA (5 eq). TATA was then dissolved in 50% ACN / H2O to prepare a buffer solution (50 mmol / L ammonium bicarbonate + 5 mmol / L ethylenediaminetetraacetic acid + 1.0 mmol / L...). TCEP was used to cool the buffer salt solution to 0±3℃ and measure the pH to be between 7.9 and 8.5. If the pH was higher than 8.5, it was adjusted to between 7.9 and 8.5 with 5% TFA / H2O. If the pH was lower than 7.9 when peptide sequence P9 and TATA solution were added, it was adjusted to between 7.9 and 8.5 with NH4HCO3 (pH value was monitored). After the raw material monitoring was completed, peptide sequence P9 and dissolved TATA were added simultaneously under N2 protection. The reaction temperature was controlled at 0±3℃. After reacting for 1 hour, the temperature was raised to 10±3℃. The reaction was monitored by HPLC (samples were taken every hour for MS and HPLC, and the sampling time was confirmed according to the reaction progress). The reaction was stopped after 9.5 hours to obtain the crude solution of compound BR009.

[0243] 9.3 Purification and preparation of acetate of compound BR009

[0244] The crude solution of compound BR009 was purified by high-performance liquid chromatography (HPLC) using a C18 silica gel matrix and a (A: 0.2% TFA / H2O; B: ACN) solution system with gradient elution, increasing the organic phase from 24% to 34%. Further purification was performed using a (A: 0.2% HAc / H2O; B: ACN) solution system with gradient elution, increasing the organic phase from 21% to 31%. Finally, the product was lyophilized to obtain the acetate of compound BR009 (product purity 97.5%, MS: 1356.1 (M+2H)). + ) / 2).

[0245] Example 10: BR010

[0246] 10.1 The thiol groups of the 3, 7, and 17 side chains of the linear peptide form a ring with 1,3,5-tris(bromomethyl)benzene.

[0247] Peptide sequence P9 was dissolved in 50% ACN / H2O at a concentration of 0.5 mmol / L to prepare a buffer solution (50 mmol / L ammonium bicarbonate + 5 mmol / L ethylenediaminetetraacetic acid + 1.0 mmol / L...) of 1,3,5-tris(bromomethyl)benzene (6 eq) dissolved in DMF. Prepare the buffer solution according to the above method and cool it to 0±3℃. Measure the pH to be between 7.9 and 8.5. Adjust the pH to between 8.5 and 9.0 with ammonia. After the raw material monitoring is completed, simultaneously add peptide sequence P9 and dissolved 1,3,5-tris(bromomethyl)benzene under N2 protection. If the pH is lower than 8.5, adjust it to between 8.5 and 9.0 with ammonia. If the pH is higher than 9.0, adjust it to between 8.5 and 9.0 with 5% TFA / H2O (monitor the pH value). Control the reaction temperature at 0±3℃. After reacting for 1 hour, raise the temperature to 10±3℃. Monitor the reaction by HPLC (sample every hour using MS and HPLC, confirming the sampling time according to the reaction progress). The reaction is completed after 24 hours, yielding the crude solution of compound BR010.

[0248] 10.2 Purification of acetate of compound BR010

[0249] The crude solution of compound BR010 was purified by high-performance liquid chromatography (HPLC) using a C18 silica gel matrix and a solution system of (A: 2 mmol / L TCEP + 0.2% TFA / H2O; B: ACN), with gradient elution of the organic phase from 25% to 35%. Further purification was performed using a solution system of (A: 0.2% HAc / H2O; B: ACN), with gradient elution of the organic phase from 25% to 35%. Finally, purification was performed using a solution system of (A: 0.5% HAc / H2O; B: ACN), with gradient elution of the organic phase from 25% to 35%. The purified product was then lyophilized to obtain the acetate of compound BR010 (product purity 97.8%, MS: 1288.6 (M+2H). + ) / 2).

[0250] Example 11: BR011

[0251] 11.1 Synthesis of peptide sequence P11

[0252] Referring to the preparation method of P9 in Example BR009, the 8th amino acid raw material in Table 3 was replaced with Fmoc-Asp(OtBu)-OH, and the 12th amino acid raw material was replaced with Fmoc-d-Asp(OtBu)-OH. After the coupling reaction of the amino acids was completed, the resin was washed with DMF, DCM, and MeOH and then dried. The dried peptide resin was added to the prepared cleavage solution (TFA:H2O:EDT:TIS:ArOH:ArSCH3 = 81.5:5:2.5:1:5:5, DTT and NH4I need to be added), shaken on a shaker for 4 hours, filtered, the filtrate was concentrated, and 12-15 times the volume of frozen methyl tert-butyl ether was added to precipitate the peptide. The precipitate was washed 5-9 times with frozen methyl tert-butyl ether. The crude peptide P11 was obtained by vacuum drying for 1-2 hours with a purity of 63.9%.

[0253] The crude linear peptide was dissolved in 30% ACN / H2O and 2 mmol / L TCEP + 0.2% TFA / H2O solution. After complete dissolution, the solution was heated in a water bath at 32°C for 2 hours. The solution was then filtered through filter paper and a filter membrane. The filtered product was labeled for further purification and then purified by high performance liquid chromatography using C18 silica gel matrix packing material and a 2 mmol / L TCEP + 0.2% TFA aqueous solution system. The organic phase was increased from 26% to 36% for gradient elution purification. Finally, the product was lyophilized to obtain the highly pure P11 linear peptide (product purity 93.4%).

[0254] 11.2 The thiol groups of the 3, 7, and 17 side chains of the linear peptide form a ring with 1,3,5-tris(bromomethyl)benzene.

[0255] Peptide sequence P11 was dissolved in 50% ACN / H2O at a concentration of 0.5 mmol / L to prepare a buffer solution (50 mmol / L ammonium bicarbonate + 5 mmol / L ethylenediaminetetraacetic acid + 1.0 mmol / L...) with 1,3,5-tris(bromomethyl)benzene (10 eq) dissolved in DMF. Prepare the buffer solution according to the above method and cool it to 0±3℃. Measure the pH to be between 7.9 and 8.5. Adjust the pH to between 8.5 and 9.0 with ammonia. After the raw material monitoring is completed, simultaneously add the peptide sequence P11 and dissolved 1,3,5-tris(bromomethyl)benzene under N2 protection. If the pH is lower than 8.5, adjust it to between 8.5 and 9.0 with ammonia. If the pH is higher than 9.0, adjust it to between 8.5 and 9.0 with 5% TFA / H2O (monitor the pH value). Control the reaction temperature at 0±3℃. After reacting for 1 hour, raise the temperature to 10±3℃. Monitor the reaction by HPLC (sample every hour using MS and HPLC, confirming the sampling time according to the reaction progress). The reaction is completed after 24.5 hours, yielding the crude solution of compound BR011.

[0256] 11.3 Purification and preparation of acetate of compound BR011

[0257] The crude solution of compound BR011 was purified by high-performance liquid chromatography (HPLC) using a C18 silica gel matrix and a solution system of (A: 2 mmol / L TCEP + 0.2% TFA / H2O; B: ACN), with gradient elution from 29% to 39% to 42% organic phase. Further purification was performed using a solution system of (A: 0.2% HAc / H2O; B: ACN), with gradient elution from 24% to 34% organic phase. The purified product was then lyophilized to obtain the acetate of compound BR011 (product purity 96.1%, MS: 1289.6 (M+2H2O)). + ) / 2).

[0258] Example 12: BR012

[0259] Following the synthesis method of compound BR011, the 8th amino acid in Table 3 was replaced with Fmoc-Asp(OtBu)-OH, the 9th amino acid with Fmoc-Lys(Me)-OH, and the 12th amino acid with Fmoc-d-Asp(OtBu)-OH; thus obtaining the crude product solution of compound BR012. The crude product solution of compound BR012 was purified by high-performance liquid chromatography (HPLC) using a C18 silica gel matrix and a solution system of (A: 2 mmol / L TCEP + 0.2% TFA / H2O; B: ACN), with gradient elution of the organic phase from 25% to 35%. Further purification was performed using a solution system of (A: 0.5% HAc / H2O; B: ACN), with gradient elution of the organic phase from 25% to 35%. The purified product was then lyophilized to obtain the acetate of compound BR012 (product purity 97.6%, MS: 850.3 (M+3H). + ) / 3).

[0260] Example 13: BR013

[0261] 13.1 Synthesis of peptide sequence P13

[0262] 13.1.1 Resin swelling

[0263] Add 1.2 g of Rink Resin resin (substitution degree S = 0.4 mmol / g) to the reactor, and add DCM (10 mL / g) to swell for 5 minutes. Vacuum dry the swollen reagent, add 20% Pip / DMF (10 mL / g), stir for 5 minutes, and then vacuum dry. Add another 20% Pip / DMF (10 mL / g) and stir for 5 minutes. Vacuum dry the deprotecting reagent, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then vacuum dry. Ninhydrin detection: a darkening of the resin color indicates a positive result, signifying successful deprotection.

[0264] 13.1.2 Condensation of the first amino acid

[0265] Dissolve Fmoc-Cys(Trt)-OH(3eq) and Oxyma(3eq) in an appropriate amount of DMF (10mL), then add DIC(3eq), activate for 5 minutes, pour into a reactor, and stir for 1 hour. Add 10mL / g capping solution (DMF:DIEA:ACE = 90:5:5), cap for 1 hour, and dry the reagent under vacuum. Wash 5 times with DMF (10mL / g), stirring for 20-30 seconds each time, and dry under vacuum.

[0266] 13.1.3 Coupling of amino acids

[0267] Repeat step af in the order shown in Table 4 until the reaction is complete.

[0268] a. Add 20% Pip / DMF (10 mL / g), stir for 5 minutes, and then dry under vacuum. Add another 20% Pip / DMF (10 mL / g) and stir for 5 minutes. b. Remove the protective agent by vacuum drying, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum.

[0269] c. Ninhydrin detection: A darkening of the resin color indicates a positive result, signifying successful deprotection.

[0270] d. Dissolve the amino acid raw material and Oxyma in an appropriate amount of DMF (10 mL), then add DIC and activate for 5 minutes. Pour the mixture into a reactor and stir to react for 1 hour.

[0271] e. Wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry.

[0272] f. Ninhydrin detection: No significant color change in the resin indicates successful condensation reaction.

[0273] Table 4 Material Feeding Table

[0274] The resin was washed five times with methanol (10 mL) and dried under vacuum. The solid-phase condensation was then complete.

[0275] 13.1.4 Resin lysis and peptide purification

[0276] The dried resin was loaded into a boat-shaped reactor, and 10 mL / g lysis buffer (TFA:H2O:EDT:TIS:phenol = 80:5:10:2.5:2.5) was added. The mixture was lysed at room temperature for 3 hours, filtered, and the filtrate was added to 10 times its volume of ice-cold diethyl ether. The precipitate was centrifuged and washed three times with 200 mL of ice-cold diethyl ether to obtain a crude peptide solid. The crude product was lyophilized and purified by high-performance liquid chromatography (HPLC) (column type: C18 20*250 mm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 25-40, 40 min) to obtain a high-purity linear peptide sequence P13 (ESI-MS m / z: 1336.2 (M+2H)). + ) / 2).

[0277] 13.2 TATA Loop Closure

[0278] 13.2.1 Reagent Preparation

[0279] 100mM NH4HCO3 solution: Weigh 79mg of NH4HCO3 solid, add 100mL of purified water to dissolve and shake well.

[0280] 13.2.2 TATA Modification

[0281] Linear peptide P13 (1 eq) was added to NH4HCO3 solution and mixed well (concentration: 1 mg / mL). The reaction solution became clear. TATA (1.3 eq) was dissolved in pure acetonitrile and slowly added dropwise to the linear peptide solution. If precipitation occurred, pure acetonitrile was added until the solution became clear. The reaction was stirred for 1 hour to obtain the cyclized crude product BR013. The reaction was monitored by HPLC (samples were taken for HPLC and MS detection).

[0282] 13.3 Synthesis of the trifluoroacetate of BR013

[0283] The crude BR013 was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 20-40, 40 min); then purified by HPLC (column type: C18 20*250mm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 20-40, 60 min), and lyophilized to obtain trifluoroacetate of BR013 (HRMS m / z: 2917.231).

[0284] Example 14: BR014

[0285] Referring to the synthesis methods 13.1 and 13.2 of compound BR013, the 16th amino acid raw material in Table 4 was replaced with Fmoc-Phe-OH, and the 17th amino acid raw material was replaced with Fmoc-Gly-OH to obtain crude product BR014. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, mobile phase: A: 30mM (NH4)2SO4 / H2O (pH 2.23); B: ACN, gradient: 28-43, 40 min); then purified by HPLC (column type: C18 20*250mm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 21-41, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR014 (HRMS m / z: 2917.234).

[0286] Example 15: BR015

[0287] Following the synthetic methods 13.1 and 13.2 for compound BR013, the 16th amino acid precursor, Fmoc-Gly-OH, in Table 4 was deleted to obtain crude BR015. The crude product was purified by high-performance liquid chromatography (HPLC) (column: C18 20*250 mm, mobile phase: A: NH4OAc / H2O (pH 4.85); B: ACN, gradient: 28-48, 60 min); followed by HPLC (column: C18 20*250 mm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 21-41, 40 min), and then lyophilized to obtain the trifluoroacetate of compound BR015 (HRMS m / z: 2860.220).

[0288] Example 16: BR016

[0289] Referring to the synthesis methods 13.1 and 13.2 of compound BR013, the 16th amino acid raw material in Table 4 was replaced with Fmoc-Val-OH, and the 17th amino acid raw material was replaced with Fmoc-Phe-OH, to obtain crude product BR016. The crude product was purified by high-performance liquid chromatography (HPLC) (column type: C18 20*250mm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 24-39, 40 min); then purified by HPLC (column type: C18 20*250mm, mobile phase: A: NH4OAc / H2O (pH 4.85); B: ACN, gradient: 30-45, 40 min); then purified by HPLC (column type: C18 20*250mm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 24-39, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR016 (HRMS m / z: 2943.249).

[0290] Example 17: BR017

[0291] 17.1 Synthesis of peptide sequence P17

[0292] 17.1.1 Resin swelling

[0293] Add 1.2 g of Rink Resin resin (substitution degree S = 0.4 mmol / g) to the reactor, and add DCM (10 mL / g) to swell for 5 minutes. Vacuum dry the swollen reagent, add 20% Pip / DMF (10 mL / g), stir for 5 minutes, and then vacuum dry. Add another 20% Pip / DMF (10 mL / g) and stir for 5 minutes. Vacuum dry the deprotecting reagent, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then vacuum dry. Ninhydrin detection: a darkening of the resin color indicates a positive result, signifying successful deprotection.

[0294] 17.1.2 Condensation of the first amino acid

[0295] Dissolve Fmoc-Cys(Trt)-OH(3eq) and Oxyma(3eq) in an appropriate amount of DMF (10mL), then add DIC(3eq), activate for 5 minutes, pour into a reactor, and stir for 1 hour. Add 10mL / g capping solution (DMF:DIEA:ACE = 90:5:5), cap for 1 hour, and dry the reagent under vacuum. Wash 5 times with DMF (10mL / g), stirring for 20-30 seconds each time, and dry under vacuum.

[0296] 17.1.3 Coupling of amino acids

[0297] Repeat step af in the order shown in Table 5 until the reaction is complete.

[0298] a. Add 20% Pip / DMF (10 mL / g), stir for 5 minutes, and then dry under vacuum. Add another 20% Pip / DMF (10 mL / g) and stir for 5 minutes.

[0299] b. Remove the protective reagent by vacuum drying, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum.

[0300] c. Ninhydrin detection: A darkening of the resin color indicates a positive result, signifying successful deprotection.

[0301] d. Dissolve the amino acid raw material and Oxyma in an appropriate amount of DMF (10 mL), then add DIC and activate for 5 minutes. Pour the mixture into a reactor and stir to react for 1 hour.

[0302] e. Wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry.

[0303] f. Ninhydrin detection: No significant color change in the resin indicates successful condensation reaction.

[0304] Table 5 Material Feeding Table

[0305] 17.1.4 Removal of the protecting group OAll

[0306] Dissolve tetra(triphenylphosphine)palladium (0.2 eq) and phenylsilane (10 eq) in DCM (15 mL), add to the resin, react for 2 hours, and then remove the solvent. Repeat the reaction once more. Prepare copper reagents (A: 0.5% sodium diethylaminothiocarbamate / DMF; B: 0.5% DIEA / DMF) to wash the resin. Wash with 50% of each reagent, changing the solution every 5-10 minutes, until the resin regains its original color.

[0307] 17.1.5 Cycloning

[0308] Dissolve DIC (2 eq) and Oxyma (2 eq) in an appropriate amount of DMF (10 mL), pour the solution into the reactor, stir and react for 1 hour, then vacuum dry. Wash five times with DMF (10 mL / g), stirring for 20-30 seconds each time, then vacuum dry. Next, dissolve HATU (2 eq) and DIEA (2 eq) in an appropriate amount of DMF (10 mL), pour the solution into the reactor, stir and react for 1 hour, then vacuum dry. Wash five times with DMF (10 mL / g), stirring for 20-30 seconds each time, then vacuum dry. Ninhydrin testing showed no significant color change in the resin, indicating successful condensation.

[0309] The resin was washed five times with methanol (10 mL) and dried under vacuum. The solid-phase condensation was then complete.

[0310] 17.1.6 Resin lysis and peptide purification

[0311] The dried resin was loaded into a boat-shaped reactor, and 10 mL / g lysis buffer (TFA:H2O:EDT:TIS:phenol = 80:5:10:2.5:2.5) was added. The mixture was lysed at room temperature for 3 hours, filtered, and the filtrate was added to 10 times its volume of ice-cold diethyl ether. The precipitate was centrifuged and washed three times with 200 mL of ice-cold diethyl ether to obtain crude peptide solid. The crude product was lyophilized and purified by high-performance liquid chromatography (HPLC) (column type: C18 20*250 mm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 24-44, 40 min) to obtain a high-purity linear peptide sequence P17 (ESI-MS m / z: 1484.5 (M+2H)). + ) / 2).

[0312] 17.2 TATA Loop Closure

[0313] 17.2.1 Reagent Preparation

[0314] 100mM NH4HCO3 solution: Weigh 79mg of NH4HCO3 solid, add 100mL of purified water to dissolve and shake well.

[0315] 17.2.2 TATA Modification

[0316] Linear peptide P17 (1 eq) was added to NH4HCO3 solution and mixed well (concentration: 1 mg / mL). The reaction solution became clear. TATA (1.3 eq) was dissolved in pure acetonitrile and slowly added dropwise to the linear peptide solution. If precipitation occurred, pure acetonitrile was added until the solution became clear. The reaction was stirred for 1 hour to obtain the cyclized crude product BR017. The reaction was monitored by HPLC (samples were taken for HPLC and MS detection).

[0317] 17.3 Synthesis of trifluoroacetate of BR017

[0318] The crude BR017 was purified by high-performance liquid chromatography (HPLC) (column: C18 20*250mm, mobile phase: A: NH4OAc / H2O (pH 4.85); B: ACN, gradient: 21-41, 60 min); then purified by HPLC (column: C18 20*250mm, mobile phase: A: NH4OAc / H2O (pH 4.85); B: ACN, gradient: 21-41, 40 min); then purified by HPLC (column: C4 20*250mm, mobile phase: A: 30mM (NH4)2SO4 / H2O (pH 2.23); B: ACN, gradient: 28-43, 40 min); and finally purified by HPLC (column: C18 20*250mm, mobile phase: A: 0.1%). (TFA / H2O; B: 0.1% TFA / ACN, gradient: 28-43, 30 min) Separation and purification, lyophilization, to obtain trifluoroacetate of BR017 (HRMS m / z: 3213.377).

[0319] Example 18: BR018

[0320] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 19th amino acid raw material in Table 5 was replaced with Fmoc-Phe-OH, and the 20th amino acid raw material was replaced with Fmoc-Phe-OH, to obtain crude product BR018. The crude product was purified by high-performance liquid chromatography (HPLC) (column type: C18 20*250mm, mobile phase: A: NH4OAc / H2O (pH 4.85); B: ACN, gradient: 21-41, 60 min); then purified by HPLC (column type: C18 20*250mm, mobile phase: A: 30mM (NH4)2SO4 / H2O (pH 2.23); B: ACN, gradient: 21-41, 40 min); then purified by HPLC (column type: C18 20*250mm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 28-43, 30 min), and lyophilized to obtain the trifluoroacetate of compound BR018 (HRMS m / z: 3213.376).

[0321] Example 19: BR019

[0322] Following the synthetic methods 17.1 and 17.2 for compound BR017, the 19th amino acid precursor, Fmoc-Gly-OH, in Table 5 was deleted to obtain crude BR019. The crude product was purified by high-performance liquid chromatography (HPLC) (column: C18 20*250 mm, mobile phase: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 20-35, 40 min); then purified by HPLC (column: C18 20*250 mm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 28-43, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR019 (HRMS m / z: 3156.353).

[0323] Example 20: BR020

[0324] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 19th amino acid raw material in Table 5 was replaced with Fmoc-Val-OH, and the 20th amino acid raw material was replaced with Fmoc-Phe-OH, to obtain crude product BR020. The crude product was purified by high-performance liquid chromatography (HPLC) (column: C18 20*250mm, mobile phase: A: NH4OAc / H2O (pH 4.85); B: ACN, gradient: 21-41, 60 min); then purified by HPLC (column: C4 20*250mm, mobile phase: A: 30mM (NH4)2SO4 / H2O (pH 2.23); B: ACN, gradient: 28-43, 40 min); then purified by HPLC (column: C18 20*250mm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 28-43, 30 min), and lyophilized to obtain the trifluoroacetate of compound BR020 (HRMS m / z: 3239.392).

[0325] Example 21: BR021

[0326] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 18th amino acid feedstock in Table 5 was replaced with Fmoc-Lys(Boc)-OH, the 19th amino acid feedstock was replaced with Fmoc-Gly-OH, and the 20th amino acid feedstock was replaced with Fmoc-Phe-OH, to obtain crude product BR021. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 25-45, 40 min); then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 25-45, 60 min), and lyophilized to obtain the trifluoroacetate of compound BR021 (HRMS m / z: 3197.355).

[0327] Example 22: BR022

[0328] 22.1 Synthesis of peptide sequence P22

[0329] 22.1.1 Resin swelling

[0330] Add 1.2 g of Rink Resin resin (substitution degree S = 0.4 mmol / g) to the reactor, and add DCM (10 mL / g) to swell for 5 minutes. Vacuum dry the swollen reagent, add 20% Pip / DMF (10 mL / g), stir for 5 minutes, and then vacuum dry. Add another 20% Pip / DMF (10 mL / g) and stir for 5 minutes. Vacuum dry the deprotecting reagent, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then vacuum dry. Ninhydrin detection: a darkening of the resin color indicates a positive result, signifying successful deprotection.

[0331] 22.1.2 Condensation of the first amino acid

[0332] Dissolve Fmoc-Cys(Trt)-OH(3eq) and Oxyma(3eq) in an appropriate amount of DMF (10mL), then add DIC(3eq), activate for 5 minutes, pour into a reactor, and stir for 1 hour. Add 10mL / g capping solution (DMF:DIEA:ACE = 90:5:5), cap for 1 hour, and dry the reagent under vacuum. Wash 5 times with DMF (10mL / g), stirring for 20-30 seconds each time, and dry under vacuum.

[0333] 22.1.3 Coupling of amino acids

[0334] Repeat step af in the order shown in Table 6 until the condensation of the 17th amino acid raw material Fmoc-Asp(Oall)-OH is completed.

[0335] a. Add 20% Pip / DMF (10 mL / g), stir for 5 minutes, and then dry under vacuum. Add another 20% Pip / DMF (10 mL / g) and stir for 5 minutes.

[0336] b. Remove the protective reagent by vacuum drying, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum.

[0337] c. Ninhydrin detection: A darkening of the resin color indicates a positive result, signifying successful deprotection.

[0338] d. Dissolve the amino acid raw material and Oxyma in an appropriate amount of DMF (10 mL), then add DIC and activate for 5 minutes. Pour the mixture into a reactor and stir to react for 1 hour.

[0339] e. Wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry.

[0340] f. Ninhydrin detection: No significant color change in the resin indicates successful condensation reaction.

[0341] Table 6 Material Feeding Table

[0342] Add 20% Pip / DMF (10 mL / g), stir for 5 minutes, and then dry under vacuum. Add another 20% Pip / DMF (10 mL / g) and stir for 5 minutes. Remove the protective reagent under vacuum, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum. A positive result (deprotection) is indicated by a darkening of the resin color in the ninhydrin test. Dissolve succinic anhydride (3 eq) in an appropriate amount of DMF (10 mL), pour into the reactor, add DIEA (6 eq), and stir for 1 hour. Remove the reagent under vacuum, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum. No significant color change in the resin indicates successful condensation in the ninhydrin test.

[0343] Dissolve Fmoc-L-Dap-OtBu·HCl (3 eq) and HBTU (3 eq) in an appropriate amount of DMF (10 mL), pour the solution into a reactor, add DIEA (6 eq), and stir for 1 hour. Vacuum dry the reagents, wash five times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum.

[0344] Repeat step af to complete the condensation of amino acid raw materials from the 20th to the 22nd amino acid raw materials in Table 6 in sequence.

[0345] Following the synthetic methods 17.1.4, 17.1.5, and 17.1.6 for compound BR017, the linear peptide sequence P22 (ESI-MS m / z: 1525.9 (M+2H)) was obtained. + ) / 2).

[0346] Following the synthetic method 17.2 for compound BR017, crude product BR022 was obtained. The crude product was purified by high-performance liquid chromatography (HPLC) (column: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 28-43, 40 min). Further purification by HPLC (column: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 27-42, 40 min) followed by lyophilization to obtain trifluoroacetate of compound BR022 (HRMS m / z: 3297.372).

[0347] Example 23: BR023

[0348] Following the synthetic method of compound BR022, the 19th amino acid raw material in Table 6 was replaced with Fmoc-D-Dap-OtBu·HCl to obtain crude product BR023. The crude product was purified by high-performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 24-44, 40 min). It was then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 25-45, 60 min), lyophilized, and the trifluoroacetate of compound BR023 was obtained (HRMS m / z: 3297.374).

[0349] Example 24: BR024

[0350] Synthesis of peptide sequence P24, 24.1

[0351] 24.1.1 Resin swelling

[0352] Add 1.2 g of Rink Resin resin (substitution degree S = 0.4 mmol / g) to the reactor, and add DCM (10 mL / g) to swell for 5 minutes. Vacuum dry the swollen reagent, add 20% Pip / DMF (10 mL / g), stir for 5 minutes, and then vacuum dry. Add another 20% Pip / DMF (10 mL / g) and stir for 5 minutes. Vacuum dry the deprotecting reagent, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then vacuum dry. Ninhydrin detection: a darkening of the resin color indicates a positive result, signifying successful deprotection.

[0353] 24.1.2 Condensation of the first amino acid

[0354] Dissolve Fmoc-Cys(Trt)-OH(3eq) and Oxyma(3eq) in an appropriate amount of DMF (10mL), then add DIC(3eq), activate for 5 minutes, pour into a reactor, and stir for 1 hour. Add 10mL / g capping solution (DMF:DIEA:ACE = 90:5:5), cap for 1 hour, and dry the reagent under vacuum. Wash 5 times with DMF (10mL / g), stirring for 20-30 seconds each time, and dry under vacuum.

[0355] 24.1.3 Coupling of amino acids

[0356] Repeat step af in the order shown in Table 7 until the condensation of the 14th amino acid raw material Fmoc-Cys(Trt)-OH is completed.

[0357] a. Add 20% Pip / DMF (10 mL / g), stir for 5 minutes, and then dry under vacuum. Add another 20% Pip / DMF (10 mL / g) and stir for 5 minutes.

[0358] b. Remove the protective reagent by vacuum drying, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum.

[0359] c. Ninhydrin detection: A darkening of the resin color indicates a positive result, signifying successful deprotection.

[0360] d. Dissolve the amino acid raw material and Oxyma in an appropriate amount of DMF (10 mL), then add DIC and activate for 5 minutes. Pour the mixture into a reactor and stir to react for 1 hour.

[0361] e. Wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry.

[0362] f. Ninhydrin detection: No significant color change in the resin indicates successful condensation reaction.

[0363] Table 7 Material Feeding Table

[0364] Add 20% Pip / DMF (10 mL / g), stir for 5 minutes, and then dry under vacuum. Add another 20% Pip / DMF (10 mL / g) and stir for 5 minutes. Remove the protective reagent under vacuum, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum. A positive result (deprotection) is indicated by a darkening of the resin color in the ninhydrin test. Dissolve succinic anhydride (3 eq) in an appropriate amount of DMF (10 mL), pour into the reactor, add DIEA (6 eq), and stir for 1 hour. Remove the reagent under vacuum, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum. No significant color change in the resin indicates successful condensation in the ninhydrin test.

[0365] Dissolve Fmoc-L-Dap-OtBu·HCl (3 eq) and HBTU (3 eq) in an appropriate amount of DMF (10 mL), pour the solution into a reactor, add DIEA (6 eq), and stir for 1 hour. Vacuum dry the reagents, wash five times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum.

[0366] Repeat step af to complete the condensation of amino acid raw materials from the 17th to the 19th amino acid raw materials in Table 7 in sequence.

[0367] The resin was washed five times with methanol (10 mL) and dried under vacuum. The solid-phase condensation was then complete.

[0368] Following the synthetic method 13.1.4 for compound BR013, the linear peptide sequence P24 (ESI-MS m / z: 1378.1 (M+2H)) was obtained. + ) / 2).

[0369] Following the synthetic method 13.2 for compound BR013, crude BR024 was obtained. The crude product was purified by high-performance liquid chromatography (HPLC) (column: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 25-40, 60 min). Further purification by HPLC (column: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 26-41, 40 min) followed by lyophilization to obtain trifluoroacetate of compound BR024 (HRMS m / z: 3001.230).

[0370] Example 25: BR025

[0371] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 7th amino acid raw material in Table 5 was replaced with Fmoc-Asn(Trt)-OH, the 18th amino acid raw material was replaced with Fmoc-Lys(Boc)-OH, and the 19th and 20th amino acid raw materials were deleted to obtain crude product BR025. The crude product was purified by high performance liquid chromatography (HPLC) (column: C18 20*250mm, 10μm; mobile phase: A: 0.025% NH3 / H2O; B: ACN; gradient: 5-45, 45 min); then purified by HPLC (column: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 26-46, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR025 (HRMS m / z: 2951.248).

[0372] Example 26: BR026

[0373] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 7th amino acid feedstock in Table 5 was replaced with Fmoc-Asn(Trt)-OH, the 8th amino acid feedstock was replaced with Fmoc-hArg(Pbf)-OH, the 18th amino acid feedstock was replaced with Fmoc-Lys(Boc)-OH, and the 19th and 20th amino acid feedstocks were deleted to obtain crude product BR026. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: 0.025% NH3 / H2O; B: ACN; gradient: 5-45, 45 min); then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 26-48, 60 min), and lyophilized to obtain the trifluoroacetate of compound BR026 (HRMS m / z: 2952.234).

[0374] Example 27: BR027

[0375] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 7th amino acid feedstock in Table 5 was replaced with Fmoc-Asn(Trt)-OH, the 8th amino acid feedstock was replaced with Fmoc-hArg(Pbf)-OH, the 11th amino acid feedstock was replaced with Fmoc-D-Asp(OtBu)-OH, the 18th amino acid feedstock was replaced with Fmoc-Lys(Boc)-OH, and the 19th and 20th amino acid feedstocks were deleted to obtain crude product BR027. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 23-43, 60 min); then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 25-45, 50 min), and lyophilized to obtain the trifluoroacetate of compound BR027 (HRMS m / z: 2908.247).

[0376] Example 28: BR028

[0377] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 7th amino acid feedstock in Table 5 was replaced with Fmoc-Asn(Trt)-OH, the 8th amino acid feedstock was replaced with Fmoc-hArg(Pbf)-OH, the 11th amino acid feedstock was replaced with Fmoc-D-Asp(OtBu)-OH, the 18th amino acid feedstock was replaced with Fmoc-Lys(Boc)-OH, and the 19th and 20th amino acid feedstocks were deleted to obtain crude product BR028. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 25-45, 50 min); then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 23-43, 60 min), and lyophilized to obtain the trifluoroacetate of compound BR028 (HRMS m / z: 2984.279).

[0378] Example 29: BR029

[0379] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 8th amino acid feedstock in Table 5 was replaced with Fmoc-hArg(Pbf)-OH, the 18th amino acid feedstock was replaced with Fmoc-Lys(Boc)-OH, and the 19th and 20th amino acid feedstocks were deleted to obtain crude product BR029. The crude product was purified by high performance liquid chromatography (column type: C18 20*250mm, 10μm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 26-46, 60 min), and then lyophilized to obtain trifluoroacetate of compound BR029 (HRMS m / z: 2966.256).

[0380] Example 30: BR030

[0381] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 9th amino acid raw material in Table 5 was replaced with Fmoc-Nle-OH, the 18th amino acid raw material was replaced with Fmoc-Lys(Boc)-OH, and the 19th and 20th amino acid raw materials were deleted to obtain crude product BR030. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 25-45, 50 min); then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 25-45, 60 min), and lyophilized to obtain the trifluoroacetate of compound BR030 (HRMS m / z: 2934.278).

[0382] Example 31: BR031

[0383] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 7th amino acid feedstock in Table 5 was replaced with Fmoc-Asn(Trt)-OH, the 8th amino acid feedstock was replaced with Fmoc-hArg(Pbf)-OH, the 18th amino acid feedstock was replaced with Fmoc-Lys(Boc)-OH, and the 19th and 20th amino acid feedstocks were deleted to obtain crude product BR031. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 26-41, 40 min); then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 24-39, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR031 (HRMS m / z: 2965.271).

[0384] Example 32: BR032

[0385] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 8th amino acid feedstock in Table 5 was replaced with Fmoc-hArg(Pbf)-OH, and an amino acid feedstock Fmoc-Val-OH was inserted between the 8th and 9th amino acid feedstocks. The 18th amino acid feedstock was replaced with Fmoc-Sar-OH, and the 19th and 20th amino acid feedstocks were deleted to obtain crude product BR032. The obtained crude product was purified by high performance liquid chromatography (column type: C18 20*250mm, 10μm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 23-53, 80 min), and then lyophilized to obtain trifluoroacetate of compound BR032 (HRMS m / z: 3051.287).

[0386] Example 33: BR033

[0387] Synthesis of peptide sequence P33 (33.1)

[0388] 33.1.1 Resin swelling

[0389] CTC Resin resin (1.2 g, substitution degree S = 0.4 mmol / g) was added to the reactor, and DCM (10 mL / g) was added to swell the resin for 5 minutes. The swollen reagent was then dried under vacuum.

[0390] 33.1.2 Resin Modification

[0391] Dissolve Fmoc-β-Ala-OH (0.5 eq) in an appropriate amount of DCM (10 mL), pour it into a reactor, add DIEA (2 eq) dropwise, and stir the reaction for 1 hour. Add 1 mL / g methanol, cap the reaction for 1 hour, and then dry the reagent under vacuum. Wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum.

[0392] 33.1.3 Coupling of amino acids

[0393] Repeat step af in the order shown in Table 8 until the reaction is complete.

[0394] a. Add 20% Pip / DMF (10 mL / g), stir for 5 minutes, and then dry under vacuum. Add another 20% Pip / DMF (10 mL / g) and stir for 5 minutes.

[0395] b. Remove the protective reagent by vacuum drying, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum.

[0396] c. Ninhydrin detection: A darkening of the resin color indicates a positive result, signifying successful deprotection.

[0397] d. Dissolve the amino acid raw material and Oxyma in an appropriate amount of DMF (10 mL), then add DIC and activate for 5 minutes. Pour the mixture into a reactor and stir to react for 1 hour.

[0398] e. Wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry.

[0399] f. Ninhydrin detection: No significant color change in the resin indicates successful condensation reaction.

[0400] Table 8 Material Feeding Table

[0401] Following the synthetic methods 17.1.4, 17.1.5, and 17.1.6 for compound BR017, the linear peptide sequence P33 (ESI-MS m / z: 1389.6 (M+2H)) was obtained. + ) / 2).

[0402] Following the synthetic method 17.2 for compound BR017, crude product BR033 was obtained. The crude product was purified by high-performance liquid chromatography (HPLC) (column: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 27-42, 40 min). It was then purified by HPLC (column: C18 20*250mm, 10μm; mobile phase: A: 30mM (NH4)2SO4 / H2O (pH adjusted to 2.25 with phosphoric acid); B: ACN; gradient: 25-45, 30 min). The purified product was then lyophilized to obtain the trifluoroacetate of compound BR033 (HRMS). m / z:3024.253).

[0403] Example 34: BR034

[0404] Following the synthetic method of compound BR033, the amino acid raw material in 33.1.2 was replaced with Fmoc-Asp(OtBu)-OH to obtain crude BR034. The crude product was purified by high-performance liquid chromatography (HPLC) (column: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 28-43, 40 min). Further purification was achieved by HPLC (column: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 24-39, 40 min), followed by lyophilization to obtain the trifluoroacetate of compound BR034 (HRMS m / z: 3068.237).

[0405] Example 35: BR035

[0406] Referring to the synthesis methods 13.1 and 13.2 of compound BR013, the 7th amino acid feedstock in Table 4 was replaced with Fmoc-Asn(Trt)-OH, the 8th amino acid feedstock was replaced with Fmoc-hArg(Pbf)-OH, the 11th amino acid feedstock was replaced with Fmoc-D-Asp(OtBu)-OH, the 15th amino acid feedstock was replaced with Fmoc-L-adamantaneglycine, and the 16th and 17th amino acid feedstocks were deleted to obtain crude product BR035. The crude product was purified by high performance liquid chromatography (column type: C18 20*250mm, 10μm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 25-55, 80 min), and then lyophilized to obtain the trifluoroacetate of compound BR035 (HRMS m / z: 2775.198).

[0407] Example 36: BR036

[0408] Referring to the synthesis methods 13.1 and 13.2 of compound BR013, the 8th amino acid feedstock in Table 4 was replaced with Fmoc-HOMOCIT-OH, the 9th amino acid feedstock was replaced with Fmoc-Nle-OH, the 15th amino acid feedstock was replaced with Fmoc-Lys(Boc)-OH, and the 16th and 17th amino acid feedstocks were deleted; the cyclization feedstock TATA in 13.2.2 was replaced with 1,3,5-tris(bromomethyl)benzene to obtain crude product BR036. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 23-43, 50 min); then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 25-45, 60 min), and lyophilized to obtain the trifluoroacetate of compound BR036 (HRMS m / z: 2558.103).

[0409] Example 37: BR037

[0410] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 7th amino acid feedstock in Table 5 was replaced with Fmoc-Asn(Trt)-OH, the 8th amino acid feedstock was replaced with Fmoc-hArg(Pbf)-OH, the 11th amino acid feedstock was replaced with Fmoc-D-Asp(OtBu)-OH, the 18th amino acid feedstock was replaced with Fmoc-Lys(Boc)-OH, and the 19th and 20th amino acid feedstocks were deleted to obtain crude product BR037. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.025% NH3 / H2O; B: ACN; gradient: 5-45, 40 min); then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 24-44, 60 min), and lyophilized to obtain the trifluoroacetate of compound BR037 (HRMS m / z: 2951.249).

[0411] Example 38: BR038

[0412] Referring to the synthesis methods 13.1 and 13.2 of compound BR013, the 15th amino acid feedstock in Table 4 was replaced with Fmoc-Lys(Boc)-OH, the 16th amino acid feedstock was replaced with Fmoc-Gly-OH, and the 17th amino acid feedstock was replaced with Fmoc-Phe-OH, to obtain crude product BR038. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: AcOH-NH4OAc (containing 0.2% AcOH, pH adjusted to 4.8±0.1 with NH3 / H2O); B: ACN; gradient: 28-43, 40 min). It was then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 28-43, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR038 (HRMS m / z: 2954.189).

[0413] Example 39: BR039

[0414] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 18th amino acid feedstock in Table 5 was replaced with Fmoc-Lys(Boc)-OH, the 19th amino acid feedstock with Fmoc-Gly-OH with Fmoc-L-2-NAL-OH, and the 20th amino acid feedstock with Fmoc-Phe-OH with Fmoc-Gly-OH to obtain crude product BR039. The crude product was purified by high-performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 30-45, 60 min), and then lyophilized to obtain the trifluoroacetate of compound BR039 (HRMS m / z: 3250.356).

[0415] Example 40: BR040

[0416] Referring to the synthesis methods 13.1 and 13.2 of compound BR013, an amino acid feedstock Fmoc-Acp-OH was inserted between the 14th and 15th amino acid feedstocks in Table 4. The 15th amino acid feedstock was replaced by Fmoc-Lys(Boc)-OH with Fmoc-Beta-Asp(OtBu)-OH, the 16th amino acid feedstock was replaced by Fmoc-Gly-OH with Fmoc-L-2-NAL-OH, and the 17th amino acid feedstock was replaced by Fmoc-Phe-OH with Fmoc-Gly-OH, to obtain crude product BR040. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: AcOH-NH4OAc (containing 0.2% AcOH, pH adjusted to 4.8±0.1 with NH3 / H2O); B: ACN; gradient: 28-43, 40 min). It was then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 28-43, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR040 (HRMS m / z: 3067.270).

[0417] Example 41: BR041

[0418] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, an amino acid feedstock Fmoc-Acp-OH was inserted between the 17th and 18th amino acid feedstocks in Table 5. The 18th amino acid feedstock was replaced by Fmoc-Lys(Boc)-OH with Fmoc-Beta-Asp(OtBu)-OH, the 19th amino acid feedstock was replaced by Fmoc-Gly-OH with Fmoc-L-2-NAL-OH, and the 20th amino acid feedstock was replaced by Fmoc-Phe-OH with Fmoc-Gly-OH, to obtain crude product BR041. The crude product was purified by high performance liquid chromatography (column type: C18 20*250mm, 10μm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 28-43, 40 min), and then lyophilized to obtain the trifluoroacetate of compound BR041 (HRMS m / z: 3363.419).

[0419] Example 42: BR042

[0420] 42.1 Synthesis of peptide sequence P42

[0421] 42.1.1 Coupling of amino acids

[0422] 1) Swell MBHA-resin (20.0 g, 100-200 mesh, 1% DVB, 0.4–0.9 mmol / g) in DMF (200 mL) for 30 minutes. Add Fmoc-Cys(Trt)-OH (2.0 eq) pre-dissolved in 20 mL of dry DMF to the resin. Add DIC (2.0 eq) and shake the reaction vessel on a shaker at room temperature for 2 hours. After draining, wash the resin sequentially with DCM (200 mL, 3 times), DMF (200 mL, 2 times), and DCM (200 mL, 3 times).

[0423] 2) Repeat step ac in the order of Table 9 until the reaction is complete.

[0424] a. Add 20% piperidine / DMF (200mL), react for 5 minutes, dry under vacuum, then add another 20% piperidine / DMF (200mL) and react for 15 minutes.

[0425] b. Drain and rinse five times with DMF (200mL), each time with nitrogen agitation for 30 seconds.

[0426] c. Add 200 mL of DMF solution of amino acid raw material to the reaction flask, add condensing agent, and react on a shaker for 2-3 hours.

[0427] Table 9 Feeding Sequence

[0428] 42.1.2 Peptide cleavage and purification:

[0429] Add DTT (1.0 g), TIS (1 mL), and H2O (1 mL) to TFA (200 mL). Add this mixture to a beaker containing the resin to be cut and stir for 1.5 hours. Filter the solution, add cold diethyl ether to the filtrate until no more solid precipitates, and centrifuge to obtain the crude product. The crude product is then purified by reverse-phase chromatography (Ultimate XB-C18, 50*250 mm, 10 μm (PARP-02), 0.1% TFA in H2O / CH3CN, 0% to 50%, 30 min) to obtain the linear peptide sequence. MS-ESI m / z: 1950.6 [M+H] + .

[0430] 42.1.3 TATA Loop Closure

[0431] The above linear peptide sequence (1.0 g, 0.5 mmol) was dissolved in acetonitrile (200 mL) and water (200 mL), and sodium bicarbonate (43 mg, 0.5 mmol) and TATA (70 mg, 0.56 mmol) were added. The reaction was carried out at room temperature for 3 hours. LC-MS showed that the starting material disappeared. The reaction solution was lyophilized, and the crude product was purified by reverse-phase chromatography (Ultimate XB-C18, 50*250 mm, 10 μm (PARP-02), 0.1% TFA in H2O / CH3CN, 0% to 50%, 30 min) to obtain P42. MS-ESI m / z: 1100.1 [M / 2+H] + .

[0432] 42.2 Synthesis of peptide sequence BR042-3

[0433] 42.2.1 Synthesis of peptide sequence BR042-1

[0434] Fmoc-Asp-OtBu (15.0 g, 36.5 mmol) and glycine benzyl ester hydrochloride (8.80 g, 43.8 mmol) were dissolved in DMF (100 mL), and DIEA (14.1 g, 109.5 mmol) and HATU (16.6 g, 43.8 mmol) were added. The reaction mixture was stirred at 25 °C for 16 hours. After the reaction was complete, an aqueous solution (200 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (100 mL x 3). The combined organic phases were washed with saturated brine (50 mL x 5), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by rapid column chromatography (120 g silica gel column, petroleum ether / ethyl acetate, gradient 0–60%). The obtained product (10.0 g, 17.9 mmol) was dissolved in DMF (100 mL), and Pd / C (10%, 966 mg, 0.895 mmol) was added. Hydrogen was then purged three times using hydrogen balloons. The reaction mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the solvent was removed by rotary evaporation. The obtained crude product and HL-Phe-Oall (4.40 g, 21.4 mmol) were dissolved in DMF (100 mL), and N-methylmorpholine (5.42 g, 53.7 mmol), HOBT (2.88 g, 21.4 mmol), and EDCI (4.10 g, 21.4 mmol) were added. The reaction mixture was stirred at 25 °C for 16 hours. After the reaction was complete, an aqueous solution (200 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (100 mL x 3). The combined organic phases were washed with saturated brine (50 mL x 5), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by rapid column chromatography (80 g silica gel column, petroleum ether / ethyl acetate, gradient 0–80%). The obtained product (5.0 g, 7.63 mmol) was dissolved in DCM (50 mL), and triisopropylsilane (1.20 g, 7.63 mmol) and trifluoroacetic acid (25 mL) were added. The reaction mixture was stirred at 25 °C for 16 hours. After the reaction was complete, toluene solution (20 mL) was added to the reaction mixture, and the solvent was removed by rotary evaporation. The crude product was purified by rapid column chromatography (40 g silica gel column, dichloromethane / methanol, gradient 0–20%) to obtain BR042-1. MS-ESI m / z: 600.2 [M+H] + .

[0435] 42.2.2 Synthesis of peptide sequence BR042-3

[0436] 1) Add DCM (20 mL) to a container containing 2-CTC-Resin resin (1.50 g, 1.50 mmol, 100-200 mesh, 1% DVB, 1.00 mmol / g) and Fmoc-Lys(Alloc)-OH (1.0 eq).

[0437] 2) Add DIEA (4.00 eq) and stir for 2 hours.

[0438] 3) Add MeOH (2.00 mL) and stir for 30 minutes.

[0439] 4) Repeat steps a and b in the order shown in Table 10 until the reaction is complete.

[0440] a. Drain and rinse three times with DMF (30 mL), each time agitated with nitrogen for 30 seconds.

[0441] b. Add 20% piperidine / DMF (20 mL) and stir for 30 minutes.

[0442] c. Drain and rinse five times with DMF (30 mL), each time with nitrogen agitation for 30 seconds.

[0443] d. Add 20 mL of DMF solution containing amino acid raw material, agitate with nitrogen for 30 seconds, then add condensing agent and organic base, and agitate with nitrogen for nearly 1 hour.

[0444] Table 10 Material Feeding Table

[0445] 5) Drain the solution, rinse three times with DMF (30mL), rinse three times with DCM (30mL), and purge with nitrogen for 30 seconds each time.

[0446] 6) De-OAll and Alloc: Add Pd(PPh3)4 (0.1 eq.) and PhSiH3 (10.0 eq.) to the DCM resin solution, purge with nitrogen for about 15 minutes, dry under vacuum, and repeat this step three times.

[0447] 7) Drain and rinse five times with DMF (30 mL), each time with nitrogen agitation for 30 seconds.

[0448] 8) Ring closure: Add 20 mL of DMF solution containing condensing agents HATU (2.85 eq.) and DIEA (6.0 eq.), and purge with nitrogen for nearly 1 hour.

[0449] 9) Drain and rinse three times with DMF (30 mL), each time with nitrogen agitation for 30 seconds.

[0450] 10) Add 20% piperidine / DMF (20 mL) and stir for 30 minutes.

[0451] 11) Drain the solution, rinse three times with DMF (30 mL), and wash three times with methanol (30 mL). Purge with nitrogen for 30 seconds each time, then dry after evacuation.

[0452] Peptide cleavage and purification: A cleavage buffer solution (30 mL of 20% hexafluoroisopropanol in DCM solution) was added to a flask containing the peptide with side chain protection, and the mixture was stirred at 25°C for 30 minutes. The filtrate was filtered, and the filtrate was collected. The cleavage buffer solution (30 mL of 20% hexafluoroisopropanol in DCM solution) was added again to the flask, and the mixture was stirred at 25°C for 30 minutes. The filtrates were filtered, collected, and combined. The mixture was dried under vacuum, and the crude product was purified by reversed-phase column chromatography (80 g C18 reversed-phase column, water (0.1% TFA) / acetonitrile, gradient 0–50%) to obtain BR042-2. MS-ESI m / z: 652.4 [M+H] + .

[0453] BR042-2 (245 mg, 0.376 mmol) and Fmoc-Sar-OSu (230 mg, 0.564 mmol) were dissolved in DMF (3 mL), and DIEA (146 mg, 1.128 mmol) was added. The reaction mixture was stirred at 25 °C for 1 hour. After the reaction was complete, most of the solvent was removed by vacuum concentration. The crude product was purified by reversed-phase column chromatography (40 g C18 reversed-phase column, water (0.1% TFA) / acetonitrile, gradient 0–60%) to obtain BR042-3. MS-ESI m / z: 945.6 [M+H] + .

[0454] 42.3 Synthesis of BR042

[0455] BR042-3 (190 mg, 0.201 mmol) and HOSu (69 mg, 0.603 mmol) were dissolved in DMF (2 mL), and EDCI (116 mg, 0.603 mmol) was added. The reaction mixture was stirred at 25 °C for 2 hours. After the reaction was completed, the reaction mixture was purified by reversed-phase preparative HPLC (column: Prime C18, 30*150 mm, 5 μm; gradient: 47-61% B; mobile phase A: 0.1% TFA / H2O; mobile phase B: CH3CN; flow rate: 25 mL / min). The obtained product (28 mg, 0.0273 mmol) and P42 (40 mg, 0.0182 mmol) were dissolved in DMF (0.5 mL), and DIEA (7 mg, 0.0546 mmol) was added. The reaction mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the reaction solution was purified by reversed-phase preparative HPLC (column: Prime C18, 30*150mm, 5μm; gradient: 40-54% B; mobile phase A: 0.1% TFA / H2O; mobile phase B: CH3CN; flow rate: 25mL / min) to obtain BR042-4. MS-ESI m / z: 1564.6 [M / 2+H] + .

[0456] BR042-4 (40 mg, 0.0128 mmol) was dissolved in DMF (1 mL), and diethylamine (0.2 mL, 141 mg, 1.93 mmol) was added dropwise under ice bath conditions. The reaction mixture was stirred at 25 °C for 30 minutes. After the reaction was complete, the solvent was removed by vacuum concentration. The obtained crude product and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidine) ester (10 mg, 0.0191 mmol) were dissolved in DMF (0.5 mL), and DIEA (5 mg, 0.0381 mmol) was added. The reaction mixture was stirred at 25 °C for 2 hours. After the reaction was completed, the reaction solution was purified by reversed-phase preparative HPLC (column: Ultimate C18, 21, 2*250mm, 5μm; gradient: 22-45% B; mobile phase A: 0.1% TFA / H2O; mobile phase B: CH3CN; flow rate: 20mL / min) to obtain trifluoroacetate of BR042. HPLC purity: 99.30%. MS-ESI m / z: 1098.1 [M / 3+H] + .

[0457] Example 43: BR043

[0458] Synthesis of peptide sequence BR043-2 (43.1)

[0459] 43.1.1 Synthesis of peptide sequence BR043-1

[0460] Boc-Gly-OH (25.0 g, 143.0 mmol) was dissolved in DMF (250 mL) and placed in an ice bath. DIEA (74.0 mL, 427.0 mmol) was slowly added, followed by allyl bromide (19.0 g, 12.6 mmol). The mixture was allowed to return to room temperature and stirred overnight. The reaction solution was diluted with ethyl acetate (1200 mL) and washed successively with water (600 mL x 2) and saturated brine (600 mL x 1). The organic phase was dried over anhydrous sodium sulfate and then evaporated to dryness. The resulting crude product (10.0 g, 46.5 mmol) was dissolved in dichloromethane (100 mL), and trifluoroacetic acid (30 mL) was added at room temperature. The mixture was stirred at room temperature for 1 hour. The solvent was removed by vacuum concentration to obtain the crude product. The crude product was dissolved in DMF (100 mL), and Fmoc-Phe-OH (21.6 g, 55.8 mmol) was added. EDCI (3.64 g, 55.8 mmol), HOBt (7.5 g, 55.8 mmol), and NMM (14.0 g, 139.5 mmol) were then added sequentially, and the mixture was stirred overnight at room temperature. The reaction solution was diluted with ethyl acetate (1200 mL) and washed sequentially with water (600 mL x 2) and saturated brine (600 mL x 1). The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to obtain the crude product. The crude product was purified by rapid column chromatography (120 g silica gel column, petroleum ether / ethyl acetate, gradient 0–35%). The obtained product (2.40 g, 4.95 mmol) was dissolved in DMF (20 mL), and DEA (5.1 mL, 49.5 mmol) was added. The mixture was stirred at room temperature for 1 hour. The solvent was removed by concentration under reduced pressure. The obtained crude product was dissolved in DMF (30 mL), and Fmoc-Asp-OtBu (2.45 g, 5.94 mmol) was added. HATU (2.26 g, 5.94 mmol) and DIEA (2.5 mL, 15.0 mmol) were then added sequentially, and the mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate (150 mL) and washed successively with water (70 mL x 2) and saturated brine (100 mL x 1). The organic phase was dried over anhydrous sodium sulfate and then evaporated to dryness. The crude product was purified by rapid column chromatography (80 g silica gel column, petroleum ether / ethyl acetate, gradient 0–50%). The obtained product (1.37 g, 2.09 mmol) was dissolved in dichloromethane (15 mL), and trifluoroacetic acid (5 mL) was added at room temperature, and the mixture was stirred at room temperature for 1 hour. The solvent was removed from the reaction solution by rotary evaporation to obtain the crude product. The crude product was dissolved in toluene (30 mL), concentrated under reduced pressure, and BR043-1 was obtained. MS-ESI m / z: 599.7 [M+H] + .

[0461] 43.1.2 Synthesis of BR043-2

[0462] 1) Swell CTC-resin (5.0 g, 100-200 mesh, 1% DVB, 1.245 mmol / g) in DMF (50 mL) for 30 minutes. Add Fmoc-Lys(Alloc)-OH (8.45 g, 18.67 mmol) pre-dissolved in 20 mL of dry DMF to the resin. Add DIEA (4.02 g, 31.12 mmol) and shake the reaction vessel on a shaker at room temperature for 2 hours. After draining, wash the resin with DCM (60 mL, 3 times), DMF (60 mL, 2 times), DCM (60 mL, 3 times), and MeOH (60 mL, 2 times). Dry the resin under high vacuum overnight for later use.

[0463] 2) Repeat step ac in the order of Table 11 until the reaction is complete.

[0464] a. Add 20% piperidine / DMF (50 mL), react for 5 minutes, dry under vacuum, add another 20% piperidine / DMF (50 mL), and react for 15 minutes.

[0465] b. Drain the solution, then rinse five times with DMF (60 mL), each time with nitrogen agitation for 30 seconds.

[0466] c. Add 50 mL of DMF solution of amino acid raw material to the reaction flask, add condensation reagent, and react on a shaker for 4-18 hours. Dry the mixture under vacuum, rinse three times with DMF (60 mL), and three times with DCM (60 mL), each time with nitrogen agitation for 30 seconds.

[0467] Table 11 Feeding Sequence

[0468] 3) Removal of OAll and Alloc: Add tetrakis(triphenylphosphine)palladium (0.1 eq.) and benzenesilane (10.0 eq.) to the DCM resin solution, purge with nitrogen for about 15 minutes, dry under vacuum, and repeat this step three times.

[0469] 4) Drain and rinse five times with DMF (60mL), each time with nitrogen agitation for 30 seconds.

[0470] 5) Ring closure: Add 50 mL of DMF solution containing condensing agents HATU (1.5 eq.) and DIEA (3 eq.) and purge with nitrogen for nearly 1 hour.

[0471] 6) Drain and rinse three times with DMF (60 mL), agitating with nitrogen for 30 seconds each time.

[0472] 7) Add 20% piperidine / DMF (50 mL) and stir for 30 minutes.

[0473] 8) Drain and rinse five times with DMF (60 mL), each time with nitrogen agitation for 30 seconds.

[0474] 9) Add 50 mL of DMF solution of Fmoc-Sar-OH (3 eq) to the reaction flask, add HOBT (3 eq) and DIC (3 eq), and purge with nitrogen for nearly 1 hour.

[0475] Peptide cleavage and purification: Cleavage buffer (60 mL of 20% hexafluoroisopropanol in DCM solution) was added to a flask containing the peptide with side chain protection, and the mixture was stirred at 25°C for 1.5 hours. The mixture was filtered, and the filtrate was evaporated to dryness. The filtrate was then purified by reversed-phase preparative HPLC (column: Prime C18, 30*150 mm, 5 μm, mobile phase: [water (0.1% TFA)-ACN], B%: 41-61%, 30 min) to obtain BR043-2.

[0476] 43.2 Synthesis of BR043

[0477] Following the synthesis method 42.3 of BR042, the starting material BR042-3 was replaced with BR043-2 to obtain crude BR043. The crude product was then purified by reversed-phase preparative HPLC (column: Prime C18, 30*150mm, 5μm; gradient: 26-46% B; mobile phase A: 0.1% TFA / H2O; mobile phase B: CH3CN; flow rate: 25mL / min) to obtain trifluoroacetate of BR043. HPLC purity: 98.18%. MS-ESI m / z: 1098.1 [M / 3+H] + .

[0478] Example 46: BR046

[0479] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 8th amino acid feedstock in Table 5 was replaced with Fmoc-HOMOCIT-OH, the 18th amino acid feedstock was replaced with Fmoc-Lys(Boc)-OH, and the 19th and 20th amino acid feedstocks were deleted to obtain crude product BR046. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: AcOH-NH4OAc (containing 0.2% AcOH, pH adjusted to 4.8±0.1 with NH3 / H2O); B: ACN; gradient: 30-45, 40 min). It was then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 28-43, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR046 (HRMS m / z: 3041.294).

[0480] Example 47: BR047

[0481] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 7th amino acid feedstock in Table 5 was replaced with Fmoc-Asn(Trt)-OH, the 8th amino acid feedstock was replaced with Fmoc-hArg(Pbf)-OH, the 18th amino acid feedstock was replaced with Fmoc-Lys(Boc)-OH, and the 19th and 20th amino acid feedstocks were deleted to obtain crude product BR047. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.025% NH3 / H2O; B: ACN; gradient: 14-34, 40 min); then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 28-48, 60 min), and lyophilized to obtain the trifluoroacetate of compound BR047 (HRMS m / z: 3040.303).

[0482] Example 48: BR048

[0483] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the amino acid raw material in 17.1.2 was replaced by Fmoc-Cys(Trt)-OH with Fmoc-Thr(tBu)-OH; two amino acid raw materials, Fmoc-Tle-OH and Fmoc-Cys(Trt)-OH, were added sequentially before the first amino acid raw material Fmoc-Trp(Boc)-OH in Table 5; the 18th amino acid raw material in Table 5 was replaced by Fmoc-Lys(Boc)-OH with Fmoc-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid; and the 19th and 20th amino acid raw materials were deleted to obtain crude product BR048. The crude product was purified by high-performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 27-42, 40 min). It was then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: AcOH-NH4OAc (containing 0.2% AcOH, pH adjusted to 4.8±0.1 with NH3 / H2O); B: ACN; gradient: 27-42, 40 min). Finally, it was purified by HPLC (column type: C4...). The sample was separated and purified using a 21.2*250mm, 5μm filter. The mobile phase was: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 27-42, 40 min. The sample was then lyophilized to obtain the trifluoroacetate of compound BR048 (HRMS m / z: 3254.448).

[0484] Example 49: BR049

[0485] Following the synthetic method of compound BR033, the amino acid raw material in 33.1.2 was replaced with Fmoc-β-Ala-OH and Fmoc-Thr(tBu)-OH; the 19th amino acid raw material in Table 8 was replaced with Fmoc-Sar-OH and Fmoc-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid to obtain crude product BR049. The crude product was purified by high performance liquid chromatography (HPLC) (column: C18 20*250mm, 10μm; mobile phase: A: AcOH-NH4OAc (containing 0.2% AcOH, pH adjusted to 4.8±0.1 with NH3 / H2O); B: ACN; gradient: 28-43, 40 min). It was then purified by HPLC (column: C4 21.2*250mm, 5μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 27-42, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR049 (HRMS m / z: 3142.338).

[0486] Example 50: BR050

[0487] Referring to the synthetic methods 17.1.1, 17.1.2, 17.1.3, 17.1.4, and 17.1.5 of BR017, replace the 11th amino acid feedstock in Table 5 with Fmoc-D-Asp(OtBu)-OH, replace the 18th amino acid feedstock with Fmoc-D-Lys(ivdde)-OH, and replace the 18th amino acid feedstock with Fmoc-Lys(Boc)-OH, replacing the 18th amino acid feedstock with Fmoc-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid. Delete the 19th and 20th amino acid feedstocks. Add three steps after 17.1.5: removal of the protecting group ivdde, condensation of N,N'-disuccinimidyl carbonate, and condensation of butylamine.

[0488] Specific operating steps: Add 5% hydrazine hydrate / DMF (10 mL / g), stir for 60 minutes, dry under vacuum, and repeat twice. Remove the protective reagent under vacuum, then wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and dry under vacuum. Dissolve N,N'-disuccinimidyl carbonate (3 eq) in DMF (10 mL), pour it into the reactor, add DIEA (6 eq), and stir for 1 hour. Dry the reaction reagent under vacuum, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and dry under vacuum. Ninhydrin detection shows no significant color change in the resin, indicating successful condensation. Dissolve HATU (3 eq) in DMF (10 mL), add DIEA (6 eq), mix well, pour into the reactor, add butylamine (3 eq), and react for 2 hours. The reaction reagents were dried under vacuum, washed five times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dried under vacuum. The resin was washed five times with methanol (10 mL) and then dried under vacuum. The solid-phase condensation was complete.

[0489] Subsequently, following the synthetic methods 17.1.6 and 17.2 for compound BR017, crude BR050 was obtained. The crude product was purified by high-performance liquid chromatography (HPLC) (column: C18 20*250mm, 10μm; mobile phase: A: AcOH-NH4OAc (containing 0.2% AcOH, pH adjusted to 4.8±0.1 with NH3 / H2O); B: ACN; gradient: 29-44, 40 min). Further purification was achieved by HPLC (column: C4 21.2*250mm, 5μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 31-46, 40 min). The purified product was then lyophilized to obtain trifluoroacetate of BR050 (HRMS m / z: 3152.445).

[0490] Example 51: BR051

[0491] Referring to synthesis methods 17.1.1, 17.1.2, and 17.1.3 of BR017, replace the 11th amino acid feedstock in Table 5 with Fmoc-D-Asp(OtBu)-OH, and delete the 18th to 21st amino acid feedstocks. Add two steps after 17.1.3: condensation of N,N'-disuccinimidyl carbonate and condensation of butylamine.

[0492] Specific operating steps: Dissolve N,N'-disuccinimidyl carbonate (3 eq) in DMF (10 mL), pour it into the reactor, add DIEA (6 eq), and stir for 1 hour. Vacuum dry the reaction reagent, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry. Ninhydrin detection: no obvious color change in the resin indicates successful condensation. Dissolve HATU (3 eq) in DMF (10 mL), add DIEA (6 eq), mix well, pour it into the reactor, add butylamine (3 eq), and react for 2 hours. Vacuum dry the reaction reagent, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry.

[0493] Then, refer to the synthetic methods for compound BR017 in 17.1.4 and 17.1.5. Three steps are added after 17.1.5: removal of the protecting group ivdde, condensation of Fmoc-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid, and condensation of DOTA-(OtBu)3.

[0494] Specific operating steps: Add 5% hydrazine hydrate / DMF (10 mL / g), stir for 60 minutes, and dry under vacuum. Repeat the addition twice. Remove the protective reagent under vacuum, then wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and dry under vacuum. Dissolve Fmoc-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid (3 eq) and Oxyma (3 eq) in an appropriate amount of DMF (10 mL), then add DIC (3 eq) and activate for 5 minutes. Pour the mixture into the reactor and stir for 1 hour. Remove the reaction reagent under vacuum, wash 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time, and dry under vacuum. Ninhydrin detection shows no significant color change in the resin, indicating successful condensation. Dissolve DOTA-(OtBu)3 (3 eq) and Oxyma (3 eq) in an appropriate amount of DMF (10 mL), then add DIC (3 eq) and activate for 5 minutes. Pour the mixture into the reactor and stir for 1 hour. Vacuum dry the reaction reagents, wash five times with DMF (10 mL / g), stirring for 20-30 seconds each time, and then dry under vacuum. Ninhydrin detection shows no significant color change in the resin, indicating successful condensation. Wash the resin five times with methanol (10 mL) and dry under vacuum. Solid-phase condensation is complete.

[0495] Subsequently, following the synthetic methods 17.1.6 and 17.2 for compound BR017, crude BR051 was obtained. The crude product was then purified by high-performance liquid chromatography (HPLC) (column: C18 20*250mm, 10μm; mobile phase: A: 0.025% NH3 / H2O; B: ACN; gradient: 14-34, 40 min). Further purification was achieved by HPLC (column: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 29-49, 60 min), followed by lyophilization to obtain trifluoroacetate of BR051 (HRMS m / z: 3152.437).

[0496] Example 52: BR052

[0497] Synthesis of intermediate BR052-1 (52.1)

[0498] Diisopropylethylamine (1.24 g, 9.6 mmol) was added to a 20 mL solution of Fmoc-Lys-OtBu (3.0 g, 4.8 mmol) and succinic anhydride (0.48 g, 7.07 mmol) in dichloromethane. The resulting mixture was stirred at room temperature for 2 hours, concentrated under reduced pressure, and purified by preparative HPLC (column: SunFire C18 19*250 mm, 10 μm; mobile phase: A: 0.1% FA / H2O; B: ACN; flow rate: 20 mL / min; gradient: 24–34; retention time: 8.4–9.1 of 17 min) to obtain BR052-1 (ESI-MS m / z: 525.0 (M+H)). + )).

[0499] 52.2 Synthesis of trifluoroacetate of BR052

[0500] Referring to the synthetic methods 17.1 and 17.2 for compound BR017, replace the condensing agent combination in 17.1.2 and Table 5 with Oxyma / DIC to HATU / DIEA; replace the 18th amino acid feedstock in Table 5 with Fmoc-Lys(Boc)-OH to Fmoc-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid; replace the 19th amino acid feedstock with Fmoc-Gly-OH to BR052-1; and replace the 20th amino acid feedstock with Fmoc-Phe-OH to Fmo c-Val-OH, add an amino acid feedstock Fmoc-Met-OH between the 20th and 21st amino acid feedstocks; replace the condensing agent combination in 17.1.5 with HATU / DIEA, and replace the lysis buffer in 17.1.6 with (TFA:H2O:EDT:TIS:phenol = 80:5:10:2.5:2.5) with (TFA:H2O:EDT:TIS = 90:2.5:5:2.5) to obtain crude product BR052. The crude product was purified by high-performance liquid chromatography (HPLC) (column: C18 20*250mm, mobile phase: A: NH4OAc / H2O (pH 4.85); B: ACN, gradient: 23-40, 50 min); then purified by HPLC (column: Boston Green ODS, C18 30*150mm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 22-42, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR052 (ESI-MS m / z: 1167.7 (M+3H)). + ) / 3).

[0501] Example 53: BR053

[0502] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 15th amino acid feedstock in Table 5 was replaced with Fmoc-hLys(Alloc)-OH, the 17th amino acid feedstock was replaced with Fmoc-Asp(Oall)-OH, the 18th amino acid feedstock was replaced with Fmoc-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid, and the 19th and 20th amino acid feedstocks were deleted. The condensing agent in 17.1.5 was replaced with HBTU to obtain crude product BR053. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 25-41, 40 min); then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 28-43, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR053 (HRMS m / z: 3068.307).

[0503] Example 54: BR054

[0504] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 18th amino acid raw material in Table 5 was replaced with Fmoc-Lys(Boc)-OH, the 19th and 20th amino acid raw materials were deleted, the 21st amino acid raw material was replaced with DOTA-(OtBu)3 with DOTAM-mono-acid, and the condensing agent in 17.1.5 was replaced with HBTU to obtain crude product BR054. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 30-45, 40 min); then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 30-45, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR054 (HRMS m / z: 2949.298).

[0505] Example 55: BR055

[0506] Referring to the synthetic methods 17.1 and 17.2 for compound BR017, the 15th amino acid feedstock in Table 5 was replaced with Fmoc-hLys(Alloc)-OH, the 17th amino acid feedstock was replaced with Fmoc-Asp(Oall)-OH, the 18th amino acid feedstock was replaced with Fmoc-hLys(Alloc)-OH, and the 19th and 2nd amino acid feedstocks were replaced with Fmoc-hLys(Alloc)-OH. The 0 amino acid feedstocks were deleted, and the 21st amino acid feedstock was replaced by DOTA-(OtBu)3 with DOTAM-mono-acid. The condensing agent in 17.1.5 was replaced by HBTU, and the lysis buffer in 17.1.6 was replaced by (TFA:H2O:EDT:TIS:phenol = 80:5:10:2.5:2.5) with (TFA:DODT:phenol:water:anisole = 87.5:5:2.5:2.5:2.5) to obtain the crude product BR055. The crude product was purified by high-performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: AcOH / H2O (containing 0.2% acetic acid, pH adjusted to 4.8±0.1 with ammonia); B: ACN; gradient: 23-38, 40 min). It was then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 23-38, 40 min). The sample was separated and purified using a 20*250mm, 10μm filter with the following mobile phases: A: 30mM (NH4)2SO4 aqueous solution (pH adjusted to 2.25±0.05 with phosphoric acid); B: ACN, gradient: 27-37, 40 min. After purification, the sample was desalted using a conventional 0.1% TFA system and lyophilized to obtain the trifluoroacetate of compound BR055 (HRMS m / z: 2977.322).

[0507] Example 56: BR056

[0508] Referring to the synthetic methods 17.1 and 17.2 for compound BR017, the 7th amino acid precursor in Table 5 was replaced with Fmoc-Asn(Trt)-OH, the 8th amino acid precursor with Fmoc-hArg(Pbf)-OH with Fmoc-HOMOCIT-OH, and the 18th amino acid precursor with Fmoc-Lys(Boc)-OH with Fmoc-Sar-OH. The 19th and 20th amino acids... The acid feedstock was removed, and the 21st amino acid feedstock was replaced by DOTA-(OtBu)3 with DOTAM-mono-acid. The condensing agent in 17.1.5 was replaced by HBTU instead of HATU. The lysis buffer in 17.1.6 was replaced by (TFA:H2O:EDT:TIS:phenol = 80:5:10:2.5:2.5) instead of (TFA:DODT:phenol:water:anisole = 87.5:5:2.5:2.5:2.5) to obtain the crude product BR056. The crude product was purified by high performance liquid chromatography (column type: C18 20*250mm, 10μm, mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, gradient: 25-35, 40 min), and then lyophilized to obtain the trifluoroacetate of compound BR056 (HRMS m / z: 2949.295).

[0509] Example 57: BR057

[0510] Referring to the synthetic methods 17.1 and 17.2 for compound BR017, the 8th amino acid precursor in Table 5 was replaced with Fmoc-HOMOCIT-OH, the 18th amino acid precursor was replaced with Fmoc-Lys(Boc)-OH, and the 19th and 20th amino acid precursors were deleted. The 21st amino acid precursor was replaced with D... OTA-(OtBu)3 was replaced with DOTAM-mono-acid, the condensing agent in 17.1.5 was replaced with HBTU, and the pyrolysis solution in 17.1.6 was replaced with (TFA:H2O:EDT:TIS:phenol = 80:5:10:2.5:2.5) = (TFA:DODT:phenol:water:anisole = 87.5:5:2.5:2.5:2.5) to obtain crude product BR057. The crude product was purified by high performance liquid chromatography (HPLC) (column type: C18 20*250mm, 10μm; mobile phase: A: AcOH / H2O (containing 0.2% acetic acid, pH adjusted to 4.8±0.1 with ammonia); B: ACN; gradient: 29-39, 40 min). It was then purified by HPLC (column type: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 28-38, 40 min), and lyophilized to obtain the trifluoroacetate of compound BR057 (HRMS m / z: 3038.317).

[0511] Example 58: BR058

[0512] Referring to the synthesis methods 17.1 and 17.2 of compound BR017, the 15th amino acid feedstock in Table 5 was replaced with Fmoc-hLys(Alloc)-OH, the 17th amino acid feedstock was replaced with Fmoc-Asp(Oall)-OH, the 18th amino acid feedstock was replaced with Fmoc-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid, the 19th and 20th amino acid feedstocks were deleted, the 21st amino acid feedstock was replaced with DOTA-(OtBu)3 and DOTAM-mono-acid, and the condensing agent in 17.1.5 was replaced with HBTU to obtain crude product BR058. The crude product was purified by high performance liquid chromatography (HPLC) (column: C18 20*250mm, 10μm; mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / ACN; gradient: 22-42, 40 min); then purified by HPLC (column: C18 20*250mm, 10μm; mobile phase: A: HCl / H2O (4L pure water with 1mL concentrated hydrochloric acid); B: ACN; gradient: 24-44, 40 min), and lyophilized to obtain the hydrochloride salt of compound BR058 (HRMS m / z: 3065.364).

[0513] 177 Preparation and purification methods of Lu-labeled compounds 1

[0514] Take a C18 separation column, rinse it slowly with 20 mL of anhydrous ethanol and 20 mL of sterile water for injection, and then dry it with 20 mL of air to complete the activation process before use.

[0515] Add approximately 74-1850 MBq to a 2 mL centrifuge tube. 177 LuCl3 solution, DMSO solution of BR001 (concentration 1 mg / mL) at a ratio of 10 μg / 37 MBq, and 20 μL of anhydrous ethanol (ethanol content 10%) were added; 0.15 M ascorbic acid-acetic acid-0.22 M sodium acetate solution was added to bring the reaction volume to 0.2 mL, mixed well, and reacted at 75 °C for 15 minutes, then cooled to room temperature for 5 minutes. After cooling, the reaction solution was drawn into a 10 mL syringe containing 9 mL of 0.15 M ascorbic acid-acetic acid-0.22 M sodium acetate solution and loaded onto an activated C18 separation column. The C18 separation column was then rinsed with 20 mL of sterile water for injection and the waste liquid was discarded. Finally, the C18 separation column was rinsed with 0.4 mL of anhydrous ethanol, and the labeled sample was collected in a sterile vacuum vial. After dilution with 3.6 mL of ascorbic acid-gentianic acid-physiological saline solution, the sample was sterilely filtered into a sterile vacuum vial to obtain the desired product.177 Lu-labeled complex injection solution, i.e. 177 Lu-BR001.

[0516] Following the exact same method and proportions described above, BR002 was added to the above reagents to prepare the following... 177 Lu-labeled complex injection solution, i.e. 177 Lu-BR002.

[0517] Furthermore, this labeling and purification method, except for the substitution of individual reagents with reagents of similar function or the adjustment of the ratio, is applicable to all compounds of the present invention.

[0518] The labeling rate and purity of the labeled compounds were characterized by Radio-HPLC, and the results are shown in Table 12.

[0519] Table 12 Radiolabeling rates and radiochemical purity of radioactive compounds

[0520] 177 Preparation and purification methods of Lu-labeled compounds 2

[0521] Take a C18 separation column, rinse it slowly with 10 mL of anhydrous ethanol and 10 mL of sterile water for injection, and then dry it with 20 mL of air to complete the activation process before use.

[0522] Add approximately 74-1850 MBq to a 2 mL centrifuge tube. 177 LuCl3 solution, DMSO solution (concentration 1 mg / mL) of the compound of the present invention added at a ratio of 10 μg / 37 MBq, and 20 μl of anhydrous ethanol (ethanol content 10%) were added; 0.22 M sodium acetate / 0.15 M ascorbic acid solution was added to bring the reaction volume to 0.2 mL, mixed well, and reacted at 75 °C for 15 minutes, then cooled to room temperature for 5 minutes. After cooling, the reaction solution was drawn into a 10 mL syringe containing 9 mL of sterile water for injection and loaded onto an activated C18 separation column. The C18 separation column was then rinsed with 10 mL of sterile water for injection and the waste liquid was discarded. Finally, the C18 separation column was rinsed with 0.3 mL of anhydrous ethanol, and the labeled substance was collected in a sterile vacuum vial. After dilution with 2.7 mL of 0.25 M ascorbic acid / 0.002 M gentianic acid solution, the solution was sterilely filtered into a sterile vacuum vial to obtain the desired product. 177 The Lu-labeled injectable solution of the compound of the present invention. The purity of the labeled compound was characterized by Radio-HPLC, and the results are shown in Table 13.

[0523] Table 13 177 Radiochemical purity of Lu-labeled compounds

[0524] 177 Preparation and purification methods of Lu-labeled compounds 3

[0525] Take a C18 separation column, rinse it slowly with 10 mL of anhydrous ethanol and 10 mL of sterile water for injection, and then dry it with 20 mL of air to complete the activation process before use.

[0526] Add approximately 74-1850 MBq to a 2 mL centrifuge tube. 177 LuCl3 solution, DMSO solution (concentration 1 mg / mL) containing the compound of the present invention added at a ratio of 8 μg / 37 MBq, and 20 μL of anhydrous ethanol (ethanol content 10%) were added; 0.22 M sodium acetate / 0.15 M ascorbic acid solution was added to bring the reaction volume to 0.2 mL, mixed well, and reacted at 75 °C for 15 minutes, then cooled to room temperature for 5 minutes. After cooling, the reaction solution was drawn into a 10 mL syringe containing 9 mL of sterile water for injection and loaded onto an activated C18 separation column. The C18 separation column was then rinsed with 10 mL of sterile water for injection and the waste liquid was discarded. Finally, the C18 separation column was rinsed with 0.5 mL of 60% anhydrous ethanol aqueous solution, and the labeled substance was collected in a sterile vacuum vial. The solution was diluted with 2.5 mL of 0.15 M ascorbic acid-2 mM gentianic acid preparation solution and sterilely filtered into a sterile vacuum vial to obtain the desired product. 177 The Lu-labeled injectable solution of the compound of the present invention. The purity of the labeled compound was characterized by Radio-HPLC, and the results are shown in Table 14.

[0527] Table 14 177 Radiochemical purity of Lu-labeled compounds

[0528] 177 Preparation and purification methods of Lu-labeled compounds 4

[0529] Take a C18 separation column, rinse it slowly with 10 mL of anhydrous ethanol and 10 mL of sterile water for injection, and then dry it with 20 mL of air to complete the activation process before use.

[0530] Add approximately 74-1850 MBq to a 2 mL centrifuge tube. 177LuCl3 solution, DMSO solution (concentration 1 mg / mL) containing the compound of the present invention added at a ratio of 8 μg / 37 MBq, and 10 μL of anhydrous ethanol (ethanol content 10%) were added; 0.22 M sodium acetate / 0.15 M ascorbic acid solution was added to bring the reaction volume to 0.1 mL, mixed well, and reacted at 75 °C for 15 minutes, then cooled to room temperature for 5 minutes. After cooling, the reaction solution was drawn into a 10 mL syringe containing 9 mL of sterile water for injection and loaded onto an activated C18 separation column. The C18 separation column was then rinsed with 10 mL of sterile water for injection and the waste liquid was discarded. Finally, the C18 separation column was rinsed with 0.5 mL of 60% anhydrous ethanol aqueous solution, and the labeled substance was collected in a sterile vacuum vial. The solution was diluted with 2.5 mL of 0.15 M ascorbic acid-2 mM gentianic acid preparation solution and sterilely filtered into a sterile vacuum vial to obtain the desired product. 177 The Lu-labeled injectable solution of the compound of the present invention. The purity of the labeled compound was characterized by Radio-HPLC, and the results are shown in Table 15.

[0531] Table 15 177 Radiochemical purity of Lu-labeled compounds

[0532] 68 Preparation and purification methods of Ga-labeled compounds

[0533] Using a syringe, draw 5 mL of 0.1 M HCl and connect it to the germanium-gallium generator. Slowly push the syringe to elute the Ga-68 solution into a 10 mL coated vial containing 1 mL of 0.6 M sodium acetate / 10 mM selenomethionine / 3 mg / mL ascorbic acid / 20% ethanol solution, and shake well. Add 50 μL of DMSO solution (1 mg / mL) of the compound of this invention to the vial, shake well, cap, and place on a 95°C constant temperature mixer (preheated for 30 minutes) for 20 minutes. After the reaction, allow it to cool to room temperature for 5 minutes. After cooling, use a syringe to draw the reaction solution and load it onto an activated C18 extraction column (pre-activated sequentially with 10 mL of ethanol and 10 mL of physiological saline). Then rinse the C18 separation column with 10 mL of physiological saline and discard the waste liquid. Finally, the C18 separation column was rinsed with 1 mL of 50% ethanol / physiological saline solution, and the labeled sample was collected in a 10 mL coated vial. After dilution with 4 mL of 0.25 M sodium ascorbate / 2 mM gentianic acid solution, the desired product was obtained. 68 Injectable solutions of Ga-labeled compounds of the present invention. The purity of the labeled compounds was characterized by Radio-HPLC, and the results are shown in Table 16.

[0534] Table 16 68 Radiochemical purity of Ga-labeled compounds

[0535] 212 Preparation and purification methods of Pb-labeled compounds

[0536] Take a C18 separation column, rinse it slowly with 10 mL of anhydrous ethanol and 10 mL of sterile water for injection, and then dry it with 20 mL of air to complete the activation process before use.

[0537] Add 1.9 mL of [[ to a 10 mL vial]] 212 Pb]Pb(OAc)2 solution, 50 μL glacial acetic acid, DMSO solution (concentration 1 mg / mL) containing the compound of the present invention added at a ratio of 1 μg / 2 μCi, and anhydrous ethanol (10% of the total reaction volume); mix well and react at 75°C for 10 minutes, then cool to room temperature for 5 minutes. After cooling, use a 10 mL syringe pre-filled with 9 mL of sterile water for injection to draw the reaction solution, and load it onto an activated C18 separation column. Then rinse the C18 separation column with 10 mL of sterile water for injection and discard the waste liquid. Finally, rinse the C18 separation column with 0.5 mL of 60% anhydrous ethanol aqueous solution, collect the labeled substance in a sterile vacuum vial, dilute with 2.5 mL of 0.25 M ascorbic acid-2 mM gentianic acid preparation solution, and sterilely filter into a sterile vacuum vial to obtain... 212 The Pb-labeled injectable solution of the compound of the present invention. The purity of the labeled compound was characterized by Radio-HPLC, and the results are shown in Table 17.

[0538] Table 17 212 Radiochemical purity of Pb-labeled compounds

[0539] Biological test data

[0540] Test Example 1: Binding ability test of the compound prepared in the embodiments of the present invention with Nectin-4 protein.

[0541] 1. Experimental Objective

[0542] The affinity of the analyte for the target protein Nectin 4 was determined using the SPR method.

[0543] 2. Materials and Instruments

[0544] Biacore 8K (GE Healthcare)

[0545] 96-well Plate(Cat#650101,greiner bio-one)

[0546] CM5 chip (Cat#BR-1005-30, GE Healthcare)

[0547] Amine Coupling Kit(Cat#BR-1000-50,GE Healthcare)

[0548] EDC

[0549] NHS

[0550] 1M ethanolamine

[0551] 10mM Sodium Acetate, pH 4.5 (Cat#BR-1003-50, GE Healthcare)

[0552] DMSO (Cat#D4540, Sigma)

[0553] P20(Cat#BR-1000-54,GE Healthcare)

[0554] PBS(Cat#BR-1006-72,GE Healthcare)

[0555] Nectin-4(Cat#1006-72,GE Healthcare)

[0556] 3. Experimental Design

[0557] This experiment employed the amino-coupled method, where the target protein Nectin 4 was directly immobilized onto a CM5 chip using a Biacore 8K microarray. The analyte was then used as the analytical compound, diluted to the desired concentration gradient with buffer (10 mM PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5% DMSO, 0.05% P2O). Multiple cycles of kinetic analysis were performed, with each cycle consisting of 180 seconds of injection followed by 180 seconds of dissociation before proceeding to the next cycle. This yielded kinetic data on the affinity between Nectin 4 and the analyte. The final data were then analyzed using Biacore Insight Evaluation Software (V 2.0.15.12933) in a 1:1 kinetic model.

[0558] 4. Experimental methods and procedures

[0559] 1) Prepare the buffer solution: 10mM PBS (pH 7.4), 137mM NaCl, 2.7mM KCl, 5% DMSO, 0.05% P2O.

[0560] 2) CM5 chip activation: Activate with 400mM EDC and 100mM NHS at a flow rate of 10μL / min for 600 seconds.

[0561] 3) Target protein coupling: Dilute the target protein to 10 μg / mL with 10 mM sodium acetate (pH 4.5) and couple for 600 s at a flow rate of 10 μL / min.

[0562] 4) CM5 chip sealing: seal with 1M ethanolamine at a flow rate of 10μL / min for 600 seconds.

[0563] 5) Analyte concentration: Dilute the analyte with running buffer (HEPES, 150mM NaCl, 0.5% P2O, 5% DMSO, pH 7.4).

[0564] 6) Sample injection analysis: Each concentration of the analyte working solution is one cycle, with a flow rate of 30 μL / min for 180 seconds for binding and 180 seconds for dissociation.

[0565] 7) All results were subjected to kinetics fitting analysis using a 1:1 model.

[0566] 5. Experimental Results

[0567] Five effective concentrations of experimental data were selected and the compounds prepared in Examples 1, 2 and 5 to 8 of this invention were subjected to Kinetics fitting analysis using Biacore Insight Evaluation Software (V 2.0.15.12933) at a 1:1 model. The results are shown in Table 18.

[0568] Table 18 Results of binding of the compounds of the present invention to Human Nectin-4 SPR

[0569] Conclusion: The compounds prepared in Examples 1 to 8 of this invention exhibit strong binding affinity to Human Nectin-4 protein. Test Example 2: Plasma stability analysis of the compounds prepared in the examples of this invention across different species.

[0570] A. Experimental Objective

[0571] The stability of the compounds prepared in the embodiments of the present invention in CD-1 mouse and human plasma was tested.

[0572] B. Experimental Procedures

[0573] 2 μL of the analyte compound (100 μM) in DMSO working solution was added to the corresponding incubation plates, including T0, T10, T30, T60, and T120 incubation plates, with three parallel wells prepared for each sample. Then, 98 μL of CD-1 mouse and human blank plasma were added to the corresponding incubation plates containing the working solution. All samples were incubated in a 37°C water bath. The final incubation concentration of the analyte compound was 2 μM. At the end of each incubation time point, the corresponding incubation plate was removed, stop solution was added, proteins were precipitated, centrifuged for 20 minutes, and 150 μL of the supernatant was analyzed by LC-MS / MS. The concentration of the analyte compound in the samples was semi-quantitatively determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0574] C. Experimental Results

[0575] The experimental results are shown in Table 19.

[0576] Table 19. Stability results of the compounds prepared in Examples 1 and 2 of this invention in mouse and human plasma, respectively.

[0577] Conclusion: The compounds prepared in Examples 1 and 2 of this invention have excellent stability in human plasma.

[0578] Test Example 3: Stability analysis of compounds prepared in different species in whole blood according to the embodiments of the present invention.

[0579] A. Experimental Objective

[0580] The stability of the compounds prepared in the embodiments of the present invention was tested in CD-1 mice and human whole blood.

[0581] B. Experimental Procedures

[0582] 2 μL of the 100 μM DMSO working solution of the test compound was added to the corresponding incubation plates, including T0, T10, T30, T60, and T120 incubation plates, with three parallel wells prepared for each sample. Then, 98 μL of CD-1 mouse and human blank whole blood were added to the corresponding incubation plates containing the working solution. All samples were incubated in a 37°C water bath. The final incubation concentration of the test compound was 2 μM. At the end of each incubation time point, the corresponding incubation plate was removed, stop solution was added, protein was precipitated, centrifuged for 20 minutes, and 100 μL of the supernatant was diluted with 300 μL of ultrapure water. After mixing, the sample was analyzed by LC-MS / MS. The concentration of the test compound in the sample was semi-quantitatively determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0583] C. Experimental Results

[0584] The experimental results are shown in Table 20.

[0585] Table 20. Stability results of the compounds prepared in Examples 1 and 2 of this invention in CD-1 mice and human whole blood.

[0586] Conclusion: The compounds prepared in Examples 1 and 2 of this invention have excellent stability in CD-1 mice and human whole blood.

[0587] Test Example 4 177 Cellular uptake and internalization of Lu-labeled compounds

[0588] 1. Experimental Objective

[0589] pass 177 The uptake and internalization of Lu-labeled compounds were measured after binding to MDA-MB-468, MDA-MB-231, PC3-Nectin4, and PC3 cells for different durations, and the uptake and internalization rates of the test substances were calculated.

[0590] 2. Experimental Methods

[0591] (1) Preparation of MDA-MB-468, MDA-MB-231, PC3-Nectin4 and PC3 cells: Cells in logarithmic growth phase were prepared into cell suspensions, and the cell density was adjusted to 4×10⁶ cells / mL. 5 Add 1 mL of the solution to each well of a 24-well cell culture plate pre-coated with poly-L-lysine solution. Incubate at 37°C in a 5% CO2 incubator. When the cell density reaches approximately 80% confluence, the plate is ready for experimentation.

[0592] (2) Compound preparation: dilution before sample addition. 177 The incubation concentration for Lu-BR001 was set at 7.74 nM. 177 The incubation concentration for Lu-BR002 was set to 7.92 nM. Before dilution, a certain volume of the stock solution was accurately transferred for activity determination, and the determination time, volume, and activity were recorded.

[0593] (3) Sample addition: On the day of the experiment, discard the old culture medium, wash once with serum-free culture medium, and add 0.5 mL of serum-free culture medium containing the compound of the present invention to each well. At the same time, transfer 0.5 mL of serum-free culture medium containing the compound of the present invention to one radioimmunoassay tube as a standard tube T.

[0594] (4) Incubation: After the sample is added, shake gently and incubate in an incubator at 37°C for 15 min, 30 min, 60 min, 75 min and 120 min, with 3 replicates at each time point.

[0595] (5) Washing: After incubation, remove the 24-well plate from the incubator, remove the culture medium, wash twice with 0.4 mL PBS (pre-cooled to 4°C), incubate the cells twice with 0.5 mL glycine-hydrochloric acid buffer (0.2 M, pH 3.0) (5 minutes each time) and collect them into an immunization tube and record them as A1. Then wash twice with 0.4 mL PBS (pre-cooled to 4°C) and collect them into an immunization tube and record them as A2.

[0596] (6) Cell lysis: Add 0.2 mL of 1 M NaOH to lyse the cells. After about 5 minutes, transfer the cell lysis buffer to a radioimmunoassay tube. Then wash twice with 0.4 mL of PBS (pre-cooled to 4 °C) and transfer the lysate to a radioimmunoassay tube, labeled B.

[0597] (7) Gamma count: The radioactive CPM values ​​of cell lysate and washing buffer were measured using a gamma counter. Blank tubes and standard tubes were tested before the samples were tested on the same day, with each sample being tested for 30 seconds.

[0598] 3. Data Processing

[0599] The results of the intake experiment are expressed as %AD, and the calculation formula is as follows:

[0600] The results of the internalization experiment are expressed as %AD, and the calculation formula is as follows:

[0601] 4. Experimental Results

[0602] Table 21 177 Results of uptake and internalization of Lu-labeled compounds in MDA-MB-468 and MDA-MB-231 cells

[0603] Table 22 177 Results of uptake and internalization of Lu-labeled compounds in PC3-Nectin4 and PC3 cells

[0604] in conclusion: 177 The Lu-labeled compound showed good uptake in Nectin-4 positive MDA-MB-468 cells and PC3-Nectin4 cells, but almost no uptake in Nectin-4 negative MDA-MB-231 cells and PC3 cells.

[0605] Test Example 5 177 Cell saturation binding assay of Lu-labeled compounds

[0606] 1. Experimental Objective

[0607] Through different concentrations 177The Lu-labeled compound binds to PC3-Nectin4 cells, and the radioactivity count of the bound compound is detected to calculate the K of the test substance. D value.

[0608] 2. Experimental Methods

[0609] (1) PC3-Nectin4 cell preparation: Logarithmic growth phase cells were prepared into a cell suspension, and the cell density was adjusted to approximately 4 × 10⁻⁶ cells / mL. 5 Add 1 mL of the solution to each well of a 24-well cell culture plate pre-coated with poly-L-lysine solution. Incubate at 37°C in a 5% CO2 incubator. When the cell density reaches approximately 80% confluence, the plate is ready for experimentation.

[0610] (2) Compound preparation: Before adding the sample, dilute the solution by three-fold serial dilution using serum-free medium to obtain seven concentrations: 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.41 nM, and 0.14 nM. Before dilution, accurately transfer a certain volume of the stock solution for activity determination, and record the determination time, volume, and activity. Take 0.5 mL of each concentration as a standard tube.

[0611] (3) Sample addition: On the day of the experiment, the old culture medium was discarded, and the plate was washed once with serum-free culture medium. 0.5 mL of the prepared serum-free culture medium solution containing different concentrations of the compound of the present invention was added to each well of the 24-well plate, with 3 wells for each concentration.

[0612] (4) Incubation: After the sample addition is completed, place the well plate in a 37°C incubator for 1 hour.

[0613] (5) Cell lysis: After incubation, remove the 24-well plate from the incubator, remove the culture medium, and wash twice with 0.4 mL PBS (pre-cooled to 4°C). Then add 0.2 mL 1M NaOH to lyse the cells. After about 5 minutes, transfer the cell lysis buffer to an immunization tube, and then wash twice with 0.4 mL PBS (pre-cooled to 4°C) and transfer the buffer to the immunization tube.

[0614] (6) Gamma count: The radioactive CPM values ​​of cell lysate and washing buffer were measured using a gamma counter. Blank tubes were tested before the samples were tested on the same day, and the testing time for each sample was 30 seconds.

[0615] 3. Data Processing

[0616] Based on the experimental results, K was calculated using Prism data processing software. D value.

[0617] 4. Experimental Results

[0618] Table 23177 Results of experiments on the saturation binding of Lu-labeled compounds to PC3-Nectin4 cells.

[0619] in conclusion: 177 The Lu-labeled compound exhibited good saturation binding ability with PC3-Nectin4 cells.

[0620] Test Example 6 177 Cell competition binding assay of Lu-labeled compounds

[0621] 1. Experimental Objective

[0622] Different concentrations of BCY8234 and the compounds of this invention were used to competitively bind to MDA-MB-468 and PC3-Nectin4 cells, and the radioactive counts of the bound cells were detected to calculate the IC50. 50 value.

[0623] BCY8234 is a compound in CN112601539A.

[0624] 2. Experimental Methods

[0625] (1) Preparation of MDA-MB-468 and PC3-Nectin4 cells: Cells in the logarithmic growth phase were prepared into a cell suspension, and the cell density was adjusted to approximately 4 × 10⁻⁶ cells / mL. 5 Add 1 mL of the solution to each well of a 24-well cell culture plate pre-coated with poly-L-lysine solution. Incubate at 37°C in a 5% CO2 incubator. When the cell density reaches approximately 80% confluence, the plate is ready for experimentation.

[0626] (2) Preparation of BCY8234 inhibitor: Weigh the inhibitor, dissolve it in DMSO, mix well to prepare a stock solution of 5 mg / mL, and store at -20℃ (shelf life 1 month) or -80℃ (shelf life 6 months).

[0627] (3) Preparation of BCY8234 stock solution: On the day of the experiment, 0.1 mmol / L stock solution was prepared using 5 mg / mL stock solution.

[0628] The inhibitor solutions were then diluted to final concentrations of 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM.

[0629] Concentration A: Take 30 μL of 0.1 mM stock solution and bring the volume to 2.4 mL with serum-free culture medium to obtain a 1250 nM inhibitor solution.

[0630] Concentration B: Take 0.2 mL of 1250 nM stock solution and dilute to 2 mL with serum-free culture medium to obtain a 125 nM inhibitor solution.

[0631] Concentration C: Take 0.2 mL of 125 nM stock solution and dilute to 2 mL with serum-free culture medium to obtain a 12.50 nM inhibitor solution.

[0632] Concentration D: Take 0.2 mL of 12.5 nM stock solution and dilute to 2 mL with serum-free culture medium to obtain a 1.25 nM inhibitor solution.

[0633] Concentration E: Take 0.2 mL of 1.25 nM stock solution and dilute to 2 mL with serum-free culture medium to obtain a 0.125 nM inhibitor solution.

[0634] Solution F: Take 0.2 mL of 0.125 nM stock solution and dilute to 2 mL with serum-free culture medium to obtain a 0.0125 nM inhibitor solution.

[0635] Solution G: Take 0.2 mL of 0.0125 nM stock solution and dilute to 2 mL with serum-free culture medium to obtain a 0.00125 nM inhibitor solution.

[0636] (4) Compound preparation: Before adding the sample, dilute the compound and prepare it to a suitable activity concentration using serum-free medium (this activity concentration is determined based on the binding experiment results and is set to 5 times K). D Before dilution, accurately transfer a certain volume of the original solution for activity determination, and record the determination time, volume, and activity.

[0637] (5) Sample addition: Add samples according to the table below and mix well. 177 The final concentration of Lu-BR001 was 8.36 nM. 177 The final concentration of Lu-BR002 was 9.72 nM, with 3 replicates per concentration.

[0638] a Based on the experimental results, the total reaction volume was determined to be 500 μL. Simultaneously, 0.1 mL of [amount missing] was transferred... 177 Lu-BR001 / 177 Lu-BR002 is placed into one radioimmunoassay tube as a standard tube. 177 Lu-BR001 / 177 Lu-BR002 competes with BCY8234.

[0639] (6) Incubation: After the sample addition is completed, place the well plate in a 37°C incubator for 1 hour.

[0640] (7) Cell lysis: After incubation, remove the 24-well plate from the incubator, remove the culture medium, and wash twice with 0.4 mL PBS (pre-cooled to 4°C). Then add 0.2 mL 1M NaOH to lyse the cells, and after about 5 minutes, transfer the cell lysis buffer to an immunization tube. Then wash twice with 0.4 mL PBS (pre-cooled to 4°C) and transfer the buffer to the immunization tube.

[0641] (8) Gamma count: The radioactive CPM values ​​of cell lysate and washing buffer were measured using a gamma counter. Blank tubes and standard tubes were tested before the samples were tested on the same day, with each sample being tested for 30 seconds.

[0642] 3. Data Processing

[0643] Based on the results of the competitive combination experiment, Prism data processing software was used for data analysis and graphing.

[0644] 4. Experimental Results

[0645] From Figures 1 and 2, we can conclude that: 177 The Lu-labeled compound competes with BCY8234 for binding to Nectin-4.

[0646] Test Example 7 177 Cellular uptake assay of Lu-labeled compounds

[0647] 1. Experimental Objective

[0648] pass 177 The uptake of radioactivity was measured after Lu-labeled compounds were bound to PC3-Nectin4 and PC3 cells for different durations, and the uptake value of the test substance was calculated.

[0649] 2. Experimental Methods

[0650] (1) Preparation of PC3-Nectin4 and PC3 cells: Logarithmic growth phase cells were prepared into a cell suspension, and the cell density was adjusted to 2×10⁴ cells / cells. 5 Add 1 mL of the solution to each well of a 24-well cell culture plate pre-coated with poly-L-lysine solution. Incubate at 37°C in a 5% CO2 incubator. When the cell density reaches approximately 80% confluence, the plate is ready for use in experiments.

[0651] (2) Compound preparation: dilute before adding the sample and set the incubation concentration to 10 nM. Before dilution, accurately transfer a certain volume of the original solution for activity determination and record the determination time, volume and activity.

[0652] (3) Sample addition: On the day of the experiment, discard the old culture medium, wash once with serum-free culture medium, and add 0.5 mL of serum-free culture medium containing the compound of the present invention to each well. At the same time, transfer 0.5 mL of serum-free culture medium containing the compound of the present invention to one radioimmunoassay tube as a standard tube.

[0653] (4) Incubation: After the sample is added, shake gently and incubate in an incubator at 37°C for 15 min, 30 min, 60 min and 120 min, with 3 replicates at each time point.

[0654] (5) Washing: After incubation, remove the 24-well plate from the incubator, remove the culture medium, and wash twice with 0.4 mL PBS (pre-cooled to 4°C).

[0655] (6) Cell lysis: Add 0.2 mL of 1 M NaOH to lyse the cells. After about 5 minutes, transfer the cell lysis buffer to a radioimmunoassay tube. Then wash twice with 0.4 mL of PBS (pre-cooled to 4°C) and transfer the lysate to a radioimmunoassay tube.

[0656] (7) Gamma count: The radioactive CPM values ​​of cell lysate and washing buffer were measured using a gamma counter. Blank tubes and standard tubes were tested before the samples were tested on the same day, with each sample being tested for 30 seconds.

[0657] 3. Data Processing

[0658] The results of the intake experiment are expressed as %AD, and the calculation formula is as follows:

[0659] 4. Experimental Results

[0660] From Figures 3, 8, 9, and 4, we can conclude that: 177 The Lu-labeled compounds showed good uptake in Nectin-4 positive PC3-Nectin4 cells, but almost no uptake in Nectin-4 negative PC3 cells.

[0661] Test Example 8 177 Biodistribution of Lu-labeled compounds in PC3-Nectin4 tumor-bearing mice

[0662] Purchase PC3-Nectin4 tumor model mice (BALBc-Nude nude mice), and feed the mice, approximately 6-9 weeks old, with 5×10 6 PC3-Nectin4 cells were subcutaneously inoculated into the right shoulder of a male animal until the tumor grew to >100 mm. 3 Mice were randomly divided into groups. 177The Lu radiolabeled compound was injected intravenously into mice at a rate of 100 μCi per mouse (nine mice per group). Mice were anesthetized by isoflurane inhalation at 2, 24, and 72 hours.

[0663] Blood was collected via the abdominal aorta and transferred to pre-weighed test tubes. Organs such as the brain, heart, liver, spleen, lungs, kidneys, bones, muscles, and tumors were collected, washed with deionized water, and then transferred to pre-weighed test tubes. Blood and tissue samples were weighed again, and the sample weight was calculated. The measurements were taken on the day of sample collection using a gamma counter. The tissue distribution results are shown in Figures 5, 6, 7, 10, and 11.

[0664] Test Example 9 68 Ga-labeled compound cellular uptake assay

[0665] 1. Experimental Objective

[0666] pass 68 The uptake of Ga-labeled compounds was measured by detecting the radioactivity count after binding to PC3-Nectin4 cells for different durations, and the uptake value of the test substance was calculated.

[0667] 2. Experimental Methods

[0668] (1) PC3-Nectin4 preparation: Prepare a cell suspension from cells in the logarithmic growth phase and adjust the cell density to 3×10⁻⁶. 5 Add 1 mL of the solution to each well of a 24-well cell culture plate pre-coated with poly-L-lysine solution. Incubate at 37°C in a 5% CO2 incubator. When the cell density reaches approximately 80% confluence, the plate is ready for experimentation.

[0669] (2) Compound preparation: dilute before adding the sample and set the incubation concentration to 1 nM; add a 1000-fold concentration of the precursor to the blocking group. Before dilution, accurately transfer a certain volume of the stock solution for activity determination and record the determination time, volume and activity.

[0670] (3) Sample addition: On the day of the experiment, discard the old culture medium, wash once with serum-free culture medium, and add 0.5 mL of serum-free culture medium containing the compound of the present invention or containing the blocking drug and the product of the present invention to each well. At the same time, transfer 0.5 mL of serum-free culture medium containing the compound of the present invention or containing the blocking drug and the product of the present invention to one radioimmunoassay tube as a standard tube.

[0671] (4) Incubation: After the sample is added, shake gently and incubate in an incubator at 37°C for 15 min, 30 min, 60 min and 120 min, with 3 replicates at each time point.

[0672] (5) Washing: After incubation, remove the 24-well plate from the incubator, remove the culture medium, and wash twice with 0.4 mL PBS (pre-cooled to 4°C).

[0673] (6) Cell lysis: Add 0.2 mL of 1 M NaOH to lyse the cells. After about 5 minutes, transfer the cell lysis buffer to a radioimmunoassay tube. Then wash twice with 0.4 mL of PBS (pre-cooled to 4°C) and transfer the lysate to a radioimmunoassay tube.

[0674] (7) Gamma count: The radioactive CPM values ​​of cell lysate and washing buffer were measured using a gamma counter. Blank tubes and standard tubes were tested before the samples were tested on the same day, with each sample being tested for 30 seconds.

[0675] 3. Data Processing

[0676] The results of the intake experiment are expressed as %IA, and the calculation formula is as follows:

[0677] 4. Experimental Results

[0678] As can be seen from Figure 12, 68 Ga-labeled compounds showed good uptake in Nectin-4 positive PC3-Nectin4 cells, but almost no uptake was observed in PC3-Nectin4 cells after blocking.

[0679] Test Case 10 177 SPECT / CT imaging of Lu-labeled compounds in PC3-Nectin4 tumor-bearing mice

[0680] PC3-Nectin4 tumor model mice (BALBc-Nude nude mice, 6-9 weeks old mice were fed 5×10 6 PC3-Nectin4 cells were subcutaneously inoculated into the right shoulder of a male animal until the tumor grew to 200-600 mm. 3 Mice were randomly divided into groups. 177 The radiolabeled compound BR005 was injected into the tail vein of mice, with 3 mice per group, at a dose of 500 μCi per mouse. 177 The Lu-BR005 was scanned at 1 hour, 5 hours, 24 hours, 48 ​​hours, 72 hours, and 120 hours.

[0681] Before scanning, animals were pre-anesthetized using a small animal anesthesia machine. The anesthetic was approximately 1%–2.5% isoflurane, with a gas flow rate of approximately 0.4–1 L / min. The anesthetized animals were placed in the small animal SPECT / CT chamber and positioned on the scanning table, with isoflurane continued to maintain anesthesia. SPECT / CT scanning was performed after anesthesia, and the scan time was recorded. The scanning energy window was 145.88–270.92 KeV. The CT scan was performed in fast mode.

[0682] Reconstruction and Analysis: Image reconstruction was performed after the scan. The reconstructed nuclide was Lu-177, and the number of iterations was 10. After reconstruction, the image and data were processed using the image processing software PMOD to delineate tissues such as tumors, brain, heart, liver, lungs, kidneys, muscles, and bones, and to calculate the %ID / g value of radioactive uptake in the target area. 177 The experimental results of Lu-BR005 are shown in Figures 13 and 14.

[0683] Test Example 11 212 Pharmacodynamic studies of Pb-labeled compounds in PC3-Nectin4 tumor-bearing mice

[0684] PC3-Nectin4 tumor model mice (BALBc-Nude nude mice, 6-9 weeks old mice were fed 5×10 6 PC3-Nectin4 cells were subcutaneously inoculated into the right shoulder of a male animal until the tumor grew to 100–300 mm. 3 Mice were randomly divided into groups. 212 The Pb radiolabeled compound BR005 was injected into the tail vein of mice. Each group consisted of 5 mice and was divided into 5 groups: saline, (5μCi), (5μCi*2), (10μCi), (10μCi*2), and (20μCi). 212 After administration of Pb-BR005, tumor volume was measured and weighed twice a week. The (5μCi*2) and (10μCi*2) groups received a second dose if tumor recurrence occurred. 212 The efficacy test results of Pb-BR005 are shown in Figures 15 and 16.

[0685] Test Example 12: Binding ability test of the compound prepared in the embodiments of the present invention with Human Nectin4 protein.

[0686] 1. Experimental Objective

[0687] The affinity of the analyte for Human Nectin4 (poliovirus receptor-associated protein 4) was determined using the SPR method.

[0688] 2. Materials and Instruments

[0689] Biacore 8K(GE Healthcareytiva)

[0690] CM5 chip (Cat#29149603, Cytiva)

[0691] Amine Coupling Kit(Cat#BR-1000-50,GE Healthcare)

[0692] 10mM Sodium Acetate (Cat#BR-1003-49, GE Healthcare)

[0693] DMSO (Cat#D5879, Sigma)

[0694] P20(Cat#BR-1000-54,GE Healthcare)

[0695] 96-well Plate(Cat#650101,greiner bio-one)

[0696] Human Nectin4 Protein(Cat#19771-H08H,Sino Biological)

[0697] 3. Experimental Design

[0698] This experiment employed the CM5 chip amino-coupling method, where Human Nectin4 protein was directly coupled to the CM5 chip as a ligand using a Biacore 8K microarray. The analyte was diluted to the desired concentration gradient using running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% P2O, 1% DMSO). Multiple cycles of kinetic analysis were performed, with each cycle consisting of 180 s of injection followed by 180 s of dissociation before the next cycle. This yielded the affinity kinetic data between Human Nectin4 protein and the analyte. The final data were analyzed using Biacore Insight Evaluation Software (V 2.0.15.12933) with a 1:1 binding model for kinetic fitting.

[0699] 4. Experimental methods and procedures

[0700] 1) Prepare running buffer: 10mM HEPES, pH 7.4, 150mM NaCl, 0.05% P2O, 1% DMSO.

[0701] 2) CM5 chip activation: The chip was activated by mixing 400mM EDC and 100mM NHS in a 1:1 ratio using the coupling kit at a flow rate of 10μL / min for 600s.

[0702] 3) Target protein coupling: Dilute Human Nectin4 protein to 5 μg / mL with 10 mM sodium acetate solution (pH 4.0) and couple to 1500 RU-3000 RU at a flow rate of 5 μL / min.

[0703] 4) Blocking the chip: Use 1M ethanolamine from the coupling kit to block the chip surface for 600 s at a flow rate of 10 μL / min.

[0704] 5) Analyte dilution: Using running buffer, the analyte was diluted 4-fold from 10 nM to 0.63 nM, for a total of 3 test concentrations.

[0705] 6) Sample injection analysis: run each cycle to test the concentration of one analyte, at a flow rate of 30 μL / min for 180 s binding and 180 s dissociation.

[0706] 7) Data analysis: Perform kinetics fitting analysis using a 1:1 binding model and calculate the dissociation constant (KD).

[0707] 5. Experimental Results

[0708] The compounds prepared in the embodiments of the present invention were subjected to Kinetics fitting analysis using Biacore Insight Evaluation software version 4.0 with a 1:1 binding model. The results are shown in Table 24.

[0709] Table 24 Results of binding of the compounds of the present invention with Human Nectin-4 SPR

[0710] Conclusion: The compounds prepared in the embodiments of the present invention have a strong binding ability with Human Nectin4 protein.

[0711] Test Example 13: Binding ability test of the compounds prepared in the embodiments of the present invention with Nectin4 proteins of different species.

[0712] 1. Experimental Objective

[0713] The affinity of the analyte for Nectin4 (poliovirus receptor-associated protein 4) of different species was determined using the SPR method.

[0714] 2. Materials and Instruments

[0715] Biacore 8K(GE Healthcareytiva)

[0716] CM5 chip (Cat#29149603, Cytiva)

[0717] Amine Coupling Kit(Cat#BR-1000-50,GE Healthcare)

[0718] 10mM Sodium Acetate (Cat#BR-1003-49, GE Healthcare)

[0719] DMSO (Cat#D5879, Sigma)

[0720] P20(Cat#BR-1000-54,GE Healthcare)

[0721] 96-well Plate(Cat#650101,greiner bio-one)

[0722] Rat Nectin4 Protein(NE4-R5255,ACRO)

[0723] Mouse Nectin4 Protein(5A7674-M08H,Sino Biological)

[0724] 3. Experimental Design

[0725] This experiment employed the CM5 chip amino-coupling method, where different species of Nectin4 proteins were directly coupled to the CM5 chip as ligands using a Biacore 8K microarray. Assays were diluted to the desired concentration gradient using running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% P2O, 1% DMSO). Multiple cycles of kinetic analysis were performed, with each cycle consisting of 180 s of injection followed by 180 s of dissociation before the next cycle. This yielded affinity kinetic data for the Nectin4 proteins from different species with the analytes. The final data were analyzed using Biacore Insight Evaluation Software (V 2.0.15.12933) with a 1:1 binding model for kinetic fitting.

[0726] 4. Experimental methods and procedures

[0727] 1) Prepare running buffer: 10mM HEPES, pH 7.4, 150mM NaCl, 0.05% P2O, 1% DMSO.

[0728] 2) CM5 chip activation: The chip was activated by mixing 400mM EDC and 100mM NHS in a 1:1 ratio using the coupling kit at a flow rate of 10μL / min for 600s.

[0729] 3) Target protein coupling: Dilute different species of Nectin4 protein to 5 μg / mL with 10 mM sodium acetate solution (pH 4.0) and couple for 600 s at a flow rate of 5 μL / min.

[0730] 4) Blocking the chip: Use 1M ethanolamine from the coupling kit to block the chip surface for 600 s at a flow rate of 10 μL / min.

[0731] 5) Analyte dilution: Using running buffer, the analyte was diluted 4-fold from 10 nM to 0.15 nM, resulting in 4 test concentrations.

[0732] 6) Sample injection analysis: run each cycle to test the concentration of one analyte, at a flow rate of 30 μL / min for 180 s binding and 180 s dissociation.

[0733] 7) Data analysis: Perform kinetics fitting analysis using a 1:1 binding model and calculate the dissociation constant (KD).

[0734] 5. Experimental Results

[0735] The compounds prepared in the embodiments of the present invention were subjected to Kinetics fitting analysis using Biacore Insight Evaluation software version 4.0 with a 1:1 binding model. The results are shown in Table 25.

[0736] Table 25. Binding results of the compounds of the present invention with SPR of Nectin4 protein from different species.

[0737] Conclusion: The compounds prepared in the embodiments of the present invention have strong binding ability with both Mouse Nectin4 and Rat Nectin4 proteins.

Claims

1. A bicyclic peptide nuclide ligand, characterized in that, The structure is represented by formula (V). in, RM is a chelating ligand, independently selected from DOTA, DO3A, DOTAGA, Bn-DOTA, Bn-Oxo-DO3A, Nota, NODAGA, Bn-NOTA, NOPO, DOTAM, Bn-TCMC, Bn-CHX-A”-DTPA, CB-TE2A, CB-DO2A, 2B3M-DTPA, Bn-PCTA, TETA, mDOTA, and nDOTA; G is selected independently. L is selected independently The left end of L* may be connected to RM or not connected to any segment including RM, and the carbonyl end on the right side** forms an amide bond with the nitrogen end of formula (V); L1 is independently selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -OCH2-, -OCH2CH2-, -OCH2CH2CH2-, -CH2OCH2CH2-, -CH2CH2OCH2CH2-, -CONHCH2-, -CONHCH2CH2-, -CH2CONHCH2-, -CH2CH2CONHCH2-, and L1 is connected in L in order from left to right; L2 is independently selected from The # terminal connects to RM, the ## terminal connects to NH, and terminals without the # terminal can exist independently. Xi, Xii, and Xiii are each independently selected from Cys, hCys, βCys, or Pen; A3 is selected independently. A4 is selected independently. A5 is selected independently. A6 is selected independently. M is independently selected from --NH2, R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from H and -C. 1-6 Alkyl, -C 1-6 Alkylene-R 21 -C 5-12 cycloalkyl, -C 1-6 alkylene-heteroatom-C 1-3 Alkyl, -C 1-6 alkylene-amino, -C 1-6 alkylene-naphthalene ring, -CO-R 21 -C 1-6 Alkyl-benzene ring; R 21 Independently selected from H, NH2, SCH3, hydroxyl, phenyl, p-hydroxyphenyl, imidazole, indole, -COOH, -CONH2, n, m, u, p, q, v, w are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

2. The bicyclic peptide nuclide ligand according to claim 1, characterized in that, R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently selected from H, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -(CH2)2CH(CH3)2, -CH(CH3)CH2CH3, -CH2CH(CH3)CH2CH3, -COOH, -CH2COOH, -(CH2)2COOH, -(CH2)3COOH, and -CH2CONH.

2. -(CH2)2CONH2, -(CH2)3CONH2, -CH2NH2, -(CH2)2NH2, -(CH2)3NH2, -(CH2)4NH2, -(CH2)5NH2, -(CH 2)2SCH3, -(CH2)3SCH3, -CH2Ph, -(CH2)2Ph, -CH2Ph(p-OH), -(CH2)2OH, -CH(CH3)OH, -CH2CH(CH3)OH, 3. The bicyclic peptide nuclide ligand according to claim 1, characterized in that, The structural fragment When u is selected from 1, the fragment is independently selected from 4. The bicyclic peptide nuclide ligand according to claim 1, characterized in that, The structural fragment Independently selected 5. The bicyclic peptide nuclide ligand according to claim 1, characterized in that, The L2 is independently selected from The # terminal connects to RM, the ## terminal connects to NH, and terminals without the # terminal can exist independently.

6. The bicyclic peptide nuclide ligand according to claim 1, characterized in that, The structural formula (V) is specifically selected from: In the Ve, A3 is selected from, The independent u is selected from 0 or 1, and L1, L2, R1, R2, R3, R4, R5, R6, R7, R8, R9, A3, A4, A5, A6, and M are as defined in claim 1.

7. The bicyclic peptide nuclide ligand according to claim 1, characterized in that, The compound is selected from the following structures:

8. A bicyclic peptide nuclide chelate, said chelate being formed by chelating the bicyclic peptide nuclide ligand according to any one of claims 1-7 with a radionuclide.

9. The bicyclic peptide nuclide chelate according to claim 8, characterized in that, The radionuclides are selected from: 18 F, 51 Cr 67 Ga、 68 Ga、 111 In、 99m Tc, 186 Re、 188 Re、 139 La、 140 La、 175 Yb、 153 Sm、 166 Ho、 86 Y、 88 Y、 90 Y、 149 Pm, 165 Dy、 169 Er、 177 Lu、 47 Sc、 142 Pr, 159 Gd, 212 Bi、 213 Bi、 72 As、 72 Se、 97 Ru、 109 Pd, 105 Rh、 101m Rh、 119 Sb, 128 Ba、 123 I, 125 I, 124 I, 131 I, 197 Hg, 211 At、 151 Eu、 153 Eu、 169 Eu、 201 Tl、 203 Pb, 212 Pb, 64 Cu、 67 Cu、 198 Au、 225 Ac、 227 Th、 199 Ag、 169 Yb、 62 Zn, 65 Zn, 89 Zr and 95 Zr.

10. A pharmaceutical composition comprising the bicyclic peptide nucleoside chelate according to any one of claims 8 to 9, and a pharmaceutically acceptable carrier.

11. A kit comprising or consisting of a bicyclic peptide nuclide chelate according to any one of claims 8-9 or a pharmaceutical composition according to claim 10, and instructions for use in diagnosing or treating a disease.

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