WO2023066314A1 - Bicyclic peptide ligand for nectin-4 and use thereof - Google Patents

Abstract

A bicyclic peptide ligand for Nectin-4 and a use thereof. The present invention specifically relates to a compound which is a peptide ligand having a high affinity for cell adhesion molecules (Nectin-4), and also relates to a drug conjugate, a stereoisomer, a pharmaceutically acceptable salt, a solvate, a co-crystal or a deuterated compound thereof, or a pharmaceutical composition containing same, and a use of the peptide ligand and the drug conjugate in the prevention and/or treatment of diseases or conditions that overexpress Nectin-4 in diseased tissue of a tumor patient.

Description

Bicyclic peptide ligands of nectin-4 and uses thereof technical field

The present invention relates to a compound containing polypeptide structure with high affinity for cell adhesion molecule (Nectin-4) and its polypeptide drug conjugate conjugated to one or more toxin molecules, and the compound and drug conjugate Related uses of the drug, including the use of preparing pharmaceutical compositions and the use of preventing and/or treating diseases or diseases in which Nectin-4 is overexpressed in diseased tissues of tumor individuals.

Background technique

Nectin is a cell adhesion molecule belonging to a Ca2 ⁺ -independent immunoglobulin (Immunoglobulin, Ig) superfamily, and is a homologue of the poliovirus receptor (PVR/CD155), also known as poliovirus Inflammatory virus receptor related protein (Pliovirus Receptor Related Protein, PVRL). Nectin family consists of four members Nectin 1-Nectin 4. Nectin is widely expressed in organisms, Nectin-1, Nectin-2 and Nectin-3 are expressed in a variety of cells, including: fibroblasts, epithelial cells and neuronal cells. Nectin-2 and Nectin-3 are also expressed in cells lacking cadherin glycoproteins, such as B cells, monocytes and sperm cells. The expression of human Nectin-4 is mainly in embryonic development stage and tumor tissue, but not in adult normal tissue.

Nectin-4 belongs to the cell membrane surface receptor, which can affect the tight junction between cells, participate in the adhesion between cells, and then regulate activities such as cell movement, differentiation and virus invasion. In recent years, the relationship between Nectin-4 and tumors has been paid close attention by researchers. For example, Takano et al. reported that Nectin-4 can promote the growth and proliferation of tumor cells through the Rac1 signaling pathway. In breast cancer, Nectin-4 was identified as an independent adverse prognostic factor in triple-negative breast cancer. Although the mechanism of action of Nectin-4 in tumor biology and cancer progression is still unclear, as a new type of tumor-associated antigen, its clinical potential is huge, which provides opportunities for the development of new therapeutic drugs (such as nucleic acid drugs, monoclonal antibodies) a great possibility. There is a significant correlation between the expression of Nectin-4 and the disease progression and prognosis of various cancer patients. Its expression in adult healthy tissues is limited, but it is overexpressed in malignant tumor cells. Therefore, targeting Nectin-4 may become an effective strategy for treating cancers with high Nectin-4 expression.

PADCEV (Enfortumab vedotin), the first ADC targeting Nectin-4, was approved by the FDA in 2019 for the treatment of locally advanced or metastatic urothelial carcinoma. -4) The human IgG1 monoclonal antibody enfortumab is conjugated with the cytotoxic agent MMAE (monomethyl auristatin E, a microtubule disruptor), and it is also the only Nectin-4 targeting medicine. Although there are not many reports on the research and development of targeted therapy targeting Nectin-4, as a potential therapeutic target in a variety of solid tumors, coupled with the successful marketing of PADCEV and the rapid expansion of indications for this target, targeting Nectin-4 Drug development will usher in new developments.

Contents of the invention

The present invention provides a compound with high affinity for cell adhesion molecule (Nectin-4), which comprises a polypeptide structure, and also provides a polypeptide drug conjugate in which the compound is conjugated to one or more toxin molecules, and Provided are pharmaceutical compositions, and their related uses in preventing and/or treating diseases or conditions in which Nectin-4 is overexpressed in diseased tissues of tumor individuals.

The present invention relates to a compound comprising a polypeptide structure and a non-aromatic molecular scaffold, the polypeptide structure comprising at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and the Cys or Hcys The residue forms a covalent bond with the non-aromatic molecular scaffold, thereby forming at least two polypeptide rings on the molecular scaffold; the condition is that the residues of Cys and Hcys contain at least one Hcys residue.

In some specific embodiments, the compound comprises a polypeptide structure and a non-aromatic molecular scaffold, the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and wherein Two are Hcys residues, and the Cys or Hcys residues form a covalent bond with the non-aromatic molecular scaffold, thereby forming at least two polypeptide loops on the molecular scaffold.

In some specific embodiments, the compound comprises a polypeptide structure and a non-aromatic molecular scaffold, the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and wherein One is a Hcys residue, and the Cys or Hcys residue forms a covalent bond with the non-aromatic molecular scaffold, thereby forming at least two polypeptide loops on the molecular scaffold.

In some specific embodiments, the non-aromatic molecular scaffold is selected from TATA and TATB.

In some specific embodiments, the compound comprises a polypeptide structure and a non-aromatic molecular scaffold, the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and wherein Two are Hcys residues, and the Cys or Hcys residues form a covalent bond with the non-aromatic molecular scaffold to form at least two polypeptide rings on the molecular scaffold, and the molecular scaffold is selected from TATA.

In some specific embodiments, the compound comprises a polypeptide structure and a non-aromatic molecular scaffold, the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and wherein One is the Hcys residue, the Cys or Hcys residue forms a covalent bond with the non-aromatic molecular scaffold to form at least two polypeptide rings on the molecular scaffold, and the molecular scaffold is selected from TATA.

In some specific embodiments, the compound comprises a polypeptide structure and a non-aromatic molecular scaffold, the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and wherein Two are Hcys residues, and the Cys or Hcys residues form a covalent bond with the non-aromatic molecular scaffold to form at least two polypeptide rings on the molecular scaffold, and the molecular scaffold is selected from TATB.

In some specific embodiments, the compound comprises a polypeptide structure and a non-aromatic molecular scaffold, the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and wherein One is the Hcys residue, the Cys or Hcys residue forms a covalent bond with the non-aromatic molecular scaffold to form at least two polypeptide rings on the molecular scaffold, and the molecular scaffold is selected from TATB.

In some specific embodiments, the polypeptide structure of the compound comprises the following amino acid sequence:

-Xa1-A1-Xa2-A2-Xa3-

Wherein, A1, A2 represent the amino acid sequence between Xa1, Xa2, and Xa3, and A1 and A2 each independently contain 3-10 amino acid residues; in some specific embodiments, the amino acid residues contained in A1, A2 are optionally is selected from Pro(P), 1Nal, D-Asp, Met(M), HArg, Asp(D), Trp(W), Ser(S), Thr(T), Hyp, Val, Ile and Gly, etc.;

Xa1, Xa2, and Xa3 are independently Cys or Hcys residues, and at least one of them is an Hcys residue, and the non-aromatic molecular scaffold forms a thioether bond with Xa1, Xa2, and Xa3 of the polypeptide respectively, thereby forming a thioether bond in the molecule Two polypeptide loops are formed on the scaffold.

In some specific embodiments, one of said A1 and A2 consists of 3 amino acid residues, and the other consists of 9 amino acid residues; in some specific embodiments, said A1 consists of 3 amino acid residues Composition, A2 consists of 9 amino acid residues; in some specific embodiments, said A1 consists of 9 amino acid residues, and A2 consists of 3 amino acid residues.

In some specific embodiments, wherein said polypeptide structure comprises the amino acid sequence shown below:

-Xa1-P(1Nal)(D-Asp)-Xa2-M(HArg)DWSTP(Hyp)W-Xa3-, said Xa1, Xa2, Xa3 are respectively selected from Cys or Hcys; the condition is Xa1, Xa2, Xa3 At least one selected from Hcys.

In some specific embodiments, wherein said polypeptide structure comprises the amino acid sequence shown in any one of SEQ ID NO:1～SEQ ID NO:7:

-Hcys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO:1);

-Cys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO:2);

-Cys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:3);

-Hcys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:4);

-Cys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO:5);

-Hcys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO:6);

-Hcys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:7).

In some specific embodiments, wherein said polypeptide structure comprises the amino acid sequence shown in any one of SEQ ID NO:8～SEQ ID NO:14:

-(β-Ala)-Sar ₁₀ -Hcys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO:8);

-(β-Ala)-Sar ₁₀ -Cys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO:9);

-(β-Ala)-Sar ₁₀ -Cys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO: 10);

-(β-Ala)-Sar ₁₀ -Hcys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO: 11);

-(β-Ala)-Sar ₁₀ -Cys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO: 12);

-(β-Ala)-Sar ₁₀ -Hcys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO: 13);

-(β-Ala)-Sar ₁₀ -Hcys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Cys- (SEQ ID NO: 14).

In some specific embodiments, wherein the compound is a bicyclic peptide containing one of the following structures, optionally containing other amino acid sequences at the N-terminus and/or C-terminus of the following structures:

Wherein, when Xa1, Xa2 or Xa3 is Cys, correspondingly, m1, m2 or m3 is 1;

When Xa1, Xa2 or Xa3 is Hcys, correspondingly, m1, m2 or m3 is 2;

n1, n2, and n3 are 1 or 2 independently.

In some specific embodiments, the compound is a bicyclic peptide having one of the following structures, wherein the bicyclic is as described above (such as I-1 or II-1), and n4 is any integer from 0 to 10, such as 3, 4, 5, 6, 7, 8, 9, 10, in some embodiments, n4 is 9:

In some embodiments, the compound is a bicyclic peptide having one of the following structures:

Wherein, n4 is selected from any integer of 0-10.

In some specific embodiments, the bicyclic peptide of formula I-2 or II-2, wherein,

(1) Xa1 is Hcys, m1 is 2, n1 is 1 or 2, and n4 is 9; or

(2) Xa2 is Hcys, m2 is 2, n2 is 1 or 2, and n4 is 9; or

(3) Xa3 is Hcys, m3 is 2, n3 is 1 or 2, and n4 is 9.

In some specific embodiments, the compound is selected from one of the structures in Table 1:

Table 1

The compound comprising at least two polypeptide ring structures of the present invention has protein stability and is stable to plasma protease, epithelial protease, gastric and intestinal protease, lung surface protease, intracellular protease, etc.; has specific targeting to Nectin-4, It is a peptide ligand specific to Nectin-4; it has a longer plasma half-life, and has good pharmacokinetic and pharmacodynamic properties.

The present invention also provides the application of the compound as a ligand of Nectin-4 in screening and/or preparing medicine.

The present invention also provides a drug conjugate or a pharmaceutically acceptable salt thereof, said conjugate comprising a compound as described above conjugated to one or more effectors and/or functional groups via a linker.

In some embodiments, wherein said effector is a cytotoxic agent.

In some specific embodiments, the present invention provides a drug conjugate represented by formula II or a pharmaceutically acceptable salt thereof,

In some specific embodiments, the cytotoxic agent is selected from one or more of the following group: alkylating agents, antimetabolites, plant alkaloids, terpenoids, podophyllotoxins, and Derivatives, taxanes and their derivatives, topoisomerase inhibitors, antitumor antibiotics.

In some embodiments, the cytotoxic agent is selected from the group consisting of alkylating agents, antimetabolites, plant alkaloids, terpenoids, podophyllotoxins and derivatives thereof, taxanes Classes and their derivatives, topoisomerase inhibitors, antitumor antibiotics.

In some embodiments, the linker is a divalent, trivalent, tetravalent or pentavalent spacer moiety linking the cytotoxic agent moiety and the peptide ligand moiety.

In some embodiments, the linker is a bivalent spacer moiety linking the cytotoxic agent moiety and the peptide ligand moiety; wherein the peptide ligand is linked to one cytotoxic agent moiety through the linker.

In some embodiments, the linker is a trivalent spacer moiety connecting the cytotoxic agent moiety and the peptide ligand moiety; wherein the peptide ligand is independently linked to the two cytotoxic agent moieties through the linker.

In some embodiments, the linker is a tetravalent spacer moiety linking the cytotoxic agent moiety and the peptide ligand moiety; wherein the peptide ligand is independently linked to the three cytotoxic agent moieties through the linker.

In some embodiments, the linker is a pentavalent spacer moiety connecting the cytotoxic agent moiety and the peptide ligand moiety; wherein the peptide ligand is independently linked to the four cytotoxic agent moieties through the linker.

In some embodiments, the peptide ligand is a compound as described above.

In some specific embodiments, wherein, the cytotoxic agent is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide, sulfur Azathioprine, mercaptopurine, pyrimidine analogs, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, paclitaxel, camptothecin and its derivatives, irinotecan, toposide Tecan, Amyridine, Etoposide, Etoposide Phosphate, Teniposide, Actinomycin D, Doxorubicin, Epirubicin, Epothilone and its derivatives, Bleomycin and its derivatives, dactinomycin and its derivatives, plicamycin and its derivatives, mitomycin C, cyspamicin, maytansine and its derivatives, auristatin and its derivatives.

In some embodiments, the cytotoxic agent is selected from maytansinoids, monomethyl auristatin, or camptothecin derivatives. In some embodiments, the cytotoxic agent is selected from maytansinoids or monomethyl auristatin. In some embodiments, the cytotoxic agent is selected from maytansine DM1, monomethylauristatin E (MMAE), or 7-ethyl-10-hydroxycamptothecin (SN38). In some embodiments, the cytotoxic agent is selected from DM1 or MMAE. In some embodiments, the cytotoxic agent is selected from MMAE.

In some specific embodiments, wherein the linker is a peptide linker, a disulfide linker or a pH-dependent linker.

In some embodiments, the disulfide linker is selected from DMDS, MDS, DSDM, NDMDS or the structure of formula III:

Wherein, R ₁ , R ₂ , R ₃ and R ₄ are independently selected from H, methyl, ethyl, propyl and isopropyl;

p and q are independently 1, 2, 3, 4 or 5;

The peptide linker is selected from: -Cit-Val-, -Phe-Lys- and -Val-Lys-;

The pH-dependent linker is selected from cis-aconitic anhydride.

The above-mentioned linkers are mainly used to connect cytotoxic agents and peptide ligands, and to release toxic substances under specific conditions. In order to control the rate of cleavage and the release of the accompanying effector molecules, the linkers can be appropriately modified, such as in its The linker of the present invention includes linker derivatives modified based on the above-mentioned links to link some groups to increase the chain length, and to increase group modification around the cleavage bond to control the hindrance of the cleavage bond.

In the present invention, the linker can theoretically be connected to the N-terminal, C-terminal and/or molecular scaffold of the peptide ligand. When linked to the C-terminal or molecular scaffold, a functional group linked to it can be modified on the C-terminal or molecular scaffold.

In some specific embodiments, wherein the linker is -PABC-Cit-Val-glutaryl-, -PABC-cyclobutyl-Ala-Cit-βAla- or

Wherein PABC represents p-aminobenzyl carbamate; k is selected from any integer of 1-20.

In some specific embodiments, wherein the linker is -PABC-Cit-Val-glutaryl- or -PABC-cyclobutyl-Ala-Cit-βAla-, wherein PABC represents p-aminobenzylcarbamate ester.

In some specific embodiments, wherein the linker is

Wherein k is selected from any integer of 1-20.

In some specific embodiments, wherein the linker is

In some specific embodiments, wherein the linker is

In respective embodiments, k is selected from any integer of 1-20; in some specific embodiments, k is selected from any integer of 1-10; in some specific embodiments, k is selected from 1, 2, 3, 4 or 5.

In some specific embodiments, wherein the drug conjugate has the structure of Formula III-1, Formula III-2, Formula III-3 or Formula III-4:

Wherein, Xa1, Xa2, and Xa3 are independently Cys or Hcys residues, and at least one of them is a Hcys residue;

When Xa1, Xa2 or Xa3 is Cys, correspondingly, m1, m2 or m3 is 1;

When Xa1, Xa2 or Xa3 is Hcys, correspondingly, m1, m2 or m3 is 2;

n1, n2, n3 are independently 1 or 2;

n4 is selected from any integer of 0-10;

k is selected from any integer of 1-10.

In some specific embodiments, the drug conjugate of formula III-1 or formula III-2 or formula III-3 or formula III-4, wherein,

(1) Xa1 is Hcys, m1 is 2, n1 is 1 or 2, and n4 is 9; or

(2) Xa2 is Hcys, m2 is 2, n2 is 1 or 2, and n4 is 9; or

(3) Xa3 is Hcys, m3 is 2, n3 is 1 or 2, and n4 is 9.

As a more specific technical solution of the present invention, it relates to a drug conjugate or a pharmaceutically acceptable salt thereof, wherein the drug conjugate is selected from one of the following structures:

The present invention also relates to a pharmaceutical composition, which comprises the aforementioned compound of the present invention (i.e. peptide ligand, or peptide compound) and/or the aforementioned drug conjugate of the present invention or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier and/or excipient.

The present invention also relates to a use of the aforementioned compound of the present invention, or the aforementioned drug conjugate of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of prevention and/or treatment of tumors Use in medicine for diseases or conditions in which Nectin-4 is overexpressed in diseased tissues of an individual. Preferably, said individual is a mammal or a human.

Typically, the disease overexpressing Nectin-4 is cancer. Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (adenomas and various types of carcinomas, including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and others ), such as bladder and urinary tract, breast, gastrointestinal (including esophagus, stomach (stomach), small intestine, colon, rectum, and anus), liver (hepatocellular), gallbladder, and biliary tract Systemic cancer, exocrine pancreatic cancer, renal cancer, lung cancer (such as adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck cancer (such as tongue cancer, buccal cavity cancer, laryngeal cancer, pharyngeal cancer , nasopharyngeal cancer, tonsil cancer, salivary gland cancer, nasal cavity cancer and paranasal sinus cancer), ovarian cancer, fallopian tube cancer, peritoneal cancer, vaginal cancer, vulvar cancer, penile cancer, cervical cancer, uterine muscle cancer, endometrial cancer , thyroid cancer (eg, follicular thyroid carcinoma), renal cancer, prostate cancer, skin and adnexal cancer (eg, melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevus); hematologic malignancies (i.e., leukemia, lymphoma) and premalignant hematologic disorders and borderline malignancies, including hematologic malignancies and associated disorders of the lymphoid lineage (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin's lymphoma, hairy cell leukemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, and post-transplantation lymphoproliferative disorder), and hematologic malignancies and myeloid-related disorders ( eg, acute myelogenous leukemia [AML], chronic myelogenous leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential platelets myelodysplastic syndrome and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome and promyelocytic leukemia); tumors of mesenchymal origin such as soft tissue sarcomas, bone or chondrosarcomas such as osteosarcoma, fibrous Sarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumor, sarcomas of benign and malignant tissues, and carina Dermatofibrosarcoma; tumors of the central or peripheral nervous system (eg, astrocytomas, gliomas, and glioblastomas, meningiomas, ependymomas, pineal tumors, and schwannomas); endocrine tumors ( such as pituitary, adrenal, islet cell, parathyroid, carcinoid, and medullary thyroid carcinomas); ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratoma, Primary cell tumor, dysgerminoma, mole, and choriocarcinoma); pediatric and embryonal tumors (eg, medulloblastoma, neuroblastoma, Wilms tumor, and primitive neuroectodermal tumor); or Congenital or other forms of syndromes that predispose patients to malignancy (eg, xeroderma pigmentosa).

Further, the disease or disease overexpressing Nectin-4 is at least one of urothelial cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, triple-negative breast cancer and bladder cancer .

The present invention also relates to a pharmaceutical composition or pharmaceutical preparation, said pharmaceutical composition or pharmaceutical preparation comprising 1-1500 mg of the aforementioned compound of the present invention or the aforementioned conjugate of the present invention or its pharmaceutically acceptable Acceptable salts, and pharmaceutically acceptable carriers and/or excipients.

The present invention also relates to a method for treating diseases in mammals or humans, the method comprising administering to a subject a therapeutically effective amount of the aforementioned compound of the present invention or the aforementioned conjugate of the present invention or its pharmaceutical The above acceptable salt, the therapeutically effective dose is preferably 1-1500 mg, and the disease is preferably tumor.

The present invention also provides a composition or pharmaceutical preparation, which contains the peptide compound, conjugate or pharmaceutically acceptable salt thereof described in any one of the preceding schemes, and a pharmaceutically acceptable carrier and/or adjuvant. The pharmaceutical composition may be in unit dosage form (a unit dosage is also referred to as a "dosage strength").

Furthermore, the composition or pharmaceutical preparation of the present invention contains 1-1500 mg of the peptide compound, conjugate or pharmaceutically acceptable salt thereof described in any one of the preceding schemes, and a pharmaceutically acceptable carrier and/or or accessories.

The present invention also provides preparations of the peptide compound, conjugate or pharmaceutically acceptable salt thereof described in any one of the preceding schemes for the prevention and/or treatment of diseases or conditions in which Nectin-4 is overexpressed in diseased tissues of tumor individuals Uses in medicine. Further, the disease or disease overexpressing Nectin-4 is preferably at least one of urothelial cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, triple-negative breast cancer and bladder cancer kind. Preferably, said individual is a mammal or a human.

The present invention also provides a method for treating diseases in mammals or humans, the method comprising administering to a subject a therapeutically effective amount of the peptide compound, conjugate or pharmaceutically acceptable amount thereof described in any one of the preceding schemes. Acceptable salts, the disease is preferably at least one of urothelial cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, triple negative breast cancer and bladder cancer, preferably the treatment is effective The amount is 1-1500 mg.

"Effective amount" or "therapeutically effective amount" in the present application refers to the administration of a sufficient amount of the compound disclosed in the present application, which will alleviate to some extent one or more symptoms of the disease or disorder being treated. In some embodiments, the result is reduction and/or alleviation of signs, symptoms or causes of disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a peptide compound disclosed herein, a conjugate, or a pharmaceutically acceptable salt thereof, required to provide a clinically significant reduction in disease symptoms. Examples of therapeutically effective amounts include, but are not limited to, 1-1500 mg, 1-1400 mg, 1-1300 mg, 1-1200 mg, 1-1000 mg, 1-900 mg, 1-800 mg, 1-700 mg, 1-600 mg, 1-500 mg, 1 -400mg, 1-300mg, 1-250mg, 1-200mg, 1-150mg, 1-125mg, 1-100mg, 1-80mg, 1-60mg, 1-50mg, 1-40mg, 1-25mg, 1-20mg , 5-1500mg, 5-1000mg, 5-900mg, 5-800mg, 5-700mg, 5-600mg, 5-500mg, 5-400mg, 5-300mg, 5-250mg, 5-200mg, 5-150mg, 5 -125mg, 5-100mg, 5-90mg, 5-70mg, 5-80mg, 5-60mg, 5-50mg, 5-40mg, 5-30mg, 5-25mg, 5-20mg, 10-1500mg, 10-1000mg 10-900mg, 10-800mg, 10-700mg, 10-600mg, 10-500mg, 10-450mg, 10-400mg, 10-300mg, 10-250mg, 10-200mg, 10-150mg, 10-125mg, 10 -100mg, 10-90mg, 10-80mg, 10-70mg, 10-60mg, 10-50mg, 10-40mg, 10-30mg, 10-20mg; 20-1500mg, 20-1000mg, 20-900mg, 20-800mg , 20-700mg, 20-600mg, 20-500mg, 20-400mg, 20-350mg, 20-300mg, 20-250mg, 20-200mg, 20-150mg, 20-125mg, 20-100mg, 20-90mg, 20 -80mg, 20-70mg, 20-60mg, 20-50mg, 20-40mg, 20-30mg; 50-1500mg, 50-1000mg, 50-900mg, 50-800mg, 50-700mg, 50-600mg, 50-500mg , 50-400mg, 50-300mg, 50-250mg, 50-200mg, 50-150mg, 50-125mg, 50-100mg; 100-1500mg, 100-1000mg, 100-900mg, 100-800mg, 100-700mg, 100 - 600mg, 100-500mg, 100-400mg, 100-300mg, 100-250mg, 100-200mg.

In some embodiments, the pharmaceutical composition or preparation of the present invention contains the aforementioned therapeutically effective amount of the peptide compound, conjugate or pharmaceutically acceptable salt thereof of the present invention.

The present invention relates to a pharmaceutical composition or pharmaceutical preparation, which comprises a therapeutically effective amount of the peptide compound, conjugate or pharmaceutically acceptable salt thereof and a carrier and/or excipient. The pharmaceutical composition may be in the form of a unit preparation (the amount of the main drug in the unit preparation is also referred to as "preparation specification"). In some embodiments, the pharmaceutical composition includes, but is not limited to, 1 mg, 1.25 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg ,70mg,75mg,80mg,85mg,90mg,95mg,100mg,110mg,120mg,125mg,130mg,140mg,150mg,160mg,170mg,180mg,190mg,200mg,210mg,220mg,230mg,240mg,250mg,275mg,300mg , 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg, 800mg, 850mg, 900mg, 950mg, 1000mg, 11 , 1200mg, 1300mg, 1400mg, 1500mg of the peptide compound, conjugate or pharmaceutically acceptable salt thereof of the present invention.

In some embodiments, the present invention provides a method for treating a disease in a mammal or a human, the method comprising administering to a subject a therapeutically effective amount of a peptide compound, conjugate, or pharmaceutically acceptable compound of the present invention salt, and pharmaceutically acceptable carriers and/or excipients, the therapeutically effective dose is preferably 1-1500 mg, and the diseases are preferably urothelial cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, At least one of pancreatic cancer, triple-negative breast cancer, and bladder cancer.

In some embodiments, the present invention provides a method for treating a disease in a mammal or a human, the method comprising administering the peptide compound of the present invention, the conjugate or a pharmaceutically acceptable salt thereof, and a pharmaceutical Acceptable carriers and/or excipients above are given to the subject at a daily dose of 1-1500 mg/day, and the daily dose can be a single dose or divided doses. In some embodiments, the daily dose includes but is not limited to 10-1500mg/day, 20-1500mg/day, 25-1500mg/day, 50-1500mg/day, 75-1500mg/day, 100-1500mg/day, 200-1500mg/day, 10-1000mg/day, 20- 1000mg/day, 25-1000mg/day, 50-1000mg/day, 75-1000mg/day, 100-1000mg/day, 200-1000mg/day, 25-800mg/day, 50-800mg/day, 100-800mg/day day, 200-800mg/day, 25-400mg/day, 50-400mg/day, 100-400mg/day, 200-400mg/day, in some embodiments, the daily dosage includes but not limited to 1mg/day, 5mg/day day, 10mg/day, 20mg/day, 25mg/day, 50mg/day, 75mg/day, 100mg/day, 125mg/day, 150mg/day, 200mg/day, 300mg/day, 400mg/day, 600mg/day, 800mg/day, 1000mg/day, 1200mg/day, 1400mg/day, 1500mg/day.

The present invention also provides a kit, which may include a composition in single dose or multi-dose form, the kit comprising a compound of the present invention, a conjugate or a pharmaceutically acceptable salt thereof, a compound of the present invention, a conjugate, or a pharmaceutically acceptable salt thereof. The amount of the conjugate or pharmaceutically acceptable salt is the same as that in the above-mentioned pharmaceutical composition.

The amounts of the compounds, conjugates or pharmaceutically acceptable salts described herein are in each case calculated as the free base.

"Preparation specification" refers to the weight of the main drug contained in each tube, tablet or other unit preparation.

synthetic route

Those skilled in the art can prepare the compounds of the present invention by combining known organic synthesis techniques, starting from commercially available chemicals and/or compounds described in the chemical literature. "Commercially available chemicals" are obtained from formal commercial sources, suppliers include: Titan Technology, Anaiji Chemical, Shanghai Demo, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, Nanjing Yaoshi, WuXi AppTec and Bailingwei Technology, etc. company.

The peptide moieties of the invention can be prepared synthetically by standard techniques and then reacted with molecular scaffolds in vitro. When doing this, standard chemistry can be used. This enables rapid large-scale preparation of soluble materials for further downstream experiments or validation. Such methods can be achieved using conventional chemistry as disclosed in, for example, Timmerman et al. (2005, ChemBioChem).

In some embodiments, the drug conjugates of the present invention are synthesized according to the following route:

For more specific synthesis steps, refer to the examples.

the term

Under the situation that the present invention does not specify otherwise, the terms of the present invention have the following meanings:

Peptide ligands refer to compounds containing amino acid sequences (peptide structures) formed by covalently binding peptides to molecular scaffolds, and can be used as ligands for cell adhesion molecules (Nectin-4) in the present invention. Typically, the peptides forming such compounds contain two or more reactive groups (i.e. cysteine residues and/or homocysteine residues) capable of forming covalent bonds (e.g. thioether bonds) with the scaffold. group), and the relative sequence between the reactive groups (in the present invention, it may also be referred to as a loop sequence). . In the context of the present invention, the peptide comprises at least three cysteine residues and/or homocysteine residues (Cys residues and/or Hcys residues), which form at least two loops on the scaffold .

Molecular scaffolds include non-aromatic molecular scaffolds. A non-aromatic molecular scaffold refers to any molecular scaffold as defined herein that does not contain an aromatic carbocyclic or aromatic heterocyclic ring system. Examples of suitable non-aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53(6) 1602-1606. Molecular scaffolds can be small molecules, such as small organic molecules. Molecular scaffolds can also be macromolecules, and in some cases, molecular scaffolds are macromolecules composed of amino acids, nucleotides, or carbohydrates. In some cases, the molecular scaffold comprises a reactive group capable of reacting with a functional group of the polypeptide to form a covalent bond. Molecular scaffolds can include chemical groups that form linkages with peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleic imides, alkyl halides and acid halides. An example of a compound containing an αβ unsaturated carbonyl group is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA ) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606).

The non-aromatic molecular scaffolds described herein can be selected from the following structures:

etc., non-aromatic skeletons in WO2018197893 can also be selected.

Polypeptide refers to a compound formed by linking three or more amino acid molecules together by peptide bonds. The unit of amino acid in a polypeptide is called an amino acid residue.

Effectors and/or functional groups are pharmacologically or functionally specific molecules or fragments that can be attached (via a linker) to, for example, the N- and/or C-termini of a polypeptide, amino acids within a polypeptide, or the molecular backbone. Suitable effectors and/or functional groups include antibodies and parts or fragments thereof, cytotoxic molecules or fragments, enzyme inhibitor molecules or fragments, metal chelators, and the like. In some cases, the effector and/or functional group is a drug, particularly a cytotoxic agent.

Derivatives refer to products derived from the substitution of hydrogen atoms or atomic groups in a compound by other atoms or atomic groups.

Unless otherwise stated, all amino acids are used in the L-configuration.

Some common amino acid names and their three-letter abbreviations and one-letter abbreviations are shown in Table 2.

Table 2

amino acid name	Three (Multiple) Letter Abbreviations	single letter abbreviation	amino acid name	Three (Multiple) Letter Abbreviations	single letter abbreviation
cysteine	Cys	C	Naphthylalanine	1 Nal	/
homocysteine	Hcys	/	aspartic acid	Asp	D.
Hydroxyproline	Hyp	/	D-aspartic acid	D-Asp	/
proline	Pro	P	Tryptophan	Trp	W
Alanine	Ala	A	Homoarginine	HArg	/
beta-alanine	β-Ala, bAla	/	serine	Ser	S
Sarcosine	Sar	/	threonine	Thr	T
Methionine	met	m	Phenylalanine	Phe	f
Asparagine	Asn	N	Tyrosine	Tyr	Y
Leucine	Leu	L	Histidine	His	h
Valine	Val	V	Isoleucine	Ile	I
Lysine	Lys	K	Glycine	Gly	G

Maytansinoids, such as DM1, are cytotoxic agents, which are thiol-containing derivatives of maytansine, and DM1 has the following structure:

Monomethyl auristatin E (MMAE) is a synthetic antineoplastic agent and has the following structure:

SN38: 7-Ethyl-10-hydroxycamptothecin.

DMDS:

MDS:

DSDM:

NDMDS:

TATB:

TATA:

The carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds of the present invention include their isotopes, and the carbon, hydrogen, oxygen, sulfur, Nitrogen or halogen is optionally further replaced by one or more of their corresponding isotopes, wherein isotopes of carbon include ¹² C, ¹³ C and ¹⁴ C, isotopes of hydrogen include protium (H), deuterium (deuterium, also known as deuterium ), tritium (T, also known as tritium), the isotopes of oxygen include ¹⁶ O, ¹⁷ O and ¹⁸ O, the isotopes of sulfur include ³² S, ³³ S, ³⁴ S and ³⁶ S, and the isotopes of nitrogen include ¹⁴ N and ¹⁵ N, the isotope of fluorine ^{is 19} F, the isotope of chlorine includes ³⁵ Cl and ³⁷ Cl, and the isotope of bromine includes ⁷⁹ Br and ⁸¹ Br.

"Pharmaceutically acceptable salt" means that the compound of the present invention maintains the biological effectiveness and characteristics of the free acid or free base, and the free acid is mixed with a non-toxic inorganic base or organic base, and the free base is mixed with a non-toxic inorganic base or organic base. Non-toxic salts obtained by reacting inorganic or organic acids.

"Pharmaceutical composition" means a mixture of one or more compounds described herein, or stereoisomers, solvates, pharmaceutically acceptable salts or co-crystals thereof, with other constituents, wherein the other constituents comprise physiological/pharmaceutical acceptable carriers and/or excipients.

"Carrier" refers to: does not cause significant irritation to the organism and does not eliminate the biological activity and characteristics of the administered compound, and can change the way the drug enters the body and its distribution in the body, controls the release rate of the drug and releases the drug. Non-limiting examples of systems for delivery to targeted organs include microcapsules and microspheres, nanoparticles, liposomes, and the like.

"Excipient" means: not itself a therapeutic agent, used as a diluent, adjuvant, binder and/or vehicle, added to a pharmaceutical composition to improve its handling or storage properties or to allow or facilitate The compound or pharmaceutical composition is presented in unit dosage form for administration. As known to those skilled in the art, pharmaceutical excipients can serve various functions and can be described as wetting agents, buffers, suspending agents, lubricants, emulsifiers, disintegrating agents, absorbing agents, preservatives , surfactants, coloring agents, flavoring agents and sweeteners. Examples of pharmaceutically acceptable excipients include, but are not limited to: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as carboxymethyl Sodium cellulose, ethyl cellulose, cellulose acetate, hydroxypropyl methylcellulose, hydroxypropyl cellulose, microcrystalline cellulose, and croscarmellose (such as croscarmellose sodium) (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, red Flower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as oil (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; ( 18) Ringer's solution; (19) ethanol; (20) pH buffer solution; (21) polyester, polycarbonate and/or polyanhydride; and (22) other nontoxic Compatible substances.

Description of drawings

Figure 1 is the tumor growth curve of the mouse NCI-H292 subcutaneous and in vivo xenograft model.

Fig. 2 is a curve of the body weight change of the mouse NCI-H292 subcutaneous and in vivo xenograft model.

Figure 3 is the tumor growth curve of the mouse NCI-H292 subcutaneous and in vivo xenograft model.

Fig. 4 is the animal body weight change curve of the mouse NCI-H292 subcutaneous and in vivo xenograft model.

Detailed ways

The content of the present invention will be described in detail below through examples. If specific conditions are not indicated in the embodiments, the experimental method of conventional conditions shall be carried out. The examples given are for better description of the content of the present invention, but it cannot be understood that the content of the present invention is limited to the examples given. Non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above content of the invention still belong to the protection scope of the present invention.

Compound preparation

Example 1

Steps 1 to 4:

CTC resin (75g, 1.0mmol/g) and Fmoc-L-citrulline (30.0g, 75.4mmol, 1.0eq) were added to dichloromethane (600mL), followed by N,N-diisopropylethyl Amine (58.4g, 453mmol, 6.0eq), reacted for 3 hours. Suction filtration, the resin was washed twice with DMF, the prepared 20% piperidine/DMF solution was added to the resin mixture, the system reacted for 2 hours, suction filtration, the resin was washed twice with DMF. DMF (600mL), Boc-L-valine (48.0g, 0.22mmol, 3.0eq) and HBTU (85.0g, 0.21mmol, 2.85eq) and N,N-diisopropylethyl were added sequentially to the resin mixture Amine (58.4g, 453mmol, 6.0eq), the system was reacted for 3 hours, filtered with suction, the resin was washed three times with DMF, three times with methanol, and twice with dichloromethane. Add the prepared 20% hexafluoroisopropanol/dichloromethane solution to the resin mixture, react the system for 30 minutes, filter with suction, and repeat the previous operation again. The mother liquors were combined and concentrated to dryness to obtain 25.0 g of intermediate 4 (yield over four steps = 88%).

LCMS m/z=374.9[M+1] ⁺

the fifth step:

Intermediate 4 (25g, 66.7mmol) was added to dichloromethane (250mL) and methanol (250mL), then p-aminobenzyl alcohol (9.84g, 80.0mmol) and EEDQ (39.5g, 80.0mmol) were added, and stirred at room temperature After 16 hours, it was concentrated to dryness and purified by column chromatography (DCM:MeOH=10:1) to obtain 10 g of intermediate 5 (yield=31%).

LCMS m/z=480.1[M+1] ⁺

Step six:

Intermediate 5 (10.0g, 20.8mmol) was added to dry dichloromethane (120mL) and dry tetrahydrofuran (60mL), under nitrogen protection, then p-nitrophenyl chloroformate (6.2g, 31.2mmol ) and pyridine (3.3g, 41.6mmol), stirred at room temperature for 5h, filtered, the mother liquor was concentrated to dryness, and purified by column chromatography (DCM:MeOH=10:1) to obtain 2.3g of intermediate 6 (yield=16.6%).

LCMS m/z=664.3[M+1] ⁺

Step seven:

Intermediate 6 (2.1g, 3.1mmol) and N,N-diisopropylethylamine (3.6g, 31mmol) were added to DMF (40mL), under nitrogen protection, stirred at room temperature for 10 minutes, cooled to 0°C, and then Add MMAE (purchased from Chengdu Huajieming Biotechnology Co., Ltd., 2.0 g, 3.1 mmol) and HOBT (0.38 g, 3.1 mmol), keep stirring for 30 minutes, move to room temperature and stir for 18 hours, and directly purify on a C18 reverse-phase column (0.1% TFA) (H ₂ O:ACN=50:50), yielding 2.7 g of intermediate 7 (yield=70%).

LCMS m/z=1223.4[M+1] ⁺

Step Eight:

To Intermediate 7 (2.0g, 1.6mmol) was added a well-mixed TFA/DCM=1:10 mixture (34mL), stirred at room temperature for 2h, concentrated the dry solvent at 25°C, added tetrahydrofuran (110mL) and potassium carbonate (2.26 g, 16mmol), stirred at room temperature for 3h. Concentrate to dryness, and directly purify by C18 reverse phase column (0.1% TFA) (H ₂ O:ACN=60:40) to obtain 1.4 g of intermediate 8 (yield=76.5%).

LCMS m/z=1123.4[M+1] ⁺

Step Nine:

Intermediate 8 (1.4g, 1.25mmol), glutaric anhydride (0.29g, 2.5mmol) and N,N-diisopropylethylamine (0.33g, 2.5mmol) were added to DMA (14mL), stirred at room temperature 18h, C18 reverse-phase preparative column purification (0.1% TFA) (H ₂ O:ACN=50:50), to obtain 1.3 g of intermediate 9 (yield=84.4%).

LCMS m/z=1237.4[M+1] ⁺

Step ten:

Intermediate 9 (1.3g, 1.05mmol) was added to DMA (58mL) and dichloromethane (20mL), under nitrogen protection, the temperature was lowered to 0°C, and N-hydroxysuccinimide (0.37g, 3.15mmol) was added ) and EDCI (0.61g, 3.15mmol), stirring at room temperature for 18h, C18 reverse phase preparative column purification (0.1%TFA) (H ₂ O:ACN=50:50), to obtain 1.0g of intermediate 10 (yield=71.4% ).

LCMS m/z=1334.5[M+1] ⁺

Eleventh step:

Intermediate 10 (10 mg, 7.5 μmol) and HSK-P18 (20 mg, 6.2 μmol) were added to DMA (1 mL), then N,N-diisopropylethylamine (2.4 mg, 18.6 μmol) was added and stirred at room temperature 18h. Separation and purification with liquid phase preparative column (liquid phase preparation conditions: C18 reverse phase preparative column, mobile phase is deionized water (A) containing 0.1% trifluoroacetic acid, acetonitrile (B) containing 0.1% trifluoroacetic acid, gradient washing B content = 5% to 70%, elution time 15min, flow rate 12mL/min, column temperature: 30°C, retention time: 4.56min) to obtain 10mg of compound 1.

LCMS m/z＝1054.8[M/4+1],1406.3[M/3+1].

Synthesis of bicyclic peptide HSK-P18:

P18 synthesis method:

Peptides were synthesized using standard Fmoc chemistry:

1. Add MBHA resin (0.5mmol, 1.85g, sub: 0.27mmol/g) and DMF solvent into the reactor and shake for 2 hours.

2. Drain and rinse three times with DMF.

3. Add 20% piperidine/DMF and mix for 30 minutes.

4. Drain and rinse with DMF five times.

5. Add the Fmoc-protected amino acid solution, mix for 30 seconds, add the coupling reagent, and bubble nitrogen for 1 hour.

6. Repeat steps 2-5 for the next amino acid coupling. The amino acid coupling reagents are shown in Table 3.

table 3

#	raw material	Coupling reagent
1	Fmoc-HCys(Trt)-OH(2.0eq.)	HATU(1.90eq.) and DIEA(4.0eq.)
2	Fmoc-Trp(Boc)-OH(3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
3	Fmoc-Hyp-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)

4	Fmoc-Pro-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
5	Fmoc-Thr(tBu)-OH(3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
6	Fmoc-Ser(tBu)-OH(3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
7	Fmoc-Trp(Boc)-OH(3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
8	Fmoc-Asp(OtBu)-OH(3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
9	Fmoc-HArg(Pbf)-OH(2.0eq.)	HATU(1.90eq.) and DIEA(4.0eq.)
10	Fmoc-Met-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
11	Fmoc-HCys(Trt)-OH(2.0eq.)	HATU(1.90eq.) and DIEA(4.0eq.)
12	Fmoc-D-Asp(OtBu)-OH(3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
13	Fmoc-1-Nal-OH (2.0eq.)	HATU(1.90eq.) and DIEA(4.0eq.)
14	Fmoc-Pro-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
15	Fmoc-HCys(Trt)-OH(2.0eq.)	HATU(1.90eq.) and DIEA(4.0eq.)
16	Fmoc-Sar-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
17	Fmoc-Sar-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
18	Fmoc-Sar-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
19	Fmoc-Sar-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
20	Fmoc-Sar-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
twenty one	Fmoc-Sar-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
twenty two	Fmoc-Sar-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
twenty three	Fmoc-Sar-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
twenty four	Fmoc-Sar-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
25	Fmoc-Sar-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)
26	Fmoc-β-Ala-OH (3.0eq.)	HATU(2.85eq.) and DIEA(6.0eq.)

7. 20% piperidine DMF solution, 30 minutes for the removal of Fmoc protective agent. The coupling reaction was monitored using ninhydrin and the resin was washed 5 times with DMF.

Fragmentation and purification of peptides:

1. Add the polypeptide protected by the side chain of the polypeptide into the reaction bottle, add the cleavage buffer (90% TFA/2.5% TIS/2.5% H ₂ O/5.0% DTT), and stir at room temperature for 2 hours.

2. Add iced isopropyl ether, the peptide is precipitated, and centrifuged (3min at 3000rpm).

3. Wash twice with isopropyl ether.

4. Vacuum-dried to obtain white solid crude peptide compound P18 (1.30 g, crude product).

The synthetic method of HSK-P18:

The crude peptide P18 was dissolved in 50% MeCN/H ₂ O (500 mL), slowly added TATA (purchased from WuXi AppTec, 270 mg, 0.60 mmol) under stirring at room temperature, and the addition time was more than 30 minutes. Ammonium bicarbonate was added to adjust the pH value to 8, and the reaction solution was stirred at room temperature for 12 hours. LC-MS showed that the reaction was complete. Purification by preparative HPLC (mobile phase, A: 0.075% TFA in H ₂ O, B: CH ₃ CN) gave a white solid HSK-P18 (94.2 mg, purity 96.3%).

According to the above method, HSK-P19, HSK-P34, HSK-P35, HSK-P36, HSK-P37, HSK-P38, HSK-P39, HSK-P40, HSK-P41, HSK-P42, HSK-P43 were synthesized respectively , HSK-P44, HSK-P45.

HSK-P19: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:1(-Hcys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Hcys-), Molecular scaffolds were selected from TATB.

HSK-P34: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:4(-Hcys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Cys-), The molecular scaffold was selected from TATA.

HSK-P35: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:3(-Cys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Cys-), The molecular scaffold was selected from TATA.

HSK-P36: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:2(-Cys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Hcys-), The molecular scaffold was selected from TATA.

HSK-P37: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:7(-Hcys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Cys-), The molecular scaffold was selected from TATA.

HSK-P38: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:6(-Hcys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Hcys-), The molecular scaffold was selected from TATA.

HSK-P39: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:5(-Cys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Hcys-), The molecular scaffold was selected from TATA.

HSK-P40: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:4(-Hcys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Cys-), Molecular scaffolds were selected from TATB.

HSK-P41: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:3(-Cys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Cys-), Molecular scaffolds were selected from TATB.

HSK-P42: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:2(-Cys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Hcys-), Molecular scaffolds were selected from TATB.

HSK-P43: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:7(-Hcys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Cys-), Molecular scaffolds were selected from TATB.

HSK-P44: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:6(-Hcys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Hcys-), Molecular scaffolds were selected from TATB.

HSK-P45: The amino acid sequence is (β-Ala)-Sar10-SEQ ID NO:5(-Cys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Hcys-), Molecular scaffolds were selected from TATB.

Example 2

The synthesis method refers to Example 1: Intermediate 10 (10 mg, 7.5 μmol) and HSK-P19 (20 mg, 6.2 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (2.4 mg , 18.6μmol), stirred at room temperature for 18h. 10 mg of compound 2 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1044.3[M/4+1],1391.8[M/3+1].

Example 3

The synthetic method refers to Example 1: Add intermediate 10 (16 mg, 12.1 μmol) and HSK-P34 (30 mg, 10.1 μmol) to DMA (1 mL), then add N,N-diisopropylethylamine (3.9 mg , 30.3μmol), stirred at room temperature for 18h. 15 mg of compound 3 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1047.8[M/4+1],1396.8[M/3+1].

Example 4

The synthetic method refers to Example 1: Add intermediate 10 (16 mg, 12.1 μmol) and HSK-P35 (30 mg, 10.1 μmol) to DMA (1 mL), then add N,N-diisopropylethylamine (3.9 mg , 30.3μmol), stirred at room temperature for 18h. 16 mg of compound 4 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1047.8[M/4+1],1396.7[M/3+1].

Example 5

The synthesis method refers to Example 1: Intermediate 10 (16 mg, 12.1 μmol) and HSK-P36 (30 mg, 10.1 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (3.9 mg , 30.3μmol), stirred at room temperature for 18h. 15 mg of compound 5 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1047.8[M/4+1],1396.8[M/3+1].

Example 6

The synthetic method refers to Example 1: Add intermediate 10 (16 mg, 12.0 μmol) and HSK-P37 (30 mg, 10.0 μmol) to DMA (1 mL), then add N,N-diisopropylethylamine (3.9 mg , 30.0μmol), stirred at room temperature for 18h. 17 mg of compound 6 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1051.4[M/4+1],1401.3[M/3+1].

Example 7

The synthetic method refers to Example 1: Intermediate 10 (16 mg, 12.0 μmol) and HSK-P38 (30 mg, 10.0 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (3.9 mg , 30.0μmol), stirred at room temperature for 18h. 14 mg of compound 7 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1051.3[M/4+1],1401.6[M/3+1].

Example 8

The synthesis method refers to Example 1: Intermediate 10 (16 mg, 12.0 μmol) and HSK-P39 (30 mg, 10.0 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (3.9 mg , 30.0μmol), stirred at room temperature for 18h. 15 mg of compound 8 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1051.3[M/4+1],1401.4[M/3+1].

Example 9

The synthesis method refers to Example 1: Intermediate 10 (16 mg, 12.3 μmol) and HSK-P40 (30 mg, 10.2 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (3.9 mg , 30.6μmol), stirred at room temperature for 18h. 18 mg of compound 9 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1037.3[M/4+1],1382.7[M/3+1].

Example 10

The synthesis method refers to Example 1: Intermediate 10 (16 mg, 12.3 μmol) and HSK-P41 (30 mg, 10.2 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (3.9 mg , 30.6μmol), stirred at room temperature for 18h. 15 mg of compound 10 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1037.3[M/4+1],1382.7[M/3+1].

Example 11

The synthetic method refers to Example 1: Add intermediate 10 (16 mg, 12.3 μmol) and HSK-P42 (30 mg, 10.2 μmol) to DMA (1 mL), then add N,N-diisopropylethylamine (3.9 mg , 30.6μmol), stirred at room temperature for 18h. 13 mg of compound 11 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1037.4[M/4+1],1382.8[M/3+1].

Example 12

The synthetic method refers to Example 1: Add intermediate 10 (16 mg, 12.3 μmol) and HSK-P43 (30 mg, 10.2 μmol) to DMA (1 mL), then add N,N-diisopropylethylamine (3.9 mg , 30.6μmol), stirred at room temperature for 18h. 15 mg of compound 12 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1040.7[M/4+1],1387.4[M/3+1].

Example 13

The synthetic method refers to Example 1: Add intermediate 10 (16 mg, 12.3 μmol) and HSK-P44 (30 mg, 10.2 μmol) to DMA (1 mL), then add N,N-diisopropylethylamine (3.9 mg , 30.6μmol), stirred at room temperature for 18h. 14 mg of compound 13 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1040.8[M/4+1],1387.3[M/3+1].

Example 14

The synthesis method refers to Example 1: Intermediate 10 (16 mg, 12.3 μmol) and HSK-P45 (30 mg, 10.2 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (3.9 mg , 30.6μmol), stirred at room temperature for 18h. 15 mg of compound 14 was obtained by separation and purification with a liquid phase preparative column. LCMS m/z＝1040.8[M/4+1],1387.3[M/3+1].

Example 15

first step:

The known compound 2-(2-(2-aminoethoxy) ethoxy) tert-butyl ethyl carbamate (25.0g, 0.1mol), ethyl bromoacetate (43.2g, 0.22mol), carbonic acid Sodium (40.5g, 0.25mol) was added to acetonitrile (1.2L), heated to 50°C and stirred for 18h, cooled to room temperature, filtered, and the mother liquor was concentrated to dryness to obtain 42g of crude product 15b. (Yield = 99%). LCMS m/z=421.3[M+1] ⁺

The second and third steps:

Compound 15b (42.0g, 0.1mol) was added to methanol (500mL) and water (500mL), then sodium hydroxide (40.3g, 1.0mol) was added, stirred at room temperature for 4h, and the pH was adjusted to 1-2 with 6N hydrochloric acid. , stirred for 0.5h. The reaction solution was directly used in the next reaction.

LCMS m/z=265.2[M+1] ⁺

the fourth step:

Add 9-fluorenylmethyl-N-succinimidyl carbonate (34.0 g, 0.1 mol) and sodium bicarbonate (42.0 g, 0.5 mol) successively to the reaction solution of 15 d, stir at room temperature for 4 h, and use a C18 reverse-phase column Purification (0.1% TFA) (H ₂ O:ACN=60:40) afforded 22 g of 15e (yield over two steps=45.2%). LCMS m/z=487.2[M+1] ⁺

the fifth step:

15e (5.0g, 0.01mol), pentafluorophenol (3.86g, 0.02mol) and N,N-diisopropylcarbodiimide (2.65g, 0.02mol) were sequentially stirred at room temperature for 2h, and purified by column chromatography (PE:EA=2:1) yielded 4.0 g of 15f (yield=47.6%).

LCMS m/z＝819.1[M+1] ⁺

Step six:

15f (140mg, 0.17mmol), intermediate 8 (390mg, 0.34mmol) and N,N-diisopropylethylamine (152mg, 1.02mmol) were sequentially added to DMF (5mL), stirred at room temperature for 12h, C18 reaction Column purification (0.1% TFA) (H ₂ O:ACN=40:60) afforded 300mg 15g (yield=65.1%). LCMS m/z=899.8[M/3+1] ⁺ , 1349.5[M/2+1] ⁺

Step seven:

Add 15g (300mg, 0.11mmol) into the mixed solution (5mL) of piperidine:DMF=1:4, stir at room temperature for 2h, C18 reverse phase column purification (0.1%TFA) (H ₂ O:ACN=50:50 ), yielding 200 mg of 15h (yield = 72.5%).

LCMS m/z=825.8[M/3+1] ⁺ , 1238.1[M/2+1] ⁺

Step Eight:

Add 15h (200mg, 0.08mmol), glutaric anhydride (19mg, 0.16mmol) and N,N-diisopropylethylamine (21mg, 0.16mmol) into DMA (2mL), stir at room temperature for 18h, C18 reverse phase Preparative column purification (0.1% TFA) (H ₂ O:ACN=50:50) afforded 140 mg of 15i (yield=66.9%). LCMS m/z=863.8[M/3+1] ⁺ , 1295.3[M/2+1] ⁺

Step Nine:

15i (46mg, 0.017mmol) was added to DMA (1mL) and dichloromethane (0.3mL), under nitrogen protection, cooled to 0°C, N-hydroxysuccinimide (6mg, 0.051mmol) and EDCI ( 10 mg, 0.051 mmol), stirred at room temperature for 18 h, purified by C18 reverse phase preparative column (0.1% TFA) (H ₂ O:ACN=50:50), and obtained 40 mg of 15j (yield=83.8%).

LCMS m/z=896.3[M/3+1] ⁺ , 1343.6+1] ⁺

Step ten:

15j (37mg, 13μmol) and HSK-P35 (30mg, 10μmol) were added to DMA (1mL), then N,N-diisopropylethylamine (5mg, 30μmol) was added, and stirred at room temperature for 18h. Separation and purification with liquid phase preparative column (liquid phase preparation conditions: C18 reverse phase preparative column, mobile phase is deionized water (A) containing 0.1% trifluoroacetic acid, acetonitrile (B) containing 0.1% trifluoroacetic acid, gradient washing B content = 5% to 70%, elution time 15min, flow rate 12mL/min, column temperature: 30°C, retention time: 5.42min) to obtain 20 mg of compound 15 (yield = 47%). LCMS m/z＝924.00[M/6+1] ⁺ ,1108.50[M/5+1] ⁺ ,1385.60[M/4+1] ⁺ .

Example 16

first step:

The known compound SN38 (4.0g, 0.01mol) and di-tert-butyl dicarbonate (3.1g, 0.013mol) were added to dichloromethane (300mL) in turn, then pyridine (26.0g, 0.3mol) was added, and stirred at room temperature After 18h, it was concentrated to dryness to obtain 5.0 g of crude product 16a. (Yield = 99%). LCMS m/z=493.1[M+1] ⁺

Step two:

16a (2.0 g, 4 mmol), N,N-diisopropylethylamine (2.63 g, 20 mmol) and 4-dimethylaminopyridine (0.5 g, 4 mmol) were sequentially added to dry dichloromethane (40 mL) , under nitrogen protection, cooled to 0°C, slowly added dropwise a solution of triphosgene (0.52g, 1.72 mmol) in dichloromethane (10mL), after dropping, stirred at 0°C for 5 minutes, stirred at room temperature for 10 minutes, then added dropwise Boc-Val - A mixed solution of Cit-PABC (1.75g, 3.6mmol) in dimethylsulfoxide (10mL) and dichloromethane (10mL), after dropping, stirred for 2 hours, added water (100mL) and dichloromethane (50mL) for extraction , dried over anhydrous sodium sulfate, filtered, concentrated to dryness and purified by column chromatography (DCM:MeOH=20:1) to obtain 1.4 g of compound 16b. (Yield = 35%). LCMS m/z=998.4[M+1] ⁺

third step:

16b (1.26g, 0.126mmol) was added to a mixed solution of dichloromethane (13mL) and trifluoroacetic acid (13mL), stirred at room temperature for 30 minutes, and concentrated to dryness at 30°C to obtain 1.0g of crude product 16c. (Yield = 99%). LCMS m/z=798.4[M+1] ⁺

the fourth step:

Add 15f (150mg, 0.18mmol) into N,N-dimethylformamide (2mL), then add N,N-diisopropylethylamine (60mg, 0.46mmol), add Val-Cit-PABC dropwise -MMAE (200mg, 0.16mmol) in N,N-dimethylformamide solution (1mL), stirred at room temperature for 30 minutes, then added N,N-diisopropylethylamine (60mg, 0.46mmol), added dropwise 18c (165mg, 0.18mmol) in N,N-dimethylformamide solution (1mL), stirred at room temperature for 2h, purified by C18 reverse-phase preparative column (0.1%TFA) (H ₂ O:ACN=35:65), and 80mg 16d (Yield = 23.2%). LCMS m/z＝1186.5[M/2+1] ⁺

the fifth step:

Add 16d (80 mg, 0.034 mmol) into 20% piperidine in N,N-dimethylformamide (1 mL), stir at room temperature for 1 minute, and purify by C18 reverse-phase preparative column (0.1% TFA) (H ₂ O :ACN=50:50), yielding 40 mg of 16e (yield=55.5%).

LCMS m/z＝1075.3[M/2+1] ⁺

Step six:

16e (40mg, 0.018mmol) and glutaric anhydride (2.4mg, 0.018mmol) were added to N,N-dimethylacetamide (1mL) solution, then N,N-diisopropylethylamine (5mg , 0.038 mmol), stirred at room temperature for 2 hours, purified by C18 reverse phase preparative column (0.1% TFA) (H ₂ O:ACN=50:50), and obtained 40 mg of 16f (yield=95%). LCMS m/z=1132.7[M/2+1] ⁺

Step seven:

16f (40 mg, 0.018 mmol), N-hydroxysuccinimide (6 mg, 0.054 mmol) and EDCI (11 mg, 0.054 mmol) were sequentially added to N,N-dimethylacetamide (1 mL) and dichloromethane (0.3mL) solution, under nitrogen protection, stirred at room temperature for 18 hours, concentrated at 20°C to remove dichloromethane, and purified by C18 reverse-phase preparative column (0.1% TFA) (H ₂ O:ACN=45:55), to obtain 40mg 16g ( Yield = 96%). LCMS m/z＝1181.1[M/2+1] ⁺

Step Eight:

Add 16g (20mg, 8.3μmol) and HSK-P35 (19mg, 6.4μmol) to DMA (1mL) sequentially, then add N,N-diisopropylethylamine (3mg, 18μmol), and stir at room temperature for 18h. Separation and purification with liquid phase preparative column (liquid phase preparation conditions: C18 reverse phase preparative column, mobile phase is deionized water (A) containing 0.1% trifluoroacetic acid, acetonitrile (B) containing 0.1% trifluoroacetic acid, gradient washing B content = 5% to 70%, elution time 15min, flow rate 12mL/min, column temperature: 30°C, retention time: 5.46min) to obtain 1.5mg of compound 16 (yield = 5%). LCMS m/z＝1043.7[M/5+1] ⁺ ,1304.2[M/4+1] ⁺ .

biological test case

1. NCI-H292 cell proliferation inhibition experiment

H292 cell culture conditions: RPMI-1640+10% FBS+1% double antibody, cultured at 37°C, 5% CO ₂ incubator. On the first day, the H292 cells in the exponential growth phase were collected and plated on 96-well culture plates, 90 μL per well, with a plating density of 500 cells/well, and cultured overnight at 37°C in a 5% CO ₂ incubator. On the second day, 10 μL of different concentrations of compounds were added to each well so that the final concentration of DMSO in each well was 0.1%, and cultured at 37° C. in a 5% CO ₂ incubator for 6 days. After the incubation, 50 μL of detection solution (Cell Viability Assay, Promega, G7573) was added to each well, mixed for 2 minutes, incubated at room temperature for 10 minutes, and detected by a microplate reader for chemiluminescence readings. The results were processed according to Inhibition%=(1-(RLU compound/RLU control)×100%, and the proliferation inhibition rate of each concentration of the compound was calculated, and the IC50 value of the concentration of the compound was calculated when the inhibition rate was 50% using origin9.2 software. Wherein RLU compound is the reading of drug treatment group, and RLU control is the mean value of solvent control group.Compound of the present invention is less than 100nM to the IC of NCI-H292 cell, and part is less than 50nM, more excellent less than 20nM, the results are shown in Table 4.

Table 4

compound	IC 50 (nM)	Max inh.%10μM
Compound 1	17	91.7
Compound 2	17	92.3
Compound 3	40	93.4
Compound 4	17	92.7
Compound 5	39	93.9
Compound 6	16	93.8
Compound 7	16	94.2
Compound 8	38	92.6
Compound 9	37	92.7
Compound 10	twenty three	94.7
Compound 11	15	96.5
Compound 12	30	95.7
Compound 13	12	95.8
Compound 14	38	90.5
Compound 16	8	100

Conclusion: the compound of the present invention has better inhibitory activity on NCI-H292 cells.

2. Rat pharmacokinetic test

2.1 Test animals: male SD rats, about 220 g, 6-8 weeks old, 3 rats/compound. purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

2.2 Experimental design: On the day of the experiment, 6 SD rats were randomly divided into groups according to body weight. One day before the administration, fasting without water for 12-14 hours, and giving food 4 hours after the administration. See Table 5 for dosing information.

Table 5. Dosing Information

Note: 5% DMA+95% (20% SBE-CD)

Before and after administration, 0.15 ml of blood was collected through the orbit under isoflurane anesthesia, placed in an EDTAK2 centrifuge tube, centrifuged at 5000 rpm, 4°C for 10 min, and plasma was collected. The time points of blood collection in the intravenous group and intragastric administration group were both: 0, 5, 15, 30min, 1, 2, 4, 6, 8, 24h. Before analysis and detection, all samples were stored at -80°C, and the samples were quantitatively analyzed by LC-MS/MS. The results are shown in Table 6.

Table 6. Pharmacokinetic parameters of test compounds in rat plasma

-:not applicable. NC: cannot be calculated

Conclusion: Compound 3 and Compound 5 have good pharmacokinetic characteristics in rats. The compound of the present invention is also observed to have excellent in vivo pharmacokinetic characteristics and pharmacodynamic characteristics in animal experiments of other species.

3. Mouse NCI-H292 cell xenograft model in vivo

Human lung cancer NCI-H292 cells were placed in RPMI-1640 medium (supplemented with 10% fetal calf serum and 1% double antibody), and cultured at 37°C. Routine digestion with trypsin was performed twice a week for passaging. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected, counted and inoculated. 0.1 mL (5×10 ⁶ ) of NCI-H292 cells were subcutaneously inoculated into the right back of female Balb/c nude mice (from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and the average tumor volume reached about 80- 120mm ³ began to group administration (recorded as Day 0). The vehicle group was given 5% dimethylacetamide, 5% Solutol HS-15 and 90% normal saline solution, and the administration group was intravenously given 3 mg/kg of compound 1, compound 2, compound 4 or compound 7, and the administration frequency was every Once a week, the administration cycle is 36 days (Day 0-Day 35) or 44 days (Day 0-Day 43). After grouping, the tumor diameter was measured twice a week with a vernier caliper. The formula for calculating the tumor volume was: V=0.5×a×b ² , where a and b represent the long and short diameters of the tumors, respectively. TGI (%)=[1-(average tumor volume at the end of administration of a certain treatment group-average tumor volume at the beginning of administration of this treatment group)/(average tumor volume at the end of treatment of solvent control group-solvent) for the antitumor efficacy of the compound The average tumor volume at the beginning of treatment in the control group)]×100% was evaluated. Tumor growth curves and animal body weight change curves are shown in Figure 1, Figure 2, Figure 3 and Figure 4, respectively.

Test results: after administration once a week for 36 days or 44 days, the TGI of compound 1, compound 2, compound 4 and compound 7 groups were 104%, 103%, 97% and 92% respectively; the body weight of the vehicle group decreased significantly, However, the body weight of animals in all administration groups showed an upward trend.

Conclusion: Compound 1, Compound 2, Compound 4 and Compound 7 of the present invention have good tumor growth inhibitory efficacy and good tolerance in the mouse NCI-H292 subcutaneous and in vivo xenograft model.

4. MDA-MB-468 and NCI-H322 cell proliferation inhibition experiment

Human breast cancer MDA-MB-468 cells (derived from ATCC) and human lung cancer NCI-H322 cells (derived from ECACC) were placed in L-15 medium (supplemented with 10% fetal bovine serum) and RPMI-1640 medium (supplemented with 10% fetal bovine serum and 2 mM glutamine), cultured at 37°C, 5% CO ₂ . Cells in the exponential growth phase were collected, and the cell suspension was adjusted to 500 cells/135 μL and 1000 cells/135 μL with medium. Add 135 μL of cell suspension to each well in a 96-well cell culture plate and incubate overnight. On the second day, different concentrations of compounds were added and cultured in an incubator for 6 days. After the incubation, according to the operating instructions of the CellTiter-Glo kit (Promega, Cat#G7573), add 75 μL of CTG solution that has been melted and equilibrated to room temperature in advance, mix well with a microplate shaker for 2 minutes, and place at room temperature for 10 minutes Chemiluminescence readings were then determined with an Envision 2104 plate reader (PerkinElmer). The cell proliferation inhibition rate was calculated according to the formula [(1–(RLUcompound–RLUblank)/(RLUcontrol–RLU blank))×100%]. IC50 values were obtained by four-parameter nonlinear fitting using GraphPad Prism software.

Conclusion: the compound of the present invention has a good inhibitory effect on MDA-MB-468 and NCI-H322 cells, and its IC50 value is less than 100nM.

5. NCI-H526 cell proliferation inhibition experiment

Human lung cancer NCI-H526 cells purchased from ATCC were placed in RPMI-1640 medium (supplemented with 10% fetal bovine serum and 1% double antibody) and cultured at 37°C and 5% CO ₂ . Cells in the exponential growth phase were collected, and the cell suspension was adjusted to 5000 cells/135 μL with medium. Add 135 μL of cell suspension to each well in a 96-well cell culture plate and incubate overnight. On the second day, different concentrations of compounds were added and cultured in an incubator for 6 days. After the incubation, according to the operating instructions of the CellTiter-Glo kit (Promega, Cat#G7573), add 75 μL of CTG solution that has been melted and equilibrated to room temperature in advance, mix well with a microplate shaker for 2 minutes, and place at room temperature for 10 minutes Chemiluminescence readings were then determined with an Envision 2104 plate reader (PerkinElmer). The cell proliferation inhibition rate was calculated according to the formula [(1–(RLUcompound–RLUblank)/(RLUcontrol–RLU blank))×100%]. IC50 values were obtained by four-parameter nonlinear fitting using GraphPad Prism software.

Conclusion: the compound of the present invention has weak inhibitory effect on NCI-H526 cells.

Claims (25)

1. A compound comprising a polypeptide structure and a non-aromatic molecular scaffold, the polypeptide structure comprising at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and the Cys or Hcys residues and The non-aromatic molecular scaffold forms a covalent bond, thereby forming at least two polypeptide rings on the molecular scaffold; the condition is that at least one Hcys residue is contained in the at least three residues selected from Cys and Hcys.
2. The compound according to claim 1, wherein said polypeptide structure comprises the following amino acid sequence:

   -Xa1-A1-Xa2-A2-Xa3-

   Wherein, A1 and A2 represent the amino acid sequences between Xa1, Xa2 and Xa3, and A1 and A2 each independently contain 3-10 amino acid residues;

   Xa1, Xa2, and Xa3 are independently Cys or Hcys residues, and at least one of them is an Hcys residue, and the non-aromatic molecular scaffold forms a thioether bond with Xa1, Xa2, and Xa3 of the polypeptide respectively, thereby forming a thioether bond in the molecule Two polypeptide loops are formed on the scaffold.
3. The compound according to claim 2, wherein one of said A1 and A2 consists of 3 amino acid residues, and the other consists of 9 amino acid residues.
4. The compound according to any one of claims 1-3, wherein the polypeptide structure comprises the following amino acid sequence:

   -Xa1-P(1Nal)(D-Asp)-Xa2-M(HArg)DWSTP(Hyp)W-Xa3-, said Xa1, Xa2, Xa3 are respectively selected from Cys or Hcys; the condition is Xa1, Xa2, Xa3 At least one selected from Hcys;

   Specifically, wherein the polypeptide structure comprises the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 7:

   -Hcys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO:1);

   -Cys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO:2);

   -Cys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:3);

   -Hcys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:4);

   -Cys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO:5);

   -Hcys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO:6);

   -Hcys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO:7).
5. The compound according to claim 4, wherein the polypeptide structure comprises the amino acid sequence shown in any one of SEQ ID NO:8～SEQ ID NO:14:

   -(β-Ala)-Sar ₁₀ -Hcys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO:8);

   -(β-Ala)-Sar ₁₀ -Cys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO:9);

   -(β-Ala)-Sar ₁₀ -Cys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO: 10);

   -(β-Ala)-Sar ₁₀ -Hcys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Cys-(SEQ ID NO: 11);

   -(β-Ala)-Sar ₁₀ -Cys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO: 12);

   -(β-Ala)-Sar ₁₀ -Hcys-P(1Nal)(D-Asp)-Cys-M(HArg)DWSTP(Hyp)W-Hcys-(SEQ ID NO: 13);

   -(β-Ala)-Sar ₁₀ -Hcys-P(1Nal)(D-Asp)-Hcys-M(HArg)DWSTP(Hyp)W-Cys- (SEQ ID NO: 14).
6. The compound according to claim 4, which is a bicyclic peptide containing one of the following structures:

   

   Wherein, when Xa1, Xa2 or Xa3 is Cys, correspondingly, m1, m2 or m3 is 1;

   When Xa1, Xa2 or Xa3 is Hcys, correspondingly, m1, m2 or m3 is 2;

   n1, n2, and n3 are 1 or 2 independently.
7. The compound according to claim 6, which is a bicyclic peptide having one of the following structures:

   

   Wherein, n4 is selected from any integer of 0-10.
8. The compound according to claim 7, wherein,

   Xa1 is Hcys, m1 is 2, n1 is 1 or 2, n4 is 9; or

   Xa2 is Hcys, m2 is 2, n2 is 1 or 2, n4 is 9; or

   Xa3 is Hcys, m3 is 2, n3 is 1 or 2, and n4 is 9.
9. The compound according to claim 1, which is selected from compounds of structures shown in Table 1.
10. Application of the compound described in any one of claims 1 to 9 as a ligand of Nectin-4 in screening and/or preparing drugs.
11. A drug conjugate represented by formula II or a pharmaceutically acceptable salt thereof,

    

    The cytotoxic agent is selected from one or more of the following groups: alkylating agents, anti-metabolites, plant alkaloids, terpenoids, podophyllotoxins and derivatives thereof, taxanes And its derivatives, topoisomerase inhibitors, antitumor antibiotics;

    The linker is a divalent, trivalent, tetravalent or pentavalent spacer moiety linking the cytotoxic agent moiety and the peptide ligand moiety;

    The peptide ligand is a compound according to any one of claims 1-9.
12. The drug conjugate or a pharmaceutically acceptable salt thereof according to claim 11, wherein the cytotoxic agent is selected from one or more of the following group: cisplatin, carboplatin, oxaliplatin, nitrogen Mustard, cyclophosphamide, chlorambucil, ifosfamide, azathioprine, mercaptopurine, pyrimidine analogs, vincristine, vinblastine, vinorelbine, vindesine, etoposide, tinib Poside, paclitaxel, camptothecin and its derivatives, irinotecan, topotecan, amyridine, etoposide, etoposide phosphate, teniposide, actinomycin D, doxorubicin, Epirubicin, epothilone and its derivatives, bleomycin and its derivatives, dactinomycin and its derivatives, plicamycin and its derivatives, mitomycin C, calicheamicin , maytansine and its derivatives, auristatin and its derivatives.
13. According to the drug conjugate or pharmaceutically acceptable salt thereof according to claim 11 or 12, the cytotoxic agent is selected from maytansinoids, monomethyl auristatin or camptothecin derivatives; or, the The cytotoxic agent is selected from maytansine DM1, monomethyl auristatin E or 7-ethyl-10-hydroxycamptothecin.
14. The drug conjugate or the pharmaceutically acceptable salt thereof according to claim 11, wherein the linker is a peptide linker, a disulfide linker or a pH-dependent linker.
15. The drug conjugate or a pharmaceutically acceptable salt thereof according to claim 14, wherein:

    The disulfide linker is selected from DMDS, MDS, DSDM, NDMDS or the structure of formula III:

    

    Wherein, R ₁ , R ₂ , R ₃ and R ₄ are independently selected from H, methyl, ethyl, propyl and isopropyl;

    p and q are independently 1, 2, 3, 4 or 5;

    The peptide linker is selected from: -Cit-Val-, -Phe-Lys- and -Val-Lys-;

    The pH-dependent linker is selected from cis-aconitic anhydride.
16. The drug conjugate or a pharmaceutically acceptable salt thereof according to claim 14, wherein the linker is -PABC-Cit-Val-glutaryl-, -PABC-cyclobutyl-Ala-Cit-βAla -or

    

    Wherein PABC represents p-aminobenzyl carbamate, and k is selected from any integer of 1-20.
17. The drug conjugate or a pharmaceutically acceptable salt thereof according to claim 16, wherein the linker is

    

    Wherein k is selected from any integer of 1-20.
18. The drug conjugate or a pharmaceutically acceptable salt thereof according to claim 11, wherein the drug conjugate has the structure of formula III-1, formula III-2, III-3 or III-4:

    

    

    Wherein, Xa1, Xa2, and Xa3 are independently Cys or Hcys residues, and at least one of them is a Hcys residue;

    When Xa1, Xa2 or Xa3 is Cys, correspondingly, m1, m2 or m3 is 1;

    When Xa1, Xa2 or Xa3 is Hcys, correspondingly, m1, m2 or m3 is 2;

    n1, n2, n3 are independently 1 or 2;

    n4 is selected from any integer of 0-10;

    k is selected from any integer of 1-10.
19. The drug conjugate or a pharmaceutically acceptable salt thereof according to claim 18, wherein:

    Xa1 is Hcys, m1 is 2, n1 is 1 or 2, n4 is 9; or

    Xa2 is Hcys, m2 is 2, n2 is 1 or 2, n4 is 9; or

    Xa3 is Hcys, m3 is 2, n3 is 1 or 2, and n4 is 9.
20. The drug conjugate or a pharmaceutically acceptable salt thereof according to claim 11, wherein the drug conjugate is selected from one of the following structures:

    

    

    

    
21. A pharmaceutical composition comprising the compound of any one of claims 1 to 9 and/or the drug conjugate of any one of claims 11 to 20 or a pharmaceutically acceptable salt thereof, and Pharmaceutically acceptable carrier and/or excipient.
22. The compound according to any one of claims 1 to 9, or the drug conjugate according to any one of claims 11 to 20 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 21, Application in preparation of medicaments for preventing and/or treating diseases or conditions in which Nectin-4 is overexpressed in diseased tissues of tumor individuals.
23. The application according to claim 22, wherein the disease or disease overexpressing Nectin-4 is urothelial cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, triple negative breast cancer and bladder cancer. at least one of the cancers.
24. A pharmaceutical composition or pharmaceutical preparation, said pharmaceutical composition or pharmaceutical preparation comprising 1-1500 mg of the compound described in any one of claims 1 to 9 or the conjugate described in any one of claims 11 to 20 compounds or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers and/or excipients.
25. A method for treating a disease in a mammal or a human, the method comprising administering to a subject a therapeutically effective amount of the compound of any one of claims 1 to 9 or any one of claims 11 to 20 The therapeutically effective dose of the drug conjugate or a pharmaceutically acceptable salt thereof is preferably 1-1500 mg, and the disease is preferably a tumor.

Images