CN118201627A - Nectin-4 bicyclic peptide ligands and uses thereof - Google Patents

Abstract

A bicyclic peptide ligand of Nectin-4 and its use. In particular to a compound which is a peptide ligand with high affinity to a cell adhesion molecule (Nectin-4), a drug conjugate, a stereoisomer, a pharmaceutically acceptable salt, a solvate, a eutectic crystal or a deuteride thereof or a pharmaceutical composition containing the same, and the application of the peptide ligand and the drug conjugate in preventing and/or treating diseases or symptoms of over-expressing Nectin-4 in diseased tissues of tumor patients.

Description

Bicyclic peptide ligand of nectin-4 and its use Technical Field

The present invention relates to a compound comprising a polypeptide structure having high affinity for a cell adhesion molecule (Nectin-4) and a polypeptide drug conjugate thereof conjugated to one or more toxin molecules, and related uses of the compound and the drug conjugate, including the use in preparing a pharmaceutical composition and the use in preventing and/or treating a disease or condition in which Nectin-4 is overexpressed in diseased tissues of tumor individuals.

Background technique

Nectin is a cell adhesion molecule belonging to the Ca ²⁺ -independent immunoglobulin (Ig) superfamily. It is a homolog of the poliovirus receptor (PVR/CD155) and is also known as the poliovirus receptor-related protein (PVRL). The 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 that lack calcium adhesion glycoproteins, such as B cells, monocytes and sperm cells. However, human Nectin-4 is mainly expressed in embryonic development and tumor tissues, and is not expressed in normal adult tissues.

Nectin-4 is a cell membrane surface receptor that can affect tight junctions between cells, participate in cell-to-cell adhesion, and then regulate cell movement, differentiation, and virus invasion. In recent years, the relationship between Nectin-4 and tumors has received close attention from 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 for 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, it has great clinical potential and provides great possibilities for the development of new therapeutic drugs (such as nucleic acid drugs and monoclonal antibodies). There is a significant correlation between the expression of Nectin-4 and the progression and prognosis of patients with various cancers. Its expression is limited in healthy tissues of adults, but it is overexpressed in malignant tumor cells. Therefore, using Nectin-4 as a therapeutic target may become an effective strategy for treating cancers with high Nectin-4 expression.

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

Summary of the invention

The present invention provides a compound having high affinity for a cell adhesion molecule (Nectin-4), which comprises a polypeptide structure, a polypeptide drug conjugate in which the compound is conjugated to one or more toxin molecules, a pharmaceutical composition, and related uses thereof 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, which comprises a polypeptide structure and a non-aromatic molecular scaffold, wherein the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and the Cys or Hcys residues form a covalent bond with the non-aromatic molecular scaffold, thereby forming at least two polypeptide rings on the molecular scaffold; provided that at least one Hcys residue is contained among the Cys and Hcys residues.

In some specific embodiments, the compound comprises a polypeptide structure and a non-aromatic molecular scaffold, wherein the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and two of them 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 rings on the molecular scaffold.

In some specific embodiments, the compound comprises a polypeptide structure and a non-aromatic molecular scaffold, wherein the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and one of them 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 rings 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, wherein the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and two of them 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 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, wherein the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and one of them 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 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, wherein the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and two of them 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 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, wherein the polypeptide structure comprises at least three residues selected from Cys and Hcys separated by at least two amino acid sequences, and one of them 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 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 and 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 and A2 are optionally 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 a Hcys residue. The non-aromatic molecular scaffold forms thioether bonds with Xa1, Xa2, and Xa3 of the polypeptide, respectively, thereby forming two polypeptide rings on the molecular scaffold.

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

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

-Xa1-P(1Nal)(D-Asp)-Xa2-M(HArg)DWSTP(Hyp)W-Xa3-, wherein Xa1, Xa2, and Xa3 are selected from Cys or Hcys respectively; provided that at least one of Xa1, Xa2, and Xa3 is selected from Hcys.

In some specific embodiments, the polypeptide structure comprises an 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).

In some specific embodiments, the polypeptide structure comprises an amino acid sequence shown in any one of SEQ ID NO: 8 to 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, 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 structure:

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 independently 1 or 2.

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), n4 is any integer from 0 to 10, such as 3, 4, 5, 6, 7, 8, 9, 10, and 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 of the present invention comprising at least two polypeptide ring structures has protein stability and is stable to plasma proteases, epithelial proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc.; has specific targeting to Nectin-4 and is a peptide ligand specific to Nectin-4; has a long plasma half-life and has good pharmacokinetic and pharmacodynamic properties.

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

The present invention also provides a drug conjugate or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises the aforementioned compound conjugated to one or more effectors and/or functional groups via a linker.

In some embodiments, the effector is a cytotoxic agent.

In some specific embodiments, the present invention provides a drug conjugate of Formula II or a pharmaceutically acceptable salt thereof,

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

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

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

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

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

In some embodiments, the linker is a tetravalent spacer moiety that connects the cytotoxic agent moiety and the peptide ligand moiety; wherein the peptide ligand is independently connected to three cytotoxic agent moieties via the linker.

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

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

In some specific embodiments, the cytotoxic agent is selected from the following group: cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide, azathioprine, mercaptopurine, pyrimidine analogs, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, paclitaxel, camptothecin and its derivatives, irinotecan, topotecan, acridine, 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.

In some embodiments, the cytotoxic agent is selected from maytansinoids, monomethyl auristatins or camptothecin derivatives. In some embodiments, the cytotoxic agent is selected from maytansinoids or monomethyl auristatins. In some embodiments, the cytotoxic agent is selected from maytansine DM1, monomethyl auristatin 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, 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 structure 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 linker mainly serves to connect the cytotoxic agent and the peptide ligand, and to cleave and release the toxic substance under specific conditions. In order to control the rate of cleavage and the accompanying release of effector molecules, the linker can be appropriately modified, such as connecting some groups to the connection with the peptide ligand or effector to increase the chain length, and adding groups around the cleavage bond to control the obstruction of the cleavage bond. The linker of the present invention includes linker derivatives modified based on the above.

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

In some specific embodiments, 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.

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

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, the drug conjugate has a 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 any integer selected from 0 to 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 compound described above in the present invention (i.e., peptide ligand, or peptide compound) and/or the drug conjugate described above in the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or excipient.

The present invention also relates to a use of the compound described in the present invention, or the drug conjugate described in the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a drug for preventing and/or treating a disease or condition in which nectin-4 is overexpressed in diseased tissues of a tumor individual. Preferably, the individual is a mammal or a human.

Typically, the disease in which nectin-4 is overexpressed 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 adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas and other carcinomas), such as bladder and urinary tract cancer, breast cancer, gastrointestinal cancer (including esophageal cancer, stomach (gastric) cancer, small intestine cancer, colon cancer, rectal cancer and anal cancer), liver cancer (hepatocellular carcinoma), gallbladder and biliary system cancer, exocrine pancreatic cancer, kidney cancer, lung cancer (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma and mesothelioma), head and neck cancer (e.g., tongue cancer, buccal cancer, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, tonsil cancer, salivary gland cancer, nasal cancer and paranasal sinus cancer), ovarian cancer, fallopian tube cancer, peritoneal cancer, Vaginal cancer, vulvar cancer, penile cancer, cervical cancer, uterine fibroids, endometrial cancer, thyroid cancer (e.g., follicular thyroid cancer), renal cancer, prostate cancer, skin and appendage cancer (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevus); hematological malignancies (i.e., leukemias, lymphomas) and premalignant hematological conditions and borderline malignant conditions, including hematological malignancies and related conditions 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, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myeloproliferative disorders, and myelofibrosis; hematologic malignancies and myeloid-related disorders (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, 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, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi sarcoma, and ulcerative colitis); due to sarcomas, synovial sarcomas, epithelioid sarcomas, gastrointestinal stromal tumors, benign and malignant histiosarcomas, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pinealomas, and schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoids, and medullary thyroid carcinomas); eye and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma); pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumors); or congenital or other forms of syndromes that predispose the patient to malignancy (e.g., xeroderma pigmentosum).

Furthermore, the disease or condition in which nectin-4 is overexpressed is at least one of urothelial carcinoma, 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, which comprises 1-1500 mg of the aforementioned compound of the present invention or the aforementioned conjugate of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or excipient.

The present invention also relates to 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 aforementioned compound of the present invention or the aforementioned conjugate of the present invention or a pharmaceutically acceptable salt thereof, the therapeutically effective amount is preferably 1-1500 mg, and the disease is preferably a 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 above schemes, and a pharmaceutically acceptable carrier and/or excipient. The pharmaceutical composition can be in the form of a unit preparation (unit preparation is also referred to as a "preparation specification").

Furthermore, the composition or pharmaceutical preparation of the present invention contains 1-1500 mg of the peptide compound, conjugate or pharmaceutically acceptable salt thereof according to any one of the aforementioned schemes, and a pharmaceutically acceptable carrier and/or excipient.

The present invention also provides the use of the peptide compound, conjugate or pharmaceutically acceptable salt thereof described in any of the above schemes in the preparation of a drug for preventing and/or treating a disease or condition in which Nectin-4 is overexpressed in diseased tissues of a tumor individual. Further, the disease or condition in which Nectin-4 is overexpressed is preferably at least one of urothelial carcinoma, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, triple-negative breast cancer and bladder cancer. Preferably, the individual is a mammal or a human.

The present invention also 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, a conjugate or a pharmaceutically acceptable salt thereof as described in any one of the aforementioned schemes, wherein the disease is preferably at least one of urothelial carcinoma, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, triple-negative breast cancer and bladder cancer, and the therapeutically effective amount is preferably 1-1500 mg.

The "effective amount" or "therapeutically effective amount" described in this application refers to the administration of a sufficient amount of the compound disclosed in this application, which will alleviate one or more symptoms of the disease or condition being treated to some extent. In some embodiments, the result is a reduction and/or alleviation of the signs, symptoms or causes of the disease, or any other desired change in the biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a peptide compound, conjugate, or a pharmaceutically acceptable salt thereof disclosed in this application that is 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, 1-400 mg, 1-300 mg, 1-250 mg, 1-200 mg, 1-150 mg, 1-125 mg, 1-100 mg, 1-80 mg, 1-60 mg, 1-50 mg, 1-40 mg, 1-25 mg, 1-20 mg, 5-1500 mg, 5-1000 mg, 5-900 mg, 5-800 mg, 5-700 mg, 5-600 mg, 5-500 5-100mg, 5-200mg, 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, 20-4 0mg, 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 formulation of the present invention contains the above-mentioned 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 according to the present invention. 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 a "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, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 125 mg, 130 mg , 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, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg of a peptide compound, a conjugate of the present invention, or a pharmaceutically acceptable salt thereof.

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, a conjugate or a pharmaceutically acceptable salt thereof of the present invention, and a pharmaceutically acceptable carrier and/or excipient, the therapeutically effective amount being preferably 1-1500 mg, and the disease being preferably at least one of urothelial carcinoma, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, 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 a peptide compound, a conjugate or a pharmaceutically acceptable salt thereof of the present invention, and a pharmaceutically acceptable carrier and/or excipient to a subject at a daily dose of 1-1500 mg/day, the daily dose may be a single dose or divided doses. In some embodiments, the daily dose includes but is not limited to 10-1500 mg/day, 20-1500 mg/day, 25-1500 mg/day, 50-1500 mg/day, 75-1500 mg/day, 100-1500 mg/day, 200-1500 mg/day, 10-1000 mg/day, 20-1000 mg/day, 25-1000 mg/day, 50-1000 mg/day, 75-1000 mg/day. In some embodiments, the daily dose includes but is not limited to 1 mg/day, 5 mg/day, 10 mg/day, 20 mg/day, 25 mg/day, 50 mg/day, 75 mg/day, 100 mg/day, 125 mg/day, 150 mg/day, 200 mg/day, 300 mg/day, 400 mg/day, 600 mg/day, 800 mg/day, 1000 mg/day, 1200 mg/day, 1400 mg/day, 1500 mg/day.

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

The amount of the compound, conjugate or pharmaceutically acceptable salt stated in the present invention is in each case calculated as the free base.

"Preparation specifications" refers to the weight of the main drug contained in each vial, 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, and the starting materials are commercially available chemicals and/or compounds described in chemical literature. "Commercially available chemicals" are obtained from regular commercial sources, and suppliers include: Titan Technology, Anage Chemical, Shanghai Demo, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, Nanjing Yaoshi, WuXi AppTec and Bailingwei Technology.

The peptide structure of the present invention can be prepared by standard technology synthesis, and then react with molecular scaffold in vitro. When doing this, standard chemical methods can be used. This enables rapid large-scale preparation of soluble materials for further downstream experiments or verification. Such methods can be realized using conventional chemical methods 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, see the Examples.

the term

Unless otherwise specified in the present invention, the terms of the present invention have the following meanings:

Peptide ligand refers to a compound containing an amino acid sequence (peptide structure) formed by the covalent binding of a peptide to a molecular scaffold, which can be used as a ligand of a cell adhesion molecule (Nectin-4) in the present invention. Generally, the peptide forming such a compound comprises two or more reactive groups (i.e., cysteine residues and/or homocysteine residues) capable of forming a covalent bond (such as a thioether bond) with the scaffold, and a sequence (also referred to as a ring sequence in the present invention) relative to the reactive groups, which is referred to as a ring sequence in the present invention because when the peptide is bound to the scaffold, it forms a ring. In the case 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 rings on the scaffold.

Molecular scaffolds include non-aromatic molecular scaffolds. Non-aromatic molecular scaffolds refer to any molecular scaffold defined herein that does not contain an aromatic carbocyclic ring or an aromatic heterocyclic ring system. Examples of suitable non-fragrant 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. In some cases, molecular scaffolds are macromolecules composed of amino acids, nucleotides or carbohydrates. In some cases, molecular scaffolds contain reactive groups that can react with the functional groups of polypeptides to form covalent bonds. Molecular scaffolds can include chemical groups that form connections with peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, olefins, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl 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., the non-aromatic skeleton in WO2018197893 can also be selected.

A polypeptide is a compound formed by three or more amino acid molecules linked together by peptide bonds. The amino acid unit in a polypeptide is called an amino acid residue.

Effectors and/or functional groups are molecules or fragments that can be connected (via a linker) to, for example, the N and/or C termini of a polypeptide, an amino acid within a polypeptide, or a molecular backbone, and have a pharmacological effect or a specific function. Suitable effectors and/or functional groups include antibodies and parts or fragments thereof, cytotoxic molecules or fragments, enzyme inhibitor molecules or fragments, metal chelators, etc. In some cases, the effector and/or functional group is a drug, particularly a cytotoxic agent.

A derivative is a product derived from a compound in which the hydrogen atoms or atomic groups in the compound are replaced by other atoms or atomic groups.

Unless otherwise specified, all amino acids were used in the L-configuration.

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

Table 2

Amino acid name Three (or more) letter abbreviations Single letter abbreviation Amino acid name Three (or more) letter abbreviations Single letter abbreviation Cysteine Cys C Naphthylalanine 1Nal / Homocysteine Hcy / Aspartic acid Asp D Hydroxyproline Hyp / D-Aspartic Acid D-Asp / Proline Pro P Tryptophan Trp W Alanine Ala A Homoarginine HArg / β-Alanine β-Ala, bAla / Serine Ser S Creatine 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 that are thiol-containing derivatives of maytansine. DM1 has the following structure:

Monomethyl auristatin E (MMAE) is a synthetic antitumor 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 described in the present invention include their isotopes, and the carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds described in the present invention are optionally further replaced by one or more of their corresponding isotopes, wherein carbon isotopes include ¹² C, ¹³ C and ¹⁴ C, hydrogen isotopes include protium (H), deuterium (deuterium, also known as heavy hydrogen), tritium (T, also known as super tritium), oxygen isotopes include ¹⁶ O, ¹⁷ O and ¹⁸ O, sulfur isotopes include ³² S, ³³ S, ³⁴ S and ³⁶ S, nitrogen isotopes include ¹⁴ N and ¹⁵ N, fluorine isotopes include ¹⁹ F, chlorine isotopes include ³⁵ Cl and ³⁷ Cl, and bromine isotopes include ⁷⁹ Br and ⁸¹ Br.

"Pharmaceutically acceptable salt" refers to a salt of the compound of the present invention which retains the biological effectiveness and properties of the free acid or free base, and the free acid is obtained by reacting with a non-toxic inorganic base or organic base, or the free base is obtained by reacting with a non-toxic inorganic acid or organic acid.

A "pharmaceutical composition" refers to a mixture of one or more compounds described herein, or stereoisomers, solvates, pharmaceutically acceptable salts or cocrystals thereof, with other ingredients, wherein the other ingredients include physiologically/pharmaceutically acceptable carriers and/or excipients.

"Carrier" refers to a system that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered compound, and can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug and deliver the drug to the targeted organ. Non-limiting examples include microcapsules and microspheres, nanoparticles, liposomes, etc.

"Excipient" refers to a substance that is not a therapeutic agent in itself but is used as a diluent, adjuvant, binder and/or vehicle that is added to a pharmaceutical composition to improve its handling or storage properties or to allow or facilitate the formation of a compound or pharmaceutical composition into a unit dosage form for administration. As known to those skilled in the art, pharmaceutical excipients can serve a variety of functions and can be described as wetting agents, buffers, suspending agents, lubricants, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavoring agents and sweeteners. Examples of pharmaceutical 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 sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, hydroxypropyl methylcellulose, hydroxypropyl cellulose, microcrystalline cellulose and cross-linked carboxymethylcellulose (e.g., cross-linked sodium carboxymethylcellulose); (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (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 solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical preparations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a tumor growth curve of the mouse NCI-H292 subcutaneous in vivo transplanted tumor model.

FIG. 2 is a curve showing changes in animal body weight in the mouse NCI-H292 subcutaneous in vivo transplanted tumor model.

FIG. 3 is a tumor growth curve of the mouse NCI-H292 subcutaneous in vivo transplanted tumor model.

FIG. 4 is a curve showing changes in animal body weight in the mouse NCI-H292 subcutaneous in vivo transplanted tumor model.

Detailed ways

The content of the present invention will be described in detail below through examples. If no specific conditions are specified in the examples, the experimental method according to conventional conditions is carried out. The examples are given to better illustrate the content of the present invention, but it should not be understood that the content of the present invention is limited to the examples. Those skilled in the art can make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still belongs to the protection scope of the present invention.

Preparation of compounds

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), and N,N-diisopropylethylamine (58.4g, 453mmol, 6.0eq) was added, and the reaction was continued for 3 hours. The mixture was filtered, and the resin was washed twice with DMF. A 20% piperidine/DMF solution was added to the resin mixture, and the system was reacted for 2 hours, filtered, and the resin was washed twice with DMF. DMF (600 mL), Boc-L-valine (48.0 g, 0.22 mmol, 3.0 eq), HBTU (85.0 g, 0.21 mmol, 2.85 eq), and N,N-diisopropylethylamine (58.4 g, 453 mmol, 6.0 eq) were added to the resin mixture in sequence. The system was reacted for 3 hours, filtered, and the resin was washed three times with DMF, three times with methanol, and twice with dichloromethane. The prepared 20% hexafluoroisopropanol/dichloromethane solution was added to the resin mixture, the system was reacted for 30 minutes, filtered, and the previous operation was repeated once. The mother liquors were combined and concentrated to dryness to obtain 25.0 g of intermediate 4 (four-step yield = 88%).

LCMS m/z＝374.9[M+1] ⁺

the fifth step:

Intermediate 4 (25 g, 66.7 mmol) was added to dichloromethane (250 mL) and methanol (250 mL), and then p-aminobenzyl alcohol (9.84 g, 80.0 mmol) and EEDQ (39.5 g, 80.0 mmol) were added, stirred at room temperature for 16 h, 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 6:

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

LCMS m/z＝664.3[M+1] ⁺

Step 7:

Intermediate 6 (2.1 g, 3.1 mmol) and N,N-diisopropylethylamine (3.6 g, 31 mmol) were added to DMF (40 mL) and stirred at room temperature for 10 minutes under nitrogen protection. The temperature was lowered to 0°C, and MMAE (purchased from Chengdu Huajieming Biotechnology Co., Ltd., 2.0 g, 3.1 mmol) and HOBT (0.38 g, 3.1 mmol) were added. The mixture was stirred for 30 minutes at this temperature, and stirred at room temperature for 18 h. The mixture was directly purified by C18 reverse phase column (0.1% TFA) ( _H2O :ACN=50:50) to obtain 2.7 g of intermediate 7 (yield=70%).

LCMS m/z＝1223.4[M+1] ⁺

Step 8:

To intermediate 7 (2.0 g, 1.6 mmol), a uniform mixture of TFA/DCM = 1:10 (34 mL) was added, and the mixture was stirred at room temperature for 2 h. The solvent was concentrated to dryness at 25°C, tetrahydrofuran (110 mL) and potassium carbonate (2.26 g, 16 mmol) were added, and the mixture was stirred at room temperature for 3 h. The mixture was concentrated to dryness, and directly purified 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 9:

Intermediate 8 (1.4 g, 1.25 mmol), glutaric anhydride (0.29 g, 2.5 mmol) and N,N-diisopropylethylamine (0.33 g, 2.5 mmol) were added to DMA (14 mL), stirred at room temperature for 18 h, and purified by C18 reverse phase preparative column (0.1% TFA) ( _H2O :ACN=50:50) to obtain 1.3 g of intermediate 9 (yield=84.4%).

LCMS m/z＝1237.4[M+1] ⁺

Step 10:

Intermediate 9 (1.3 g, 1.05 mmol) was added to DMA (58 mL) and dichloromethane (20 mL) under nitrogen protection, the temperature was lowered to 0°C, N-hydroxysuccinimide (0.37 g, 3.15 mmol) and EDCI (0.61 g, 3.15 mmol) were added, and the mixture was stirred at room temperature for 18 h. The mixture was purified by C18 reverse phase preparative column (0.1% TFA) ( _H2O :ACN=50:50) to obtain 1.0 g of intermediate 10 (yield = 71.4%).

LCMS m/z＝1334.5[M+1] ⁺

Step 11:

Intermediate 10 (10 mg, 7.5 μmol) and HSK-P18 (20 mg, 6.2 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (2.4 mg, 18.6 μmol) was added, and stirred at room temperature for 18 h. The mixture was separated and purified using a liquid phase preparation column (liquid phase preparation conditions: C18 reverse phase preparation column, mobile phase was deionized water containing 0.1% trifluoroacetic acid (A), acetonitrile containing 0.1% trifluoroacetic acid (B), gradient elution, B content = 5% to 70%, elution time 15 min, flow rate 12 mL/min, column temperature: 30°C, retention time: 4.56 min) to obtain 10 mg 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 (0.5 mmol, 1.85 g, sub: 0.27 mmol/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 five times with DMF.

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

6. Repeat steps 2-5 for the next amino acid coupling. See Table 3 for the amino acid coupling reagents.

table 3

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

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

7.20% piperidine DMF solution, 30 minutes for the removal of Fmoc protecting agent. Ninhydrin was used to monitor the coupling reaction, and the resin was washed with DMF 5 times.

Peptide fragmentation and purification:

1. Add the polypeptide with protected side chains to the reaction bottle, add 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 ice-cold isopropyl ether to precipitate the polypeptide and centrifuge (3 min at 3000 rpm).

3. Wash twice with isopropyl ether.

4. Vacuum drying gave a white solid crude peptide compound P18 (1.30 g, crude product).

Synthesis method of HSK-P18:

The crude peptide P18 was dissolved in 50% MeCN/ _H2O (500 mL), and TATA (purchased from WuXi AppTec, 270 mg, 0.60 mmol) was slowly added under stirring at room temperature for more than 30 minutes. After the addition was completed, the mixture was stirred at room temperature for 30 minutes, and ammonium bicarbonate was added to adjust the pH value to 8. The reaction solution was stirred at room temperature for 12 hours. LC-MS showed that the reaction was complete. The mixture was purified by preparative HPLC (mobile phase, A: 0.075% TFA in _H2O , B: _CH3CN ) to obtain 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, HSK-P44, and HSK-P45 were synthesized respectively.

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-), and the molecular scaffold is 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-), and the molecular scaffold is 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-), and the molecular scaffold is 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-), and the molecular scaffold is 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-), and the molecular scaffold is 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-), and the molecular scaffold is 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-), and the molecular scaffold is 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-), and the molecular scaffold is 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-), and the molecular scaffold is 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-), and the molecular scaffold is 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-), and the molecular scaffold is 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-), and the molecular scaffold is 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-), and the molecular scaffold is selected from TATB.

Example 2

The synthesis method is as in 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, 18.6 μmol) was added, and stirred at room temperature for 18 h. 10 mg of compound 2 was obtained by separation and purification using a liquid phase preparative column. LCMS m/z=1044.3[M/4+1], 1391.8[M/3+1].

Example 3

Synthesis method: refer to Example 1: Intermediate 10 (16 mg, 12.1 μmol) and HSK-P34 (30 mg, 10.1 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (3.9 mg, 30.3 μmol) was added, and stirred at room temperature for 18 h. 15 mg of compound 3 was obtained by separation and purification using a liquid phase preparation column. LCMS m/z=1047.8[M/4+1],1396.8[M/3+1].

Example 4

Synthesis method: refer to Example 1: Intermediate 10 (16 mg, 12.1 μmol) and HSK-P35 (30 mg, 10.1 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (3.9 mg, 30.3 μmol) was added, and stirred at room temperature for 18 h. 16 mg of compound 4 was obtained by separation and purification using a liquid phase preparation column. LCMS m/z=1047.8 [M/4+1], 1396.7 [M/3+1].

Example 5

Synthesis method: refer 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, 30.3 μmol) was added, and stirred at room temperature for 18 h. 15 mg of compound 5 was obtained by separation and purification using a liquid phase preparative column. LCMS m/z=1047.8 [M/4+1], 1396.8 [M/3+1].

Example 6

The synthesis method is as in Example 1: Intermediate 10 (16 mg, 12.0 μmol) and HSK-P37 (30 mg, 10.0 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (3.9 mg, 30.0 μmol) was added, and stirred at room temperature for 18 h. 17 mg of compound 6 was obtained by separation and purification using a liquid phase preparation column. LCMS m/z=1051.4 [M/4+1], 1401.3 [M/3+1].

Example 7

Synthesis method: refer 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, 30.0 μmol) was added, and stirred at room temperature for 18 h. 14 mg of compound 7 was obtained by separation and purification using a liquid phase preparative column. LCMS m/z=1051.3 [M/4+1], 1401.6 [M/3+1].

Example 8

The synthesis method is as in 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, 30.0 μmol) was added, and stirred at room temperature for 18 h. 15 mg of compound 8 was obtained by separation and purification using a liquid phase preparative column. LCMS m/z=1051.3 [M/4+1], 1401.4 [M/3+1].

Example 9

Synthesis method: refer 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, 30.6 μmol) was added, and stirred at room temperature for 18 h. 18 mg of compound 9 was obtained by separation and purification using a liquid phase preparative column. LCMS m/z=1037.3 [M/4+1], 1382.7 [M/3+1].

Example 10

Synthesis method: refer 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, 30.6 μmol) was added, and stirred at room temperature for 18 h. 15 mg of compound 10 was obtained by separation and purification using a liquid phase preparative column. LCMS m/z=1037.3 [M/4+1], 1382.7 [M/3+1].

Embodiment 11

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

Example 12

Synthesis method: refer to Example 1: Intermediate 10 (16 mg, 12.3 μmol) and HSK-P43 (30 mg, 10.2 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (3.9 mg, 30.6 μmol) was added, and stirred at room temperature for 18 h. 15 mg of compound 12 was obtained by separation and purification using a liquid phase preparation column. LCMS m/z=1040.7 [M/4+1], 1387.4 [M/3+1].

Example 13

The synthesis method is as in Example 1: Intermediate 10 (16 mg, 12.3 μmol) and HSK-P44 (30 mg, 10.2 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (3.9 mg, 30.6 μmol) was added, and stirred at room temperature for 18 h. 14 mg of compound 13 was obtained by separation and purification using a liquid phase preparation column. LCMS m/z=1040.8 [M/4+1], 1387.3 [M/3+1].

Embodiment 14

Synthesis method: refer 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, 30.6 μmol) was added, and stirred at room temperature for 18 h. 15 mg of compound 14 was obtained by separation and purification using a liquid phase preparation column. LCMS m/z=1040.8[M/4+1],1387.3[M/3+1].

Embodiment 15

first step:

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

Step 2 and 3:

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

LCMS m/z＝265.2[M+1] ⁺

the fourth step:

9-Fluorenylmethyl-N-succinimidyl carbonate (34.0 g, 0.1 mol) and sodium bicarbonate (42.0 g, 0.5 mol) were added to the reaction solution of 15d in sequence, stirred at room temperature for 4 h, and purified by C18 reverse phase column (0.1% TFA) ( _H2O :ACN=60:40) to obtain 22 g of 15e (two-step yield=45.2%). LCMS m/z=487.2[M+1] ⁺

the fifth step:

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

LCMS m/z＝819.1[M+1] ⁺

Step 6:

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

Step 7:

15g (300mg, 0.11mmol) was added to a mixed solution of piperidine:DMF=1:4 (5mL), stirred at room temperature for 2h, and purified by C18 reverse phase column (0.1% TFA) ( _H2O :ACN=50:50) to obtain 200mg 15h (yield=72.5%).

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

Step 8:

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

Step 9:

15i (46 mg, 0.017 mmol) was added to DMA (1 mL) and dichloromethane (0.3 mL) under nitrogen protection, and the temperature was lowered to 0°C. N-hydroxysuccinimide (6 mg, 0.051 mmol) and EDCI (10 mg, 0.051 mmol) were added, and the mixture was stirred at room temperature for 18 h. The mixture was purified by C18 reverse phase preparative column (0.1% TFA) ( _H2O :ACN=50:50) to obtain 40 mg of 15j (yield=83.8%).

LCMS m/z＝896.3[M/3+1] ⁺ ，1343.6+1] ⁺

Step 10:

15j (37 mg, 13 μmol) and HSK-P35 (30 mg, 10 μmol) were added to DMA (1 mL), and N,N-diisopropylethylamine (5 mg, 30 μmol) was added, and stirred at room temperature for 18 h. The mixture was separated and purified by liquid phase preparation column (liquid phase preparation conditions: C18 reverse phase preparation column, mobile phase is deionized water containing 0.1% trifluoroacetic acid (A), acetonitrile containing 0.1% trifluoroacetic acid (B), gradient elution, B content = 5% to 70%, elution time 15 min, flow rate 12 mL/min, column temperature: 30 ° C, retention time: 5.42 min) 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 (4.0 g, 0.01 mol) and di-tert-butyl dicarbonate (3.1 g, 0.013 mol) were added to dichloromethane (300 mL) in sequence, and then pyridine (26.0 g, 0.3 mol) was added, stirred at room temperature for 18 h, and concentrated to dryness to obtain 5.0 g of crude product 16a. (Yield = 99%). LCMS m/z = 493.1 [M+1] ⁺

Step 2:

16a (2.0 g, 4 mmol), N,N-diisopropylethylamine (2.63 g, 20 mmol) and 4-dimethylaminopyridine (0.5 g, 4 mmol) were added to dry dichloromethane (40 mL) in sequence, and the temperature was lowered to 0°C under nitrogen protection. A dichloromethane solution (10 mL) of triphosgene (0.52 g, 1.72 mmol) was slowly added dropwise. After the addition, the mixture was stirred at 0°C for 5 minutes and at room temperature for 10 minutes. A mixed solution of Boc-Val-Cit-PABC (1.75 g, 3.6 mmol) in dimethyl sulfoxide (10 mL) and dichloromethane (10 mL) was then added dropwise. After the addition, the mixture was stirred for 2 hours, and water (100 mL) and dichloromethane (50 mL) were added for extraction. The mixture was 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 (1.26 g, 0.126 mmol) was added to a mixed solution of dichloromethane (13 mL) and trifluoroacetic acid (13 mL), stirred at room temperature for 30 minutes, and concentrated to dryness at 30°C to obtain 1.0 g of crude product 16c. (Yield = 99%). LCMS m/z = 798.4 [M+1] ⁺

the fourth step:

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

the fifth step:

16d (80 mg, 0.034 mmol) was added to a 20% piperidine solution in N,N-dimethylformamide (1 mL), stirred at room temperature for 1 minute, and purified by C18 reverse phase preparative column (0.1% TFA) (H ₂ O:ACN=50:50) to obtain 40 mg of 16e (yield=55.5%).

LCMS m/z＝1075.3[M/2+1] ⁺

Step 6:

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

Step 7:

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

Step 8:

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

Biological test cases

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, 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 in a 37 ° C, 5% CO ₂ incubator. On the second day, 10 μL of different concentrations of compounds were added to each well, so that the final DMSO concentration in each well was 0.1%, and cultured in a 37 ° C, 5% CO ₂ incubator for 6 days. After the culture, 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 the chemiluminescence readings were detected by an enzyme reader. The results were treated according to Inhibition% = (1-(RLU compound/RLU control) × 100%, and the proliferation inhibition rate of each concentration of the compound was calculated. The origin9.2 software was used to calculate the concentration IC50 value of the compound when the inhibition rate was 50%. RLU compound is the reading of the drug treatment group, and RLU control is the average value of the solvent control group. The IC50 of the compounds of the present invention for NCI-H292 cells is less than 100nM, some are less than 50nM, and more excellent ones are 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 compounds of the present invention have good inhibitory activity against NCI-H292 cells.

2. Pharmacokinetics test in rats

2.1 Experimental 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 their body weight. They were fasted but not watered for 12-14 hours one day before administration and fed 4 hours after administration. The administration information is shown in Table 5.

Table 5. Dosing Information

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

Before and after administration, 0.15 ml of blood was collected from the eye socket under isoflurane anesthesia, placed in an EDTAK2 centrifuge tube, and centrifuged at 5000 rpm at 4°C for 10 min to collect plasma. The blood collection time points for the intravenous group and the gavage group were: 0, 5, 15, 30 min, 1, 2, 4, 6, 8, 24 h. Before analysis and testing, all samples were stored at -80°C and 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 compounds of the present invention were also observed to have excellent pharmacokinetic and pharmacodynamic characteristics in other animal species.

3. Mouse NCI-H292 cell subcutaneous transplant tumor model

Human lung cancer NCI-H292 cells were placed in RPMI-1640 medium (supplemented with 10% fetal bovine serum and 1% double antibody) and cultured at 37°C. Routine digestion and passage were performed with trypsin twice a week. When the cell saturation was 80%-90% and the number reached the requirement, the cells were collected and inoculated after counting. 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 group administration began when the average tumor volume reached about 80-120 mm ³ (recorded as Day 0). The vehicle group was given 5% dimethylacetamide, 5% Solutol HS-15 and 90% saline solution, and the drug administration group was intravenously administered 3 mg/kg of compound 1, compound 2, compound 4 or compound 7, and the administration frequency was once a week, and the administration period was 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 tumor volume was calculated as follows: V = 0.5 × a × b ² , where a and b represent the long diameter and short diameter of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated by TGI (%) = [1 – (average tumor volume at the end of administration of a treatment group – average tumor volume at the beginning of administration of the treatment group) / (average tumor volume at the end of treatment of the solvent control group – average tumor volume at the beginning of treatment of the solvent control group)] × 100%. The tumor growth curve and the animal weight change curve are shown in Figures 1, 2, 3 and 4, respectively.

Test results: After once-weekly administration for 36 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 animals in the vehicle group decreased significantly, but the body weight of animals in all drug-administered groups showed an upward trend.

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

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

Human breast cancer MDA-MB-468 cells (from ATCC) and human lung cancer NCI-H322 cells (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 2mM glutamine), respectively, and cultured at 37°C and 5% CO _2. Cells in the exponential growth phase were collected, and the cell suspensions were adjusted to 500/135μL and 1000/135μL respectively with culture medium. 135μL of cell suspension was added to each well of a 96-well cell culture plate and incubated overnight. The next day, different concentrations of compounds were added and placed in an incubator for 6 days. After the culture, according to the instructions of the CellTiter-Glo kit (Promega, Cat#G7573), 75 μL of CTG solution pre-melted and equilibrated to room temperature was added to each well, mixed with a microplate shaker for 2 minutes, and placed at room temperature for 10 minutes before measuring the chemiluminescence reading using an Envision 2104 plate reader (PerkinElmer). The cell proliferation inhibition rate was calculated according to the formula [(1–(RLUcompound–RLUblank)/(RLUcontrol–RLU blank))×100%]. The IC50 value was obtained by four-parameter nonlinear fitting using GraphPad Prism software.

Conclusion: The compounds of the present invention have a good inhibitory effect on MDA-MB-468 and NCI-H322 cells, and their IC50 values are less than 100 nM.

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 5000/135 μL with culture medium. 135 μL of cell suspension was added to each well of a 96-well cell culture plate and incubated overnight. On the second day, different concentrations of compounds were added and placed in an incubator for incubation for 6 days. After the culture was completed, 75 μL of CTG solution that had been pre-melted and equilibrated to room temperature was added to each well according to the operating instructions of the CellTiter-Glo kit (Promega, Cat#G7573), mixed with a microplate shaker for 2 minutes, and placed at room temperature for 10 minutes before measuring the chemiluminescence reading using an Envision 2104 plate reader (PerkinElmer). The cell proliferation inhibition rate was calculated according to the formula [(1–(RLUcompound–RLUblank)/(RLUcontrol–RLU blank))×100%]. The IC50 value was obtained by four-parameter nonlinear fitting using GraphPad Prism software.

Conclusion: The inhibitory effect of the compounds of the present invention on NCI-H526 cells is relatively weak.

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, hcys separated by at least two amino acid sequences, and the Cys or Hcys residues forming a covalent bond with the non-aromatic molecular scaffold, thereby forming at least two polypeptide loops on the molecular scaffold; provided that at least one Hcys residue is contained in the at least three residues selected from Cys, hcys.
2. The compound of claim 1, wherein the polypeptide structure comprises the amino acid sequence:

   -Xa1-A1-Xa2-A2-Xa3-

   Wherein A1, A2 represent an amino acid sequence between Xa1, xa2, xa3, and A1 and A2 each independently comprise 3 to 10 amino acid residues;

   Xa1, xa2, xa3 are independently Cys or Hcys residues and at least one is Hcys residues, and the non-aromatic molecular scaffold forms a thioether bond with Xa1, xa2, xa3 of the polypeptide, respectively, thereby forming two polypeptide loops on the molecular scaffold.
3. The compound of claim 2, wherein one of the A1, A2 consists of 3 amino acid residues and the other consists of 9 amino acid residues.
4. A compound according to any one of claims 1-3, wherein the polypeptide structure comprises the following amino acid sequence:

   -Xa1-P (1 Nal) (D-Asp) -Xa2-M (HArg) DWSTP (Hyp) W-Xa3-, said Xa1, xa2, xa3 being selected from Cys or Hcys respectively; provided that at least one of Xa1, xa2, xa3 is selected from Hcys;

   specifically, the polypeptide structure comprises an 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)。
5. the compound of claim 4, wherein the polypeptide structure comprises the amino acid sequence shown in any one of SEQ ID NOs 8 to 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 of claim 4, which is a bicyclic peptide comprising one of the following structures:

   Wherein, when Xa1, xa2 or Xa3 is Cys, respectively, m1, m2 or m3 is 1;

   when Xa1, xa2 or Xa3 is Hcys, respectively, m1, m2 or m3 is 2;

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

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

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

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

   Xa3 is Hcys, m3 is 2, n3 is 1 or 2, and n4 is 9.
9. The compound of claim 1, selected from the group consisting of compounds of the structures shown in table 1.
10. Use of a compound according to any one of claims 1 to 9 as a ligand for Nectin-4 in the screening and/or preparation of a medicament.
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 group: alkylating agents, antimetabolites, plant alkaloids, terpenoids, podophyllotoxins and derivatives thereof, taxanes and derivatives thereof, topoisomerase inhibitors, antitumor antibiotics;

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

    the peptide ligand is a compound of any one of claims 1 to 9.
12. The drug conjugate or pharmaceutically acceptable salt thereof according to claim 11, wherein the cytotoxic agent is selected from one or more of the following groups: cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide, azathioprine, mercaptopurine, pyrimidine analogs, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, paclitaxel, camptothecine and derivatives thereof, irinotecan, topotecan, an Ya, etoposide phosphate, teniposide, actinomycin D, doxorubicin, epirubicin, epothilone and derivatives thereof, bleomycin and derivatives thereof, dactinomycin and derivatives thereof, plicamycin and derivatives thereof, mitomycin C, spinocetin, maytansine and derivatives thereof, oretin and derivatives thereof.
13. The drug conjugate or pharmaceutically acceptable salt thereof according to claim 11 or 12, wherein the cytotoxic agent is selected from maytansinoids, monomethyl auristatin or camptothecin derivatives; or the cytotoxic agent is selected from maytansinoid DM1, monomethyl auristatin E or 7-ethyl-10-hydroxycamptothecin.
14. The drug conjugate or 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 pharmaceutically acceptable salt thereof according to claim 14, wherein:

    The disulfide linker is selected from DMDS, MDS, DSDM, NDMDS or a 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 the group consisting of: -Cit-Val-, -Phe-Lys-and-Val-Lys-;

    the pH-dependent linker is selected from the group consisting of cis-aconitic anhydride.
16. The drug conjugate of claim 14, or a pharmaceutically acceptable salt thereof, wherein the linker is-PABC-Cit-Val-glutaryl-, -PABC-cyclobutyl-Ala-Cit- βala-orWherein PABC represents p-aminobenzyl carbamate and k is selected from any integer from 1 to 20.
17. The drug conjugate of claim 16, or a pharmaceutically acceptable salt thereof, wherein the linker is

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

    Wherein Xa1, xa2, xa3 are independently Cys or Hcys residues and at least one is Hcys residue;

    When Xa1, xa2 or Xa3 is Cys, respectively m1, m2 or m3 is 1;

    when Xa1, xa2 or Xa3 is Hcys, respectively, m1, m2 or m3 is 2;

    n1, n2, n3 are independently 1 or 2;

    n4 is selected from any integer from 0 to 10;

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

    xa1 is Hcys, m1 is 2, n1 is 1 or 2, n4 is 9; or alternatively

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

    Xa3 is Hcys, m3 is 2, n3 is 1 or 2, and n4 is 9.
20. The drug conjugate or 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 a compound according to any one of claims 1 to 9 and/or a pharmaceutical conjugate according to any one of claims 11 to 20 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or excipient.
22. Use of a compound according to any one of claims 1 to 9, or a pharmaceutical conjugate according to any one of claims 11 to 20, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 21, for the manufacture of a medicament for the prevention and/or treatment of a disease or condition in which Nectin-4 is overexpressed in diseased tissue in a tumour individual.
23. The use of claim 22, wherein the disease or disorder that overexpresses Nectin-4 is at least one of urothelial cancer, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, hepatocellular cancer, pancreatic cancer, triple negative breast cancer, and bladder cancer.
24. A pharmaceutical composition or formulation comprising 1-1500mg of a compound of any one of claims 1 to 9 or a conjugate of any one of claims 11 to 20 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or excipient.
25. A method for treating a disease in a mammal or human, the method comprising administering to a subject a therapeutically effective amount of a compound of any one of claims 1 to 9 or a pharmaceutical conjugate of any one of claims 11 to 20, or a pharmaceutically acceptable salt thereof, preferably 1-1500mg, the disease preferably being a tumor.