Double-target-head cyclic peptide coupling medicine, medicine composition and application of double-target-head cyclic peptide coupling medicine

Abstract

The present invention relates to a drug conjugate comprising two different cyclic peptide ligands conjugated to an effector, such as a drug molecule, a pharmaceutical composition comprising the drug conjugate, and the use of the drug conjugate in the prevention or treatment of a disease, such as cancer.

Description

Technical Field

[0001] The present invention relates to a drug conjugate comprising two different cyclic peptide ligands conjugated to an effector such as a drug molecule, a pharmaceutical composition comprising the drug conjugate, and use of the drug conjugate in preventing or treating diseases such as cancer. Background Art

[0002] The full name of Nectin-4 is Nectin cell adhesion molecule 4, which belongs to the Nectin family of Ig superfamily proteins. Nectin-4 is a type I membrane protein that is overexpressed in a variety of tumor cells, including urothelial carcinoma, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, and bladder cancer. Studies have found that Nectin-4 promotes tumor cell proliferation, differentiation, migration, and invasion by activating the PI3K / Akt pathway, and is believed to play an important role in the occurrence and metastasis of cancer. It is based on these research backgrounds that drug research targeting this target has also emerged.

[0003] EphA2 is mainly restricted to adult proliferating epithelial cells. Adult EphA2 is only expressed in normal tissues when there are highly proliferative epithelial cells, and the importance and function of EphA2 are not well understood. However, increasing evidence shows that human EphA2 is abundantly expressed in prostate, lung, esophageal, colorectal, cervical, ovarian, breast, and skin cancers. The expression of EphA2 is associated with poor prognosis, increased metastatic potential, and decreased survival in tumor patients. In addition, EphA2 is not only a biomarker of malignant features, but also an active participant in malignant progression. Therefore, the transcriptional pattern of EphA2 and its functional relevance in malignant tumors make this protein an attractive target for cancer therapy.

[0004] The matrix metallopeptidase (MMPs) family is a class of zinc-dependent endopeptidases that can degrade most extracellular matrices. Membrane type 1-matrixmetalloproteinase (MT1-MMP) is an important member of the MMPs family, located on the cell membrane and highly expressed in a variety of tumors. MT1-MMP promotes tumor metastasis by affecting processes such as extracellular matrix remodeling, angiogenesis, lipid metabolism, and inflammatory response. Therefore, MT1-MMP has become an attractive target for tumor research and the development of anti-tumor drugs.

[0005] There are many reports on ADC drugs targeting various anti-tumor drugs, but few reports on PDC. The structure of PDC is similar to ADC, except that the homing device is different. PDC consists of three important components: peptide, linker and cytotoxic payload. The three work synergistically to deliver cytotoxins by targeting specific receptors of tumor cells.

[0006] Cyclic peptides are polypeptide chains with a cyclic structure, most of which are composed of 5-14 amino acids and have a molecular weight of about 500-2000Da. Compared with linear peptides, cyclic peptides have multiple advantages: ① improving the structural characteristics of the peptide chain; ② improving the pharmacokinetic properties of absorption and the biomembrane permeability required to achieve protein targets.

[0007] Specifically, in terms of structural properties, the introduction of cyclization can make the conformation of the peptide chain more stable, thereby improving the binding affinity to the target protein and reducing nonspecific binding due to fewer alternative conformations; reduced conformational flexibility can reduce the chance of the molecule fitting into the protease catalytic site and increase proteomic resistance; and it can intervene in protein-protein interactions (PPIs) by forming a larger interaction surface, thereby improving the efficacy of the peptide chain.

[0008] In terms of improving the pharmacokinetic properties of absorption and the biomembrane permeability required to reach the protein target, cyclic peptides have a lower energy barrier to adapt to the membrane environment and bind to transport proteins, thereby increasing passive diffusion and active transport; further improve the pharmacokinetic / pharmacodynamic properties by introducing non-canonical elements; introduce lipophilic side chains to greatly increase the affinity for plasma carrier proteins, thereby avoiding renal clearance and improving pharmacokinetic properties; and extend the half-life and reduce the frequency of dosing through alternative strategies, including binding to albumin or immunoglobulins.

[0009] In the prior art, there have been reports of cyclic peptide ligands targeting Nectin-4, EphA2, and MT1-MMP targets. For example:

[0010] CN 112566651A (Baisco Technology Development Co., Ltd.) discloses a cyclic peptide ligand that specifically binds to nectin-4 and a drug conjugate comprising the peptide ligand;

[0011] CN 112566651A (Baisco Technology Development Co., Ltd.) discloses a cyclic peptide ligand that specifically binds to EphA2 and a drug conjugate comprising the peptide ligand;

[0012] CN 110506049 A (Baisco Technology Development Co., Ltd.) discloses a cyclic peptide ligand that specifically binds to MT1-MMP and a drug conjugate comprising the peptide ligand.

[0013] The above-mentioned cyclic peptide ligands and drug conjugates are single-target drugs, each of which is suitable for different patient groups. Therefore, the patient group covered by each drug is relatively limited.

[0014] In order to expand the applicable population of the drug, the present invention has developed a dual-target drug, which contains two cyclic peptide ligands that target different targets. Compared with single-target drugs, the dual-target drug of the present invention is applicable to a wider population, and one dual-target PDC molecule can cover the same population as two single-target PDC molecules. Summary of the invention

[0015] The present invention provides a dual-target compound, which comprises a combination of any two of three targeting bicyclic peptides targeting NECTIN-4, MT1-MMP and EPHA2, namely:

[0016] NECTIN-4+EPHA2 or NECTIN-4+MT1-MMP or MT 1-MMP+EPHA2.

[0017] In the double-target compound of the present invention, the two bicyclic peptides are coupled by Click coupling (coupling of azide functional groups with DBCO or other acetylene functional groups, etc.), wherein the linker can be connected to the C-terminus or N-terminus of the bicyclic peptide.

[0018] In the double-target compound of the present invention, when the linker is connected to the N-terminus of the bicyclic peptide, the C-terminal cysteine ​​of the bicyclic peptide is amidated or cysteine ​​is replaced by mercaptoethylamine; when the linker is connected to the C-terminus of the bicyclic peptide, the N-terminus of the bicyclic peptide is acylated (acetylated, etc.) or cysteine ​​is replaced by mercaptopropionic acid; the cysteine ​​therein can be L-cysteine ​​or D-cysteine.

[0019] In particular, the present invention provides a drug conjugate of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof,

[0020]

[0021] in:

[0022] D is a drug, such as a cytotoxic agent used in cancer treatment,

[0023] L is a cleavable linking group,

[0024] Y is a linking group formed by coupling an azide functional group with an alkyne functional group,

[0025] X1 and X2 are each independently a molecular spacer group, and

[0026] P1 and P2 are each independently selected from cyclic peptide ligands, such as nectin-4 specific bicyclic peptide ligands, MT1-MMP specific bicyclic peptide ligands and EPHA2 specific bicyclic peptide ligands, with the proviso that P1 and P2 are not the same.

[0027] Animal experiments show that the dual-target drug of the present invention can significantly inhibit the growth of NCI-N87 nude mouse transplanted tumors, and is dose-dependent. In addition, experiments show that the dual-target drug of the present invention has equivalent efficacy to an equimolar dose of a single-target drug. The dual-target PDC drug of the present invention greatly expands the applicable population. A dual-target PDC molecule of the present invention can reach the coverage of two drugs (such as Nectin4 and EPHA2, Nectin4 and MT1-MMP) The population, the cost is also lower, and it has very good application prospects in clinical practice.

[0028] The present invention also provides a pharmaceutical composition comprising the drug conjugate of the present invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.

[0029] The present invention also provides use of the drug conjugate of the present invention or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating cancer, wherein the cancer is preferably selected from: prostate cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), breast cancer (e.g., triple-negative breast cancer), gastric cancer, ovarian cancer, esophageal cancer, multiple myeloma and fibrosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a high-resolution mass spectrum of the dual-target conjugate A1 of the present invention.

[0031] Figure 2 It is a high-resolution mass spectrum of the dual-target conjugate B1 of the present invention.

[0032] Figure 3 It is a high-resolution mass spectrum of the dual-target conjugate B2 of the present invention.

[0033] Figure 4 The results of the binding experiment of BP188e and BP188f polypeptides to Nectin-4 are shown.

[0034] Figure 5 The results of the binding affinity experiment of BP188e and BP188f polypeptides to Nectin-4 are shown.

[0035] Figure 6 The effects of BT8009, BT5528, B1 and B2 on tumor volume in a nude mouse xenograft model of human prostate cancer PC-3 are shown.

[0036] Figure 7 The effects of BT8009, BT5528, B1 and B2 on the relative tumor volume of the human prostate cancer PC-3 nude mouse xenograft model are shown.

[0037] Figure 8 The effects of BT8009, BT5528, B1 and B2 on the body weight of human prostate cancer PC-3 nude mouse xenograft model animals are shown.

[0038] Fig. 9 The effects of BT8009, BT5528, B1 and B2 on the relative body weight of human prostate cancer PC-3 nude mouse xenograft tumor model animals are shown.

[0039] Fig.10 It is a bar graph showing the effects of BT8009, BT5528, B1 and B2 on tumor weight in a human prostate cancer PC-3 nude mouse xenograft tumor model.

[0040] Fig.11 Shown are the effects of BT8009, BT5528, B1 and B2 on tumor volume in the human breast cancer MDA-MB-468 mouse xenograft model.

[0041] Fig.12 Shown are the effects of BT8009, BT5528 and B2 on relative tumor volume in the human breast cancer MDA-MB-468 mouse xenograft model.

[0042] Fig.13 The effects of BT8009, BT5528 and B2 on the body weight of the human breast cancer MDA-MB-468 mouse transplant tumor model are shown.

[0043] Fig.14 The effects of BT8009, BT5528 and B2 on the relative body weight of the human breast cancer MDA-MB-468 mouse xenograft model animals are shown.

[0044] Fig.15 The effects of BT8009, BT5528 and B2 on tumor volume in the human gastric cancer NCI-N87 nude mouse xenograft model are shown.

[0045] Fig.16 The effects of BT8009, BT5528 and B2 on the relative tumor volume of the human gastric cancer NCI-N87 nude mouse xenograft model are shown.

[0046] Fig.17 The effects of BT8009, BT5528 and B2 on the body weight of animals in the human gastric cancer NCI-N87 nude mouse transplant tumor model are shown.

[0047] Fig.18The effects of BT8009, BT5528 and B2 on the relative body weight of animals in the human gastric cancer NCI-N87 nude mouse xenograft model are shown.

[0048] Fig.19 It is a bar graph showing the effects of BT8009, BT5528 and B2 on tumor weight in the human gastric cancer NCI-N87 nude mouse xenograft tumor model.

[0049] Fig. 20 The effects of BT8009, BT5528 and A1 on tumor volume in the human gastric cancer NCI-N87 nude mouse xenograft model are shown.

[0050] Fig.21 The effects of BT8009, BT5528 and A1 on the relative tumor volume of the human gastric cancer NCI-N87 nude mouse xenograft model are shown.

[0051] Fig. 22 The effects of BT8009, BT5528 and A1 on the body weight of animals in the human gastric cancer NCI-N87 nude mouse transplant tumor model are shown.

[0052] Fig.23 The effects of BT8009, BT5528 and A1 on the relative body weight of animals in the human gastric cancer NCI-N87 nude mouse xenograft model are shown. DETAILED DESCRIPTION OF THE INVENTION

[0054] definition

[0055] The following words, phrases and symbols used in this application have the meanings described below, unless the context in which they are used indicates otherwise.

[0056] The term "alkyl" refers to a straight or branched saturated hydrocarbon group consisting of carbon atoms and hydrogen atoms. The term "C1-C6 alkyl" refers to a straight or branched saturated hydrocarbon group having 1 to 6 carbon atoms, examples of which include, for example, methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl or tert-butyl), pentyl (including n-pentyl, isopentyl, etc.), hexyl (including n-hexyl), etc.

[0057] The term "alkylene" refers to a divalent group obtained by removing two hydrogen atoms from the same or two different carbon atoms of a straight or branched saturated alkane. Specifically, the alkylene has 1-10 carbon atoms, such as 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 carbon atoms. For example, as used herein, the terms "C3-C8 alkylene" and "C4-C8 alkylene" refer to straight or branched alkylenes having 3 to 8 carbon atoms and 4 to 8 carbon atoms, respectively, including, but not limited to, propylene, butylene, pentylene, hexylene, heptylene, octylene, etc.

[0058] The term "cycloalkyl" refers to a saturated or partially unsaturated cyclic hydrocarbon group having a specified number of ring atoms; it may have one or more rings, such as 1, 2 or 3, preferably 1 or 2 rings. Cycloalkyl groups may include fused or bridged rings and spiro rings. The ring of the cycloalkyl group may be saturated, and it may also contain one or more, such as one or two double bonds (i.e., partially unsaturated) on the ring, but it is not completely conjugated and is not an "aryl" as defined in the present invention. For example, as used herein, the term "5- to 8-membered cycloalkyl" refers to a saturated or partially unsaturated cyclic hydrocarbon group having 5 to 8 carbon atoms, examples of which include, but are not limited to, cyclopentyl, cyclohexyl, spiro[2.2]pentyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, and the like.

[0059] The terms "heterocyclyl" or "heterocycle" as used herein are used interchangeably and refer to: a saturated or partially unsaturated ring with 3-12 ring atoms, the ring atoms including one or more (e.g., 1, 2 or 3, preferably 1 or 2) heteroatoms independently selected from N, O and S, and the remaining ring atoms are carbon atoms; it may have one or more rings, such as 1 or 2 rings. Wherein, N and S may be optionally oxidized to various oxidation states. The point of attachment of the heterocyclyl group may be on the N heteroatom or on the carbon atom. For example, as used herein, the term "5 to 8 membered heterocyclyl" means a heterocyclyl group with 5-8 (5, 6, 7 or 8) ring atoms, which contains at least one, such as 1, 2, 3 or 4, preferably 1, 2 or 3 heteroatoms independently selected from N, O and S. The heterocyclyl group may include fused or bridged rings and spirocycles. The ring of the heterocyclic group may be saturated, and may contain one or more, for example one or two, double bonds (i.e., partially unsaturated), but it is not completely conjugated, nor is it a "heteroaryl" as defined in the present invention. Examples of heterocyclic groups include, but are not limited to, pyrrolidinyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, oxazolidinyl, tetrahydropyridinyl, dihydropyrimidinyl, dihydropyrazinyl, pyrazolidinyl, and the like.

[0060] The terms "aryl" and "aromatic ring" as used herein are used interchangeably and refer to a carbocyclic hydrocarbon group consisting of one ring or multiple rings such as two fused rings having 6-14 carbon atoms (e.g., 6-12 carbon atoms (6-12-membered aryl), 6-10 carbon atoms (6-10-membered aryl)), wherein at least one ring is an aromatic ring. Examples of aryl include, but are not limited to, phenyl, naphthyl, etc.

[0061] As used herein, the terms "heteroaryl" and "heteroaromatic ring" are used interchangeably and refer to a mono-, bi- or tricyclic ring system having 5-12, preferably 5-10, and more preferably 5-6 ring atoms, wherein at least one ring is a 5- or 6-membered aromatic ring containing one or more, for example 1 to 4, heteroatoms selected from N, O and S. For example, the heteroaryl includes, but is not limited to, 5-6 membered monocyclic heteroaryl, such as pyridyl, N-oxypyridyl, pyrazinyl, pyrimidinyl, triazinyl (e.g., 1,2,4-triazinyl, 1,3,5-triazinyl), pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl and 1,3,4-oxadiazolyl), thiazolyl, isothiazolyl, thiadiazolyl, tetrazolyl, triazolyl, thienyl, furanyl, pyranyl, imidazolyl, oxazolyl, isoxazolyl, oxadi ... 1,2,4-triazine), oxazolyl, thiadiazolyl, more preferably pyridyl (e.g. pyridin-4-yl, pyridin-3-yl), pyrazinyl, pyridazinyl, pyrimidinyl; and 8-10 membered bicyclic heteroaryl, such as benzoxazolyl, benzisoxazolyl, benzothiophenyl, benzothiazolyl, benzisothiazolyl, etc.

[0062] In some cases, positional isomers will exist during the synthesis of the compounds of the present invention. In such cases, the structural formula provided herein only clearly describes one of the possible positional isomers. It should be understood that the structural formula provided herein is intended to represent any possible positional isomeric form of the compound, including mixtures thereof, and is not limited to the specific positional isomer described by the structural formula.

[0063] For example, the conjugate B2 prepared in Example 3 of the present invention may have a structure as shown in the following structural formula B2, or may have a structure as shown in the following structural formula B2-1, or may be a mixture of the two in any proportion.

[0064]

[0065] Similarly, the conjugate B3 in the present application may have a structure as shown in the following structural formula B3, or may have a structure as shown in the following structural formula B3-1, or may be a mixture of the two in any proportion.

[0066]

[0067] It can be understood that since the Y structure has two positional isomers, other compounds involved in the present invention, such as A1, A2, A3, A4, B4, C1, and C2, all have the above two positional isomers.

[0068] Unless otherwise indicated, all such positional isomeric forms and mixtures thereof in any ratio are included in the structures provided herein.

[0069] The term "drug" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (eg, immunosuppressants).

[0070] As used herein, "treat," ...

[0071] As used herein, "prevention" includes inhibition of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

[0072] In this article, the term "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0073] As used herein, the term "effective amount" refers to the amount or dosage of the conjugate of the present invention or a composition thereof that produces the desired effect in a patient in need of treatment or prevention after administration to the patient in single or multiple doses.

[0074] "Pharmaceutically acceptable salt" refers to a salt of the conjugate of the present invention that is non-toxic, biologically tolerable or otherwise biologically suitable for administration to a subject for treatment or prevention. For a general description of pharmaceutically acceptable salts, see, for example, SM Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977, 66: 1-19, and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, eds., Wiley-VCH and VHCA, Zurich, 2002.

[0075] As used herein, "pharmaceutically acceptable excipient" includes any solvent, dispersion medium, adjuvant, carrier, etc. that is physiologically compatible.

[0076] The pharmaceutical compositions of the present invention may be present in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. For example, a medicament comprising the conjugate described herein may be prepared by mixing the conjugate of the present invention having the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized preparation or an aqueous solution.

[0077] The pharmaceutical compositions of the present invention can be administered by any suitable method, including parenteral administration, intratumoral administration and intranasal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending on whether the medication is short-term or long-term, the medication can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various medication schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration and pulse infusion.

[0078] The pharmaceutical composition or formulation of the present invention may also contain additional active ingredients, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists), etc. The active ingredients are suitably combined in an amount effective for the intended use.

[0079] Non-limiting examples of chemotherapeutic agents include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecin and its analogs or metabolites, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, mitoxantrone, demethoxydaunorubicin, and daunomycin); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, nitrosocarbamide, lomustine, methyllomustine, streptozotocin); The invention also includes but is not limited to sedative agents (e.g., cyclophosphamide, oxaliplatin, methotrexate, mitomycin C and cyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin and carboplatin); free radical generators such as bleomycin; nucleoside analogs (e.g., 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine, azacitidine, mercaptopurine, thioguanine, pentostatin and hydroxyurea); paclitaxel, docetaxel and related analogs; vincristine, vinblastine and related analogs.

[0080] Non-limiting examples of immune checkpoint inhibitors or agonists include PD-1 inhibitors, such as anti-PD-1 antibodies, for example, pembrolizumab, nivolumab, and spartalizumab; PD-L1 inhibitors, such as anti-PD-L1 antibodies, for example, atezolizumab, durvalumab, and avelumab; CTLA-4 inhibitors, such as anti-CTLA-4 antibodies, for example, ipilimumab; and BTLA inhibitors, LAG-3 inhibitors, TIM3 inhibitors, TIGIT inhibitors, VISTA inhibitors, OX-40 agonists, and the like. DETAILED DESCRIPTION

[0081] Embodiment 1. A drug conjugate of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof,

[0082]

[0083] in:

[0084] D is for medicine,

[0085] L is a cleavable linking group,

[0086] Y is a linking group formed by coupling an azide functional group with an alkyne functional group,

[0087] X1 and X2 are each independently a molecular spacer group, and

[0088] P1 and P2 are each independently selected from cyclic peptide ligands.

[0089] Embodiment 2. A drug conjugate according to embodiment 1, wherein D is selected from cytotoxic agents used in cancer treatment, such as alkylating agents such as cisplatin and carboplatin, and oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites, including purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids, including vinca alkaloids, such as vincristine, vinblastine, vinorelbine and vindesine; podophyllotoxin and its derivatives etoposide and teniposide; taxanes, including paclitaxel; topoisomerase inhibitors, including camptothecin, irinotecan and topotecan, and type II inhibitors, including amridine, etoposide, etoposide phosphate and teniposide, antitumor antibiotics, including immunosuppressants actinomycin D, doxorubicin, epirubicin, bleomycin, calicheamicin, etc., maytansinoids, such as DM1 or monomethyl auristatins such as MMAE.

[0090] Embodiment 3. The drug conjugate according to embodiment 1, wherein D is selected from DM1 of the following formula

[0091] and monomethyl auristatin (MMAE) of the formula

[0092]

[0093] Embodiment 4. A drug conjugate according to any one of embodiments 1 to 3, wherein L is a linker group comprising one or more amino acid residues selected from alanine (Ala), citrulline (Cit), lysine (Lys), tryptophan (Trp) and valine (Val), such as -Val-Cit-, -Trp-Cit-, -Val-Lys-, -D-Trp-Cit-, -PAB-Cit-Val-glutaryl- or -PAB-cyclobutyl -Ala-Cit-βAla-, wherein PAB represents p-aminobenzyloxycarbonyl; or L is a linking group containing a cleavable disulfide bond, such as -SS-, -SS(SO3H)-, -SS-(Me)-, -(Me)-SS-(Me)-, -SS-(Me2)- or -SS-(Me)-SO3H-; preferably, L is -PAB-Cit-Val-glutaryl- or -PAB-cyclobutyl-Ala-Cit-βAla-. .

[0094] Embodiment 5. A drug conjugate according to any one of embodiments 1-4, wherein the cytotoxic agent is MMAE, and L is selected from -Val-Cit-, -Val-Lys-, -PAB-Cit-Val-glutaryl- or -PAB-cyclobutyl-Ala-Cit-βAla-; preferably, the cytotoxic agent is MMAE, and L is selected from -PAB-Cit-Val-glutaryl-.

[0095] Embodiment 6. A drug conjugate according to any one of embodiments 1 to 5, wherein the DL- portion has a structure of the following formula, the right-hand bond of which is connected to N in formula (I):

[0096]

[0097] Embodiment 7. A drug conjugate according to any one of embodiments 1-4, wherein the cytotoxic agent is DM1 and L is selected from -SS-, -SS(SO3H)-, -SS-(Me)-, -(Me)-SS-(Me)-, -SS-(Me2)- and -SS-(Me)-SO3H-.

[0098] Embodiment 8. The drug conjugate according to any one of embodiments 1-7, wherein Y is selected from or a mixture of two isomers, wherein

[0099] R1 is C3-C8 alkylene,

[0100] R2 is -(C4-C8 alkylene)-CO-NH-, wherein one or more carbon atoms in the C4-C8 alkylene are optionally replaced by an atom or group selected from N, O, S and (C=O),

[0101] R3 is selected from hydrogen, C1-C6 alkyl, aryl and heteroaryl, or

[0102] R3 is connected to the carbon atom or optional N atom in -(C4-C8 alkylene)- of R2 to form a 5- to 8-membered cyclic hydrocarbon group or a 5- to 8-membered heterocyclic ring, and the 5- to 8-membered cyclic hydrocarbon group or the 5- to 8-membered heterocyclic ring is optionally condensed with one or two rings independently selected from aromatic rings or heteroaromatic rings.

[0103] Embodiment 9. The drug conjugate according to any one of embodiments 1 to 8, wherein the drug conjugate is a drug conjugate comprising one of the following Y groups or a mixture of two drug conjugates: Wherein n is an integer of 3-6, preferably, the left bond of the group is connected to the carbon atom in formula (I), and the right bond is connected to the X2 part.

[0104] Embodiment 10. The drug conjugate according to embodiment 9, which is a drug conjugate comprising one of the following Y groups or a mixture of two drug conjugates: Its left bond is connected to the carbon atom in formula (I), and its right bond is connected to the X2 part.

[0105] Embodiment 11. The drug conjugate according to any one of embodiments 1-10, wherein X1 and X2 are each independently selected from -(CH2) p -(C=O)-(Sar) q -、-(CH2CH2O) k -(Sar) q -、 --(CH2CH2O) k -CH2-(C=O)-β-Ala-(Sar) q -, wherein Sar is sarcosine, k = 1-8, p = 1-4, q = 1-10.

[0106] Embodiment 12. The drug conjugate according to embodiment 11, wherein X1 is selected from X2 is selected from -(CH2CH2O) k -CH2-(C=O)-β-Ala-(Sar)q -, wherein Sar is sarcosine, k = 1-8, q = 1-10.

[0107] Embodiment 13. A drug conjugate according to any one of embodiments 1-12, wherein P1 and P2 are each independently selected from a bicyclic peptide ligand specific for bindin-4, a bicyclic peptide ligand specific for MT1-MMP, and a bicyclic peptide ligand specific for EPHA2, wherein X1 and X2 are connected to the C-terminus or N-terminus of P1 and P2, respectively, with the proviso that P1 and P2 are not the same.

[0108] Embodiment 14. The drug conjugate according to embodiment 13, wherein the binding protein-4 specific bicyclic peptide ligand is selected from

[0109]

[0110] Embodiment 15. The drug conjugate according to embodiment 13, wherein the MT1-MMP specific bicyclic peptide ligand portion has the following structure

[0111]

[0112] Embodiment 16. The drug conjugate according to embodiment 13, wherein the EPHA2-specific bicyclic peptide ligand portion has the following structure

[0113]

[0114] Embodiment 17. The drug conjugate according to any one of embodiments 1-16, wherein the drug conjugate is selected from:

[0115]

[0116]

[0117]

[0118]

[0119] Embodiment 18. A drug conjugate selected from the group consisting of:

[0120]

[0121]

[0122]

[0123] Embodiment 19. A pharmaceutical composition comprising the drug conjugate of any one of Embodiments 1 to 18 and one or more pharmaceutically acceptable excipients.

[0124] Embodiment 20. Use of the drug conjugate according to any one of Embodiments 1 to 19 in the preparation of a medicament for preventing or treating cancer.

[0125] Embodiment 21. The use according to embodiment 22, wherein the cancer is selected from: prostate cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), breast cancer (such as triple-negative breast cancer), gastric cancer, ovarian cancer, esophageal cancer, multiple myeloma and fibrosarcoma.

[0126] Embodiment 22. A drug combination comprising the drug conjugate of any one of embodiments 1 to 18 and one or more other therapeutic agents.

[0127] Embodiment 23. A kit of parts comprising:

[0128] - a first container containing the drug conjugate according to any one of embodiments 1 to 18;

[0129] - A second container of the pharmaceutical composition containing the other therapeutic agent.

[0130] Embodiment 24. A pharmaceutical combination according to embodiment 22 or a kit of parts according to embodiment 23, wherein the other therapeutic agent is selected from a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, other antibodies, a small molecule drug or an immunomodulator (e.g., an immune checkpoint inhibitor or agonist).

[0131] Embodiment 25. A method for preventing or treating a disease, comprising administering to an individual in need thereof an effective amount of the drug conjugate of any one of embodiments 1 to 18, the pharmaceutical composition of embodiment 19, or the pharmaceutical composition of embodiment 22, wherein the disease is cancer, such as prostate cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), breast cancer (e.g., triple-negative breast cancer), gastric cancer, ovarian cancer, esophageal cancer, multiple myeloma, and fibrosarcoma.

[0132] Embodiment 26. A bicyclic peptide ligand selected from the group consisting of:

[0133]

[0134]

[0135]

[0136] The various embodiments described in the present invention (including the examples below) and the features in the various embodiments should be understood to be able to be combined with each other arbitrarily, and the various schemes obtained by these combinations are included in the scope of the present invention, just as the schemes obtained by these combinations are specifically and one by one listed in this document, unless the context clearly shows otherwise.

[0137] Example

[0138] Amino acids and their abbreviations and English names are shown in the following table:

[0139]

[0140] Example 1. Synthesis of dual-target conjugate A1:

[0141]

[0142]

[0143] Step 1: Synthesis of direct-linked peptide BP188g01:

[0144] Lys(N3)-β-Ala-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Cys-Pro-1Nal-(D-Asp)-Cys-Met-HArg-Asp-Trp-Ser-Thr-Pro-HyP-Trp-Cys-NH2

[0145] The synthesis of the direct-linked peptide BP188g01 was carried out by solid phase synthesis using the Fmoc method, using Rinke Amide Resin, using 20% ​​piperidine / DMF to remove Fmoc, using HOBT / DIC as a coupling reagent, using HCTU / DIEA to condense difficult amino acids, using DMF as a reaction solvent, and using the ninhydrin detection method to monitor the reaction. The following protected amino acids were connected to the resin in sequence: Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-HArg(Pbf)-OH, Fmoc-Me The resin was washed with t-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Asp(OtBu)-OH, Fmoc-1-Nal-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-β-Ala-OH, Fmoc-Lys(N3)-OH, DMF, DCM, methanol and then dried. The resin was cleaved using TFA / EDT / phenol / TIS / H2O, followed by precipitation and washing with ice methyl tert-butyl ether (MTBE). The crude product was prepared and purified by reverse phase HPLC and then freeze-dried to obtain pure product BP188g01.

[0146] MS of BP188g01: [M+2] 2+ is 1430.31, [M+3] 3+ It is 953.72.

[0147] Step 2: Synthesis of bicyclic peptide BP188g02:

[0148] Under nitrogen protection, 490 mg BP188g01 (1.0 eq) was dissolved in ml acetonitrile / water (1:1), 34.2 mg TATA (1.0 eq) was weighed and dissolved in 150 ml acetonitrile / water (1:1), and added to the reaction solution, 244 mg NH4HCO3 (20.0 eq) was weighed and dissolved in 10 ml purified water, and added to the reaction bottle, and the reaction was stirred. After the reaction was monitored by HPLC, it was purified by reverse phase high performance liquid chromatography, the pure product was collected, and 314 mg BP188g02 was obtained as a white powder after freeze drying.

[0149] MS of BP188g02: [M+2] 2+ is 1554.85, [M+3] 3+ It is 1036.75.

[0150] Step 3: Synthesis of BP188g03:

[0151] 200 mg BP188g02 (1.0 eq) was dissolved in 5 ml phosphate buffer solution, 94.5 mg BP188m01 (1.1 eq) was dissolved in 0.3 ml DMF, and then added to the phosphate buffer solution of BP188g02 to start the reaction. After the reaction was completed, it was purified by reverse phase high performance liquid chromatography, and the pure product was collected and freeze-dried to obtain 138 mg BP188g03 as a white powder.

[0152] MS of BP188g03: [M+3] 3+ is 1444.37, [M+4] 4+ It is 1083.13.

[0153] Step 4: Synthesis of direct-linked peptide BP188g11:

[0154] β-Ala-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Ala-Cys-(D-Ala)-Asn-Glu-(1-Nal)-(D-Ala)-Cys-G1u-Asp-Phe-Tyr-Asp-(tBuGly)-Cys-NH2

[0155] The synthesis of the direct-linked peptide BP188g11 was carried out by Fmoc solid phase synthesis, using Rinke Amide Resin, 20% piperidine / DMF to remove Fmoc, HOBT / DIC as coupling reagent, HCTU / DIEA condensation of difficult amino acids, DMF as reaction solvent, and ninhydrin detection method for reaction monitoring. The following protected amino acids were connected to the resin in sequence: Fmoc-Cys(Trt)-OH, Fmoc-tBuGly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Ala-OH, Fmoc- Fmoc-1-Nal-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-D-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-β-Ala-OH, washed with DMF, washed with DCM, washed with methanol and then dried. The resin was cleaved using TFA / EDT / phenol / TIS / H2O, followed by precipitation and washing with ice methyl tert-butyl ether (MTBE). The crude product was prepared and purified by reverse phase HPLC and then freeze-dried to obtain pure BP188g11.

[0156] MS of BP188g11: [M-2] 2- is 1271.47, [M+2] 2+ is 1272.99, [M+3] 3+ It is 809.36.

[0157] Step 5: Synthesis of bicyclic peptide BP188g12:

[0158] Under nitrogen protection, 1000 mg BP188g11 (1.0 eq) was dissolved in 380 ml acetonitrile / water (1:1), 98.2 mg 1,3,5-tri(bromomethyl)benzene (1.0 eq) was weighed and dissolved in 5 ml acetonitrile / water (3:21), and added to the reaction solution, 560 mg NH4HCO3 (20.0 eq) was weighed and dissolved in 10 ml purified water, and added to the reaction bottle, stirred for reaction, and monitored by HPLC. After completion of the reaction, it was purified by reverse phase high performance liquid chromatography, the pure product was collected, and 434 mg BP188g12 was obtained as a white powder after freeze drying.

[0159] MS of BP188g12: [M+3] 3+ It is 887.43.

[0160] Step 6: Synthesis of BP188g13:

[0161] 100 mg BP188g12 (1.0 eq) was dissolved in 5 ml phosphate buffer solution, and 18.2 mg DBCO-Osu (1.1 eq) was dissolved in 0.1 ml DMF. The mixture was then added to the phosphate buffer solution of BP188g12 to initiate the reaction. After the reaction was completed, the mixture was purified by reverse phase HPLC, and the pure product was collected and freeze-dried to obtain 57.2 mg BP188g13 as a white powder.

[0162] MS of BP188g13: [M+2] 2+ is 1473.98, [M+3] 3+ It is 983.59.

[0163] Step 7: Synthesis of dual-target conjugate A1:

[0164] 58.8 mg BP188g03 (1.0 eq) was dissolved in 1 ml phosphate buffer solution, and 40 mg BP188g13 (1.0 eq) was dissolved in 1 ml phosphate buffer solution. Then, the mixture was added to the phosphate buffer solution of BP188g13 to start the reaction. After the reaction was completed, the reaction was monitored by HPLC and purified by reverse phase high performance liquid chromatography. The pure product was collected and freeze-dried to obtain 71 mg A1 as a white powder.

[0165] A1's MS: [M+4] 4+ is 1819.80, [M+5] 5+ The high-resolution mass spectrum of compound A1 is shown in Figure 1 shown.

[0166] Example 2: Synthesis of dual-target conjugate B1:

[0167]

[0168] Step 1: Synthesis of direct-linked peptide BP188k11:

[0169] β-Ala-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Ala-HArg-Asp-Cys-Hyp-Leu-Val-Asn-Pro-Leu-Cys-Leu-His-Pro-(D-Asp)-Trp-HArg-Cys-NH2

[0170] The synthesis of the direct peptide BP188k11 was carried out by Fmoc solid phase synthesis, using RinkeAmide Resin, 20% piperidine / DMF to remove Fmoc, HOBT / DIC as coupling reagent, HCTU / DIEA condensation of difficult amino acids, DMF as reaction solvent, and ninhydrin detection method for reaction monitoring. The following protected amino acids were connected to the resin in sequence:

[0171] Fmoc-Cys(Trt)-OH, Fmoc-HArg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-D-Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-His(Boc)-OH, Fmoc-L eu-OH, Fmoc-Cys(Trt)-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Hyp(tBu)-OH, Fmo c-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-HArg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-B-Ala-OH, washed with DMF, washed with DCM, washed with methanol and then dried. The resin was cleaved using TFA / EDT / phenol / TIS / H2O, followed by precipitation and washing with ice methyl tert-butyl ether (MTBE). The crude product was prepared and purified by reverse phase HPLC and then freeze-dried to obtain pure BP188k11.

[0172] MS of BP188k11: [M+3] 3+ is 978.65, [M+4] 4+ It is 734.20.

[0173] Step 2: Synthesis of bicyclic peptide BP188k12:

[0174] Under nitrogen protection, 1000 mg BP188k11 (1.0 eq) was dissolved in 300 ml acetonitrile / water (1:1), 68 mg TATA (1.0 eq) was weighed and dissolved in 2 ml acetonitrile / water (1:1), and added to the reaction solution, 485 mg NH4HCO3 (20.0 eq) was weighed and dissolved in 5 ml purified water, and added to the reaction bottle, and the reaction was stirred. After the reaction was monitored by HPLC, it was purified by reverse phase high performance liquid chromatography, the pure product was collected, and 725 mg BP188k12 was obtained as a white powder after freeze drying.

[0175] MS of BP188k12: [M+3] 3+ is 1062.07, [M+4] 4+ It is 796.26.

[0176] Step 3: Synthesis of bicyclic peptide BP188k13:

[0177] 100 mg BP188k12 (1.0 eq) was dissolved in 4 ml phosphate buffer solution, and 15.2 mg DBCO-Osu (1.2 eq) was dissolved in 0.2 ml DMF. The mixture was then added to the phosphate buffer solution of BP188k12 to start the reaction. After the reaction was completed, the mixture was purified by reverse phase HPLC, and the pure product was collected and freeze-dried to obtain 49 mg BP188k13 as a white powder.

[0178] MS of BP188k13: [M+3] 3+ is 1157.95, [M+4] 4+ It is 868.41.

[0179] Step 4: Synthesis of dual-target conjugate B1:

[0180] 59 mg BP188g03 (1.0 eq) was dissolved in 3 ml phosphate buffer solution, and 47 mg BP188k13 (1.1 eq) was dissolved in 3 ml phosphate buffer solution. Then, the mixture was added to the phosphate buffer solution of BP188k13 to start the reaction. After the reaction was completed, the mixture was purified by reverse phase high performance liquid chromatography and the pure product was collected and freeze dried to obtain 65 mg B1 as a white powder.

[0181] MS of B1: 7798.09[M+4] 4+ is 1950.86, [M+5] 5+ is 1560.83, [M+6] 6+ 1300.97, [M+7] 7+ is 1115.08, [M+8] 8+ The high-resolution mass spectrum of compound B1 is shown in Figure 2 shown.

[0182] Example 3: Synthesis of dual-target conjugate B2:

[0183]

[0184]

[0185]

[0186] Step 1: Synthesis of direct-linked peptide BP188b11:

[0187] Mpa-Pro-1Nal-(D-Asp)-Cys-Met-HArg-Asp-Trp-Ser-Thr-Pro-HyP-Trp-Cys-Sar-Sar-Sar-Sar-Sar-Sa r-Sar-Sar-Sar-Sar-PEG6-Lys(Lys(N3))-NH2

[0188] The synthesis of the direct peptide BP188b11 was carried out by solid phase synthesis using the Fmoc method, using RinkeAmide Resin, using 20% ​​piperidine / DMF to remove Fmoc, using HOBT / DIC as the coupling reagent, using HCTU / DIEA to condense the difficult amino acids, using DMF as the reaction solvent, and using the ninhydrin detection method to monitor the reaction. The following protected amino acids were connected to the resin in sequence:

[0189] Fmoc-Lys(Dde)-OH, Fmoc-PEG6-CH2COOH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc- Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Pro-OH, Fmoc-T hr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-HArg(Pbf)-OH, Fmoc-Met-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Asp(OtBu)-OH, Fmoc-1-Nal-OH, Fmoc-Pro-OH, Mpa(Trt), remove the side chain protecting group Dde of Lys with hydrazine hydrate, then condense Fmoc-Lys(N3)-OH, wash with DMF, wash with DCM, wash with methanol and then dry. The resin was cleaved by TFA / EDT / phenol / TIS / H2O, followed by precipitation and washing with ice methyl tert-butyl ether (MTBE). The crude product was prepared and purified by reverse phase HPLC and then freeze-dried to obtain pure BP188b11.

[0190] MS of BP188b11: [M+3] 3+ is 1075.35, [M+4] 4+ It is 806.80.

[0191] Step 2: Synthesis of bicyclic peptide BP188b12:

[0192] Under nitrogen protection, 600 mg BP188b11 (1.0 eq) was dissolved in 180 ml acetonitrile / water (1:1), 37 mg TATA (0.8 eq) was weighed and dissolved in 1 ml acetonitrile / water (1:1), and added to the reaction solution, 295 mg NH4HCO3 (20.0 eq) was weighed and dissolved in 5 ml purified water, and added to the reaction bottle, and the reaction was stirred. After the reaction was monitored by HPLC, it was purified by reverse phase high performance liquid chromatography, the pure product was collected, and 340 mg BP188b12 was obtained as a white powder after freeze drying.

[0193] MS of BP188b12: [M+2] 2+ is 1737.34, [M+3] 3+ is 1158.93, [M+4] 4+ It is 869.02.

[0194] Step 3: Synthesis of BP188b13:

[0195] 100 mg BP188b12 (1.0 eq) was dissolved in 5 ml phosphate buffer solution, and 77 mg BP188m01 (2.0 eq) was dissolved in 1 ml DMF. The mixture was then added to the phosphate buffer solution of BP188b12 to start the reaction. After the reaction was completed, the reaction was monitored by HPLC and purified by reverse phase HPLC. The pure product was collected and freeze-dried to obtain 34 mg BP188b13 as a white powder.

[0196] MS of BP188b13: [M+4] 4+ is 1173.61, [M+3] 3+ It is 1565.04.

[0197] Step 4: Synthesis of bicyclic peptide BP188k22:

[0198] Step 4.1 Synthesis of fully protected direct peptide BP188k20:

[0199] PEG6-β-Ala-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Sar-Ala-HArg-Asp-Cys-Hyp-Leu-Val-Asn-Pro-Leu-Cys-Leu-His-Pro-(D-Asp)-Trp-HArg-Cys-NH2

[0200] The synthesis of the direct peptide BP188k20 was carried out by solid phase synthesis using the Fmoc method, using 2Cl-Trt Resin, using 20% ​​piperidine / DMF to remove Fmoc, using HOBT / DIC as the coupling reagent, using HCTU / DIEA to condense the difficult amino acids, using DMF as the reaction solvent, and using the ninhydrin detection method to monitor the reaction. The following protected amino acids were connected to the resin in sequence:

[0201] Fmoc-HArg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-D-Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-His(Boc)-OH, Fmoc-Leu-OH, Fmoc-Cys(Trt)-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu) -OH, Fmoc-HArg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-Sar-OH, Fmoc-β-Ala-OH, Fmoc-NH-PEG6-CH2COOH, after removing Fmoc, use Boc anhydride to protect the amino group, wash with DMF, wash with DCM, wash with methanol and then dry. The resin was cleaved using 1% TFA / DCM, filtered and neutralized with pyridine, concentrated to remove most of the DCM, then precipitated with ice methyl tert-butyl ether (MTBE), washed, and dried to obtain the crude product BP188k20, which was directly subjected to the next condensation reaction without purification.

[0202] Step 4.2 Synthesis of fully protected straight-linked peptide BP188k21:

[0203] The fully protected straight peptide BP188k20 (1.0 eq) and S-tritylcysteamine hydrochloride (1.2 eq) were dissolved in DMF, cooled to about 0°C, DIEA (3.0 eq) was added, and TBTU (1.1 eq) was added after stirring for 5 min. After the reaction was completed by TLC monitoring, the reaction solution was poured into purified water, the solid precipitated, centrifuged, the supernatant was removed, and washed with purified water 3 times. The precipitate was collected and vacuum dried to obtain the crude product BP188k21, which was directly subjected to the next deprotection reaction without purification.

[0204] Step 4.3 Synthesis of direct-linked peptide BP188k22:

[0205] The crude fully protected straight peptide BP188k21 was cleaved by TFA / EDT / phenol / TIS / H2O, followed by precipitation and washing with ice-cold methyl tert-butyl ether (MTBE). The crude product was purified by reverse phase HPLC and lyophilized to obtain pure BP188k22.

[0206] MS of BP188k22: [M+3] 3+ is 1071.95, [M+4] 4+ 803.71, [M+5] 5+ It is 643.59.

[0207] Step 5: Synthesis of bicyclic peptide BP188k23:

[0208] Under nitrogen protection, 1000 mg BP188k22 (1.0 eq) was dissolved in 300 ml acetonitrile / water (1:1), 62 mg TATA (0.8 eq) was weighed and dissolved in 2 ml acetonitrile / water (1:1), and added to the reaction solution, 493 mg NH4HCO3 (20.0 eq) was weighed and dissolved in 5 ml purified water, and added to the reaction bottle, and the reaction was stirred. After the reaction was monitored by HPLC, it was purified by reverse phase high performance liquid chromatography, the pure product was collected, and 702 mg BP188k23 as a white powder was obtained after freeze drying.

[0209] MS of BP188k23: [M+3] 3+ is 1154.90, [M+4] 4+ is 865.90, [M+5] 5+ It is 692.93.

[0210] Step 6: Synthesis of BP188k24:

[0211] 100 mg BP188k23 (1.0 eq) was dissolved in 3 ml phosphate buffer solution, and 14 mg DBCO-Osu (1.2 eq) was dissolved in 0.1 ml DMF. The mixture was then added to the phosphate buffer solution of BP188k23 to start the reaction. After the reaction was completed, the mixture was purified by reverse phase high performance liquid chromatography, and the pure product was collected and freeze dried to obtain 82 mg BP188k24 as a white powder.

[0212] MS of BP188k24: [M+2] 2+ is 1875.63, [M+3] 3+ It is 1250.4.

[0213] Step 7: Synthesis of dual-target conjugate B2:

[0214] 35 mg BP188b13 (1.0 eq) was dissolved in 1 ml phosphate buffer solution, and 28 mg BP188k24 (1.1 eq) was dissolved in 1 ml phosphate buffer solution. Then the mixture was added to the phosphate buffer solution of BP188b13 to start the reaction. After the reaction was completed, the reaction was monitored by HPLC and purified by reverse phase high performance liquid chromatography. The pure product was collected and freeze dried to obtain 56 mg B2 as a white powder.

[0215] B2's MS: [M+5] 5+ is 1689.21, [M+6] 6+ 1408.03, [M+7] 7+ is 1206.99, [M+8] 8+ is 1056.29, [M+9] 9+ The high-resolution mass spectrum of compound B2 is shown in Figure 3 shown.

[0216] Comparative example: single target compound (positive control)

[0217] The following single-target compounds were synthesized as positive controls:

[0218] BT8009 (target NECTIN-4) positive control

[0219]

[0220] BT5528 (target EPHA2) positive control

[0221]

[0222] Synthesis of compound BT8009:

[0223]

[0224] Synthesis of compound BT5528:

[0225]

[0226] Synthesis of compound BP188e:

[0227] Synthesis of compound BP188f:

[0228]

[0229] Experimental Example 1: Binding of cyclic peptide and nectin4 - FACS detection

[0230] NCI-N87 cell line with high expression of Nectin4 was selected to detect the binding of BP188e and BP188f peptides; 293 cells (full name HEK293) were used as controls to determine the specificity of binding between peptides and Nectin4; the results are shown in Tables 1 and Figure 4 shown.

[0231] Table 1

[0232]

[0233] Cells: 3X10^5 / tube

[0234] Primary antibody: BP188e, BP188f 50ug / ml 5-fold dilution 3 points

[0235] Secondary antibody: PE-Streptavidin

[0236] Results: According to the FACS test results, BP188e (BT8009 cyclic peptide-Biotin) and BP188f (B2-Nectin4 cyclic peptide-Biotin) both bound to CHO-Nectin4 with basically the same binding strength. Figure 4 shown.

[0237] Experimental Example 2 Affinity Detection

[0238] Purpose

[0239] The binding affinity of BP188e and BP188f polypeptides to nectin-4 was detected.

[0240] Experimental protocol:

[0241] The two biotinylated cyclic peptides were immobilized using a streptavidin sensor, and the immobilization height was about 0.2 nM.

[0242] The buffer was PBST (PBS+0.02% tween 20), and the Nectin4-His protein samples were diluted to 1000, 500, 250, 125, 62.5, and 0 nM.

[0243] Affinity detection: equilibrium 60s, binding 180s, dissociation 180s, detection temperature 25℃.

[0244] Instrument: ForteBio OCTETR2

[0245] Consumables: 96-well blackboard

[0246] The experimental results are as follows Figure 5 shown.

[0247] The results showed that the KD values ​​of BT8009 and B2 cyclic peptide structures binding to Nectin-4 were 3.219x10 7 M and 3.859x10 7 M, both have comparable binding affinities to nectin-4.

[0248] Experimental Example 3. Determination of the inhibitory effect on tumor growth in vivo in a human prostate cancer PC-3 nude mouse transplant tumor model

[0249] 1. Purpose of the experiment

[0250] The inhibitory effects of four test samples, BT8009 (positive control), BT5528 (positive control), and B2 compound, on the in vivo growth of human prostate cancer PC-3 nude mouse transplanted tumor model were evaluated.

[0251] 2. Experimental Materials

[0252] 2.1. Compound information

[0253]

[0254] Experimental animals

[0255] Male Nu / Nu nude mice (number: 50; age: 6-8 weeks) were purchased from Weitong Lihua Experimental Animal Technology Co., Ltd. and kept in the SPF animal room of Suzhou Shengsu New Drug Development Co., Ltd., with a temperature of 20-25°C, a relative humidity of 40%-70%, and 12 hours of light and dark lighting respectively; the animals were free to drink water and eat. After about 1 week of normal feeding, mice with good physical signs were selected for this experiment after veterinary examination. Before grouping, the animal was marked at the base of the tail with a marker pen, and after grouping, each animal was marked by ear cut.

[0256] 2.3. Transplantable Tumor Cells

[0257] Human prostate cancer cells PC-3 were obtained from the cell bank of Type Culture Collection Committee of Chinese Academy of Sciences (CAS, frozen in liquid nitrogen in our laboratory).

[0258] 3. Experimental Methods

[0259] 3.1 Cell culture

[0260] PC-3 cells were routinely cultured in F-12K medium containing 10% fetal bovine serum under 5% CO2 and 37°C conditions; they were digested and passaged with 0.25% trypsin; and the passages were performed according to the cell growth conditions, with a passage ratio of 1:3 to 1:6.

[0261] 3.2 Animal model preparation

[0262] PC-3 cells in the logarithmic growth phase were collected, counted, and resuspended in serum-free F-12K medium to adjust the cell concentration to 5×10 7 cells / mL; pipette the cells to disperse them evenly and then put them into a 50-mL centrifuge tube, which was placed in an ice box; use a 1-mL syringe to draw up the cell suspension and inject it into the subcutaneous part of the right axilla of nude mice. Each animal was inoculated with 100 μL (6×10 6The PC-3 nude mouse transplant tumor model was established. The animal status and tumor growth were observed regularly after inoculation. The tumor diameter was measured with an electronic vernier caliper and the data was entered into an Excel spreadsheet to calculate the tumor volume. When the tumor volume reached 100-300 mm 3 35 animals with good health and similar tumor volume were selected and divided into 7 groups (n=5) by random block method. The day of grouping was the first day of the experiment (D1). After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal weight was weighed and recorded.

[0263] The tumor volume (TV) was calculated as follows:

[0264] TV(mm 3 )=l×w 2 / 2

[0265] Wherein, l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).

[0266] 3.3 Drug delivery preparation

[0267] Note: The volume of solvent added in the following preparation process can be adjusted proportionally according to the actual weight measured.

[0268] 3.3.1 Preparation of BT8009 drug delivery preparation

[0269] Before each administration, an appropriate amount of BT8009 was accurately weighed and added to an appropriate volume of PBS (pH 7.4). The drug was vortexed and sonicated to completely dissolve the drug and then filtered to obtain a concentration of 0.15 mg mL -1 The drug preparation can be prepared and used immediately.

[0270] 3.3.2 Preparation of BT5528 drug delivery preparation

[0271] Before each administration, accurately weigh an appropriate amount of BT5528, add an appropriate volume of ethanol, vortex to completely dissolve the compound, add an appropriate volume of ultrapure water, vortex to mix, filter to obtain a dosage preparation with a concentration of 0.16 mg / ml, and use it immediately. The ratio of ethanol to water in the dosage preparation is 10% and 90%.

[0272] 3.3.3 Preparation of Compounds B1 and B2 for Administration

[0273] Before each administration, accurately weigh appropriate amounts of B1 and B2, add an appropriate volume of ethanol, vortex to completely dissolve the compound, add an appropriate volume of ultrapure water, vortex to mix, filter to obtain administration preparations with concentrations of 0.28 and 0.32 mg / ml (low concentrations can be prepared by diluting high concentrations), and use them immediately. The ratio of ethanol to water in the administration preparation is 10% and 90%.

[0274] 3.3.4 Blank solvent

[0275] Take appropriate volumes of ethanol and ultrapure water respectively, mix them evenly, use them as blank solvents, and store them at room temperature for future use. The ratio of ethanol to water in the mixed solution is 10% and 90%.

[0276] 3.4 Animal grouping and drug administration

[0277] Animal grouping and dosing schedule are shown in Table 2. Dosing began on the day of grouping (D1), and the dosing volume was 10 mL kg -1 Each group was injected via tail vein (iv) once a week (QW), and the specific number of administrations and the administration cycle were determined according to the actual situation.

[0278] Table 2. Animal groups and drug administration

[0279]

[0280] 3.5 End of the experiment

[0281] On the last day of the experiment, the animal weights were recorded, the tumor diameters were measured, all experimental animals were euthanized (CO2), the tumor tissues were removed, weighed, and photographed, and the animals were grossly dissected to observe whether there were any abnormalities in the internal organs. The animal carcasses and tumor tissues were placed in the carcass storage refrigerator for unified treatment.

[0282] 4. Data recording and calculation formula

[0283] The relative tumor volume (RTV) was calculated as:

[0284] RTV=TV t / TV initial

[0285] Among them, TV initial TV is the tumor volume measured at the time of group administration; t It is the tumor volume at each measurement during the drug administration period.

[0286] The calculation formula of relative tumor proliferation rate (%T / C) is:

[0287] %T / C=100%×(RTV T / RTV C )

[0288] Among them, RTV T represents the treatment group RTV; RTV C represents the solvent control group RTV.

[0289] The calculation formula of tumor growth inhibition rate TGI (%) is:

[0290] TGI = 100% × [1-(TV t(T) -TV initial(T) ) / (TV t(C) -TV initial(C) )]

[0291] Among them, TV t(T) TV represents the tumor volume of each treatment group; initial(T) TV represents the tumor volume of the treatment group at the time of group administration; t(C) TV represents the tumor volume of the vehicle control group at each measurement; initial(C) It represents the tumor volume of the solvent control group at the time of group administration.

[0292] The formula for calculating the animal weight loss rate is:

[0293] Animal weight loss rate = 100% × (BW initial -BW t ) / BW initial

[0294] Among them, BW t Indicates the animal body weight measured at each time during the dosing period; BW initial Indicates the body weight of animals at the time of grouping and dosing.

[0295] The calculation formula of tumor weight inhibition rate IR (%) is:

[0296] IR(%)=100%×(W C -W T ) / W C

[0297] Among them, W C W represents the tumor weight of the control group; T Indicates tumor weight in treatment group.

[0298] Tumor growth delay time (TGD): The calculation formula is:

[0299] TGD=T T -T C

[0300] Among them, T T It indicates the time required for the relative tumor volume of animals in the experimental group to increase to 4 times the volume at the beginning of the experiment;

[0301] T C It indicates the time required for the relative tumor volume of animals in the vehicle control group to increase to 4 times the volume at the beginning of the experiment.

[0302] 5. Statistical analysis methods

[0303] The experimental data were calculated and statistically processed using Microsoft Office Excel software. Unless otherwise specified, the data were expressed as mean ± standard error (Mean ± SE), and the comparison between the two groups was performed using t-test.

[0304]

[0305]

[0306]

[0307]

[0308] In this experiment, we evaluated the in vivo efficacy of BT8009, BT5528, and compounds B1 and B2 in a human prostate cancer PC-3 nude mouse xenograft tumor model. The tumor volumes of each group at different time points are shown in Tables 3-4 and Figure 6-7 22 days after the start of drug administration, the average tumor volume of the solvent control group reached 1048mm 3 The average tumor volume of the positive control group BT8009 was 124 mm 3 , T / C value was 12%; the average tumor volume of the positive control group BT5528 was 42.2 mm 3 , T / C value was 4.1%; the average tumor volume of the B2 group was 35.8 mm 3 , T / C value was 3.4%; the average tumor volume of the test substance B1 group was 41.0 mm 3 , T / C value was 4.1%; compared with the solvent control group, BT8009, BT5528, B2, and B1 groups all had certain anti-tumor effects; the anti-tumor effects of the test substances B1 and B2 groups were significantly better than those of the positive control group BT8009, and slightly better than those of the positive control group BT5528.

[0309] From Table 5-6 and Figure 8-9 The changes in animal body weight over time showed that the body weight of mice treated with test compounds B1 and B2 did not decrease by more than 10%, indicating good safety. Fig.10 The bar graphs show the effects of BT8009, BT5528, B1 and B2 on tumor weight in human prostate cancer PC-3 nude mouse transplanted tumor model. It can be seen that the final tumor weight of the tested B1 and B2 compounds was significantly lower than that of the BT8009 group and was equivalent to that of the BT5528 group.

[0310] Experimental Example 2. Determination of the inhibitory effect on tumor growth in vivo in a mouse transplanted tumor model of human breast cancer cells MDA-MB-468 Experimental Purpose

[0311] The inhibitory effects of the four test samples, including the control group BT8009, the control group BT5528, and the B1 and B2 compounds, on the in vivo growth of tumors in the human breast cancer cell MDA-MB-468 mouse transplant tumor model were evaluated.

[0312] 1. Experimental Materials

[0313] 1.1. Compound information

[0314]

[0315] 1.2. Experimental animals

[0316] Female NOD / SCID mice (number: 45; age: 6-8 weeks) were purchased from Weitong Lihua Experimental Animal Technology Co., Ltd. and raised in the SPF animal room of Suzhou Shengsu New Drug Development Co., Ltd. The temperature in the animal breeding room was 20-25°C, the relative humidity was 40%-70%, and the light and dark lighting were 12 hours each; the animals were free to drink water and eat. After about 1 week of normal feeding, mice with good physical signs were selected for this experiment after veterinary examination. Before the animals were grouped, they were marked at the base of the tail with a marker pen. After grouping, each animal was marked by ear cut.

[0317] 1.3. Transplantable tumor lines

[0318] Human breast cancer cell line MDA-MB-468 was obtained from the cell bank of Type Culture Collection Committee of Chinese Academy of Sciences (CAS, cryopreserved in liquid nitrogen in our laboratory).

[0319] 2. Experimental Methods

[0320] 3.1 Cell culture

[0321] MDA-MB-468 cells were routinely cultured in MEM medium containing 10% fetal bovine serum under conditions of 5% CO2, 37°C, and saturated humidity. Depending on the growth of the cells, they were digested and passaged with 0.25% trypsin, 1 to 2 times a week, with a passage ratio of 1:3 to 1:6.

[0322] 3.2 Animal model preparation

[0323] MDA-MB-468 cells in the logarithmic growth phase were collected, counted, and resuspended in matrix gel to adjust the cell concentration to 5×10 7 cells / mL; pipette the cells to disperse them evenly and then put them into a 50-mL centrifuge tube, which was placed in an ice box; use a 1-mL syringe to draw up the cell suspension and inject it into the subcutaneous part of the right axilla of the mouse. Each animal was inoculated with 200 μL (10×10 6The MDA-MB-468 mouse transplant tumor model was established. The animal status and tumor growth were observed regularly after inoculation. The tumor diameter was measured using an electronic vernier caliper. The data was directly entered into an Excel spreadsheet to calculate the tumor volume. When the tumor volume reached 100-300 mm 3 30 animals with good health and similar tumor volume were selected and divided into 5 groups (n=6) by random block method. The day of grouping was the first day of the experiment (D1). After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal weight was weighed and recorded.

[0324] The tumor volume (TV) was calculated as follows:

[0325] TV(mm 3 )=l×w 2 / 2

[0326] Wherein, l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).

[0327] 3.3 Preparation of drug delivery preparations

[0328] Note: The volume of solvent added in the following preparation process can be adjusted proportionally according to the actual weight measured.

[0329] 3.3.1 Preparation of BT8009 drug delivery preparation

[0330] Before each administration, an appropriate amount of BT8009 was accurately weighed, and an appropriate volume of PBS (pH 7.4) was added. The drug was completely dissolved by vortexing and sonication, and then filtered to obtain a concentration of 0.1 mg mL -1 The drug preparation can be prepared and used immediately.

[0331] 3.3.2 Preparation of BT5528 drug delivery preparation

[0332] Before each administration, accurately weigh an appropriate amount of BT5528, add an appropriate volume of ethanol, vortex to completely dissolve the compound, add an appropriate volume of ultrapure water, vortex to mix, filter to obtain a dosage preparation with a concentration of 0.11 mg / ml, and use it immediately. The ratio of ethanol to water in the dosage preparation is 10% and 90%.

[0333] 3.3.3 Preparation of B2 drug delivery preparation

[0334] Before each administration, accurately weigh an appropriate amount of B2, add an appropriate volume of ethanol, vortex to completely dissolve the compound, add an appropriate volume of ultrapure water, vortex to mix, filter to obtain administration preparations with concentrations of 0.07 and 0.2 mg / ml (low concentrations can be prepared by diluting high concentrations), and use them immediately. The ratio of ethanol to water in the administration preparation is 10% and 90%.

[0335] 3.3.4 Blank solvent

[0336] Take appropriate volumes of ethanol and ultrapure water respectively, mix them evenly, use them as blank solvents, and store them at room temperature for future use. The ratio of ethanol to water in the mixed solution is 10% and 90%.

[0337] 3.4 Animal grouping and drug administration

[0338] Animal grouping and dosing schedule are shown in Table 7. Dosing began on the day of grouping (D1), and the dosing volume was 10 mL·kg -1 Each group was injected via tail vein (iv) once a week (QW), and the specific number of administrations and the administration cycle were determined according to the actual situation.

[0339] Table 7. Animal grouping and dosing

[0340]

[0341] 3.5 End of the experiment

[0342] On the last day of the experiment, the animal weights were recorded, the tumor diameters were measured, all experimental animals were euthanized (CO2), the tumor tissues were removed, weighed, and photographed, and the animals were grossly dissected to observe whether there were any abnormalities in the internal organs. The animal carcasses and tumor tissues were placed in the carcass storage refrigerator for unified treatment.

[0343] 3. Data recording and calculation formula

[0344] The relative tumor volume (RTV) was calculated as:

[0345] RTV=TV t / TV initial

[0346] Among them, TV initial TV is the tumor volume measured at the time of group administration; t It is the tumor volume at each measurement during the drug administration period.

[0347] The calculation formula of relative tumor proliferation rate (%T / C) is:

[0348] %T / C=100%×(RTV T / RTV C )

[0349] Among them, RTV T represents the treatment group RTV; RTV C represents the solvent control group RTV.

[0350] The calculation formula of tumor growth inhibition rate TGI (%) is:

[0351] TGI = 100% × [1-(TV t(T) -TV initial(T) ) / (TV t(C) -TV initial(C) )]

[0352] Among them, TV t(T) TV represents the tumor volume of each treatment group; initial(T) TV represents the tumor volume of the treatment group at the time of group administration; t(C) TV represents the tumor volume of the vehicle control group at each measurement; initial(C) It represents the tumor volume of the solvent control group at the time of group administration.

[0353] The formula for calculating the animal weight loss rate is:

[0354] Animal weight loss rate = 100% × (BW initial -BW t ) / BW initial

[0355] Among them, BW t Indicates the animal body weight measured at each time during the dosing period; BW initial Indicates the body weight of animals at the time of grouping and dosing.

[0356] The calculation formula of tumor weight inhibition rate IR (%) is:

[0357] IR(%)=100%×(W C -W T ) / W C

[0358] Among them, W C W represents the tumor weight of the control group; T Indicates tumor weight in treatment group.

[0359] Tumor growth delay time (TGD): The calculation formula is:

[0360] TGD=T T -T C

[0361] Among them, T T It indicates the time required for the relative tumor volume of animals in the experimental group to increase to 4 times the volume at the beginning of the experiment;

[0362] T C It indicates the time required for the relative tumor volume of animals in the vehicle control group to increase to 4 times the volume at the beginning of the experiment.

[0363] 4. Statistical analysis methods

[0364] The experimental data were calculated and statistically processed using Microsoft Office Excel software. Unless otherwise specified, the data were expressed as mean ± standard error (Mean ± SE), and the comparison between the two groups was performed using t-test.

[0365] The experimental results are shown in the following table:

[0366]

[0367]

[0368] In this experiment, we evaluated the in vivo efficacy of BT8009, BT5528 and B2 compounds in the human breast cancer cell MDA-MB-468 mouse transplant tumor model. The tumor volumes of each group at different time points are shown in Tables 8-9 and Figure 11-12 36 days after the start of drug administration, the average tumor volume of the solvent control group reached 625mm 3 The average tumor volume of the positive control group BT8009 was 371 mm 3 , T / C value was 58%; the average tumor volume of the positive control group BT5528 was 368mm 3 , T / C value was 57%; the average tumor volume of the test substance B2 group (0.7 mg / kg, 1 / 3 molar amount of MMAE in the positive control group) was 463 mm 3 , T / C value was 73%; the average tumor volume of the test substance B2 group (2.0 mg / kg, MMAE with the same molar amount as the positive control group) was 430 mm 3 , T / C value was 68%; compared with the solvent control group, BT8009, BT5528 and B2 groups all had certain anti-tumor effects; the anti-tumor effect of the test substance B2 group was equivalent to that of the positive control group.

[0369] Figure 13-14 The changes in animal weight over time showed that the mice maintained good weight and had good safety.

[0370] Experimental Example 3. Determination of the inhibitory effect on tumor growth in vivo in the human gastric cancer NCI-N87 nude mouse transplant tumor model

[0371] Purpose

[0372] The inhibitory effects of BT8009, BT5528 and B23 test samples on the in vivo growth of human gastric cancer NCI-N87 nude mouse transplant tumor model were evaluated.

[0373] 1. Experimental Materials

[0374] 1.1. Compound information

[0375]

[0376] 1.2. Experimental animals

[0377] Female BALB / c nude mice (number: 45; age: 6-7 weeks) were purchased from Weitong Lihua Experimental Animal Technology Co., Ltd. and raised in the SPF animal room of Suzhou Shengsu New Drug Development Co., Ltd. The temperature in the animal breeding room was 20-25°C, the relative humidity was 40%-70%, and the light and dark lighting were 12 hours each; the animals were free to drink water and eat. After about 1 week of normal feeding, mice with good physical signs were selected for this experiment after veterinary examination. Before the animals were grouped, they were marked with a marker pen at the base of the animal's tail. After grouping, each animal was marked by ear cut.

[0378] 1.3. Transplantable tumor lines

[0379] Human gastric cancer cells NCI-N87 were obtained from the cell bank of Type Culture Collection Committee of Chinese Academy of Sciences (CAS, frozen in liquid nitrogen in our laboratory).

[0380] 2. Experimental Methods

[0381] 3.1 NCI-N87 cell culture

[0382] NCI-N87 cells were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum under conditions of 5% CO2, 37°C, and saturated humidity. Depending on cell growth, cells were digested and passaged with 0.25% trypsin, 1 to 2 times a week, with a passage ratio of 1:3 to 1:6.

[0383] 3.2 Animal model preparation

[0384] Collect NCI-N87 cells in the logarithmic growth phase, count the cells and resuspend them in serum-free RPMI-1640 medium, and adjust the cell concentration to 5×10 7 cells / mL; pipette the cells to disperse them evenly and then put them into a 50-mL centrifuge tube, which was placed in an ice box; use a 1-mL syringe to draw up the cell suspension and inject it into the subcutaneous part of the right axilla of the nude mice. Each animal was inoculated with 100 μL (5×10 6 The NCI-N87 nude mouse transplant tumor model was established. The animal status and tumor growth were observed regularly after inoculation. The tumor diameter was measured using an electronic vernier caliper. The data was directly entered into an Excel spreadsheet to calculate the tumor volume. When the tumor volume reached 100-300mm 3 30 animals with good health and similar tumor volume were selected and divided into 5 groups (n=6) by random block method. The day of grouping was the first day of the experiment (D1). After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal weight was weighed and recorded.

[0385] The tumor volume (TV) was calculated as follows:

[0386] TV(mm 3 )=l×w 2 / 2

[0387] Wherein, l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).

[0388] 3.3 Preparation of drug delivery preparations

[0389] Note: The volume of solvent added in the following preparation process can be adjusted proportionally according to the actual weight measured.

[0390] 3.3.1 Preparation of BT8009 drug delivery preparation

[0391] Before each administration, an appropriate amount of BT8009 was accurately weighed, and an appropriate volume of PBS (pH 7.4) was added. The drug was completely dissolved by vortexing and sonication, and then filtered to obtain a concentration of 0.15 mg mL-1. 1 The drug preparation can be prepared and used immediately.

[0392] 3.3.2 Preparation of BT5528 drug delivery preparation

[0393] Before each administration, accurately weigh an appropriate amount of BT5528, add an appropriate volume of ethanol, vortex to completely dissolve the compound, add an appropriate volume of ultrapure water, vortex to mix, filter to obtain a dosage preparation with a concentration of 0.16 mg / ml, and use it immediately. The ratio of ethanol to water in the dosage preparation is 10% and 90%.

[0394] 3.3.3 Preparation of B2 drug delivery preparation

[0395] Before each administration, accurately weigh an appropriate amount of B2, add an appropriate volume of ethanol (low concentration can be prepared by diluting high concentration), vortex to completely dissolve the compound, add an appropriate volume of ultrapure water, vortex to mix, filter to obtain drug preparations with concentrations of 0.1 and 0.3 mg / ml, respectively, and use them immediately. The ratio of ethanol to water in the drug preparation is 10% and 90%.

[0396] 3.3.4 Blank solvent

[0397] Take appropriate volumes of ethanol and ultrapure water respectively, mix them evenly, use them as blank solvents, and store them at room temperature for future use. The ratio of ethanol to water in the mixed solution is 10% and 90%.

[0398] 3.4 Animal grouping and drug administration

[0399] Animal grouping and dosing schedule are shown in Table 10. Dosing began on the day of grouping (D1), and the dosing volume was 10 mL kg -1Each group was injected via tail vein (iv) once a week (QW), and the specific number of administrations and the administration cycle were determined according to the actual situation.

[0400] Table 10. Animal grouping and dosing

[0401]

[0402] 3.5 Accompanying PK

[0403] On the last day of the experiment (D22), the mice were weighed and the tumor diameter was measured.

[0404] Whole blood and tumor tissues were collected from animals in group A as blank matrix. Whole blood and tumors were collected from animals in groups B, C, D, and E at different time points after the last administration. The collection time points are detailed in Table 11.

[0405] Table 11. Blood sample and tumor collection schedule

[0406]

[0407] Blood sample collection method: At the set time point, the animal is deeply anesthetized with CO2, and about 300 μL of blood is collected by cardiac puncture and placed in an EDTA-K2 anticoagulant tube. After blood collection, it is immediately placed on wet ice (the storage time shall not exceed 15 minutes).

[0408] Blood sample processing and storage (for test sample concentration determination): Whole blood samples were centrifuged at 1500-1600 g and 4°C for 10 min, and plasma was collected. Plasma samples were stored in a refrigerator below -60°C for future use.

[0409] Tissue collection and preservation: Tumors were collected at different time points after drug administration, cleaned with cold PBS, weighed, quickly frozen in liquid nitrogen, and then stored at low temperature (below -60°C).

[0410] 4. Data recording and calculation formula

[0411] The relative tumor volume (RTV) was calculated as:

[0412] RTV=TV t / TV initial

[0413] Among them, TV initial TV is the tumor volume measured at the time of group administration; t It is the tumor volume at each measurement during the drug administration period.

[0414] The calculation formula of relative tumor proliferation rate (%T / C) is:

[0415] %T / C=100%×(RTV T / RTVC )

[0416] Among them, RTV T represents the treatment group RTV; RTV C represents the solvent control group RTV.

[0417] The calculation formula of tumor growth inhibition rate TGI (%) is:

[0418] TGI = 100% × [1-(TV t(T) -TV initial(T) ) / (TV t(C) -TV initial(C) )]

[0419] Among them, TV t(T) TV represents the tumor volume of each treatment group; initial(T) TV represents the tumor volume of the treatment group at the time of group administration; t(C) TV represents the tumor volume of the vehicle control group at each measurement; initial(C) It represents the tumor volume of the solvent control group at the time of group administration.

[0420] The formula for calculating the animal weight loss rate is:

[0421] Animal weight loss rate = 100% × (BW initial -BW t ) / BW initial

[0422] Among them, BW t Indicates the animal body weight measured at each time during the dosing period; BW initial Indicates the body weight of animals at the time of grouping and dosing.

[0423] The calculation formula of tumor weight inhibition rate IR (%) is:

[0424] IR(%)=100%×(W C -W T ) / W C

[0425] Among them, W C W represents the tumor weight of the control group; T Indicates tumor weight in treatment group.

[0426] Tumor growth delay time (TGD): The calculation formula is:

[0427] TGD=T T -T C

[0428] Among them, T T It indicates the time required for the relative tumor volume of animals in the experimental group to increase to 4 times the volume at the beginning of the experiment;

[0429] T C It indicates the time required for the relative tumor volume of animals in the vehicle control group to increase to 4 times the volume at the beginning of the experiment.

[0430] 5. Statistical analysis methods

[0431] The experimental data were calculated and statistically processed using Microsoft Office Excel software. Unless otherwise specified, the data were expressed as mean ± standard error (Mean ± SE), and the comparison between the two groups was performed using t-test.

[0432] Table 12 Changes of tumor volume over time

[0433]

[0434] Table 13 Changes of relative tumor volume over time

[0435]

[0436] In this experiment, we evaluated the in vivo efficacy of BT8009, BT5528 and B2 compounds in the human gastric cancer NCI-N87 nude mouse xenograft tumor model. The tumor volumes of each group at different time points are shown in Tables 12-13 and Figure 15-16 22 days after the start of drug administration, the average tumor volume of the solvent control group reached 960mm 3 The average tumor volume of the positive control group BT8009 was 330 mm 3 , T / C value was 34%; the average tumor volume of the positive control group BT5528 was 293mm 3 , T / C value was 30%; the average tumor volume of the test substance B2 group (1.0 mg / kg, 1 / 3 molar amount of MMAE in the positive control group) was 664 mm 3 , T / C value was 68%; the average tumor volume of the test substance B2 group (3.0 mg / kg, MMAE with the same molar amount as the positive control group) was 351 mm 3 , T / C value is 36%. Equimolar doses (MMAE) of BT8009, BT5528, and B2 (3.0 mg / kg) have significant growth inhibitory effects on gastric cancer NCI-N87 nude mouse transplanted tumors, and the anti-tumor activities of the three are comparable; B2 compound can inhibit the growth of NCI-N87 nude mouse transplanted tumors in a dose-dependent manner.

[0437] Figure 17-18 The changes in animal weight over time showed that the relative weight loss rate of mice was less than 10%, and their weight was well maintained, with good safety. Fig.19It is a bar graph showing the effects of BT8009, BT5528 and B2 on tumor weight in the human gastric cancer NCI-N87 nude mouse xenograft tumor model.

[0438] Experimental Example 4.

[0439] 1. Experimental purpose: To evaluate the inhibitory effect of three test samples, BT8009, BT5528 and A1 compound, on the growth of human gastric cancer NCI-N87 nude mouse transplanted tumor model in vivo.

[0440] 2. Experimental Materials

[0441] 2.1 Compound information

[0442]

[0443] A1 Drug delivery preparation

[0444] Before each administration, accurately weigh an appropriate amount of compound A1, add an appropriate volume of DMSO, vortex to completely dissolve the compound, add an appropriate volume of PEG400, vortex to add an appropriate volume of ultrapure water, mix the liquid evenly, filter to obtain a dosage preparation with a concentration of 0.26 mg / ml, and use it immediately. The proportions of DMSO, PEG400 and ultrapure water in the dosage preparation are 10%, 20% and 70%.

[0445] For other specific experiments and calculation methods, please refer to the above Experimental Example 3, which will not be repeated here. The results are as follows:

[0446]

[0447]

[0448]

[0449]

[0450] In this experiment, we evaluated the in vivo efficacy of BT8009, BT5528 and A1 compounds in the human gastric cancer NCI-N87 nude mouse xenograft tumor model. The tumor volumes of each group at different time points are shown in Tables 14-15 and Figure 20-21 25 days after the start of drug administration, the average tumor volume of the solvent control group reached 1171mm 3 The average tumor volume of the positive control group BT8009 was 440 mm 3 , T / C value was 37%; the average tumor volume of the positive control group BT5528 was 357mm 3 , T / C value was 30%; the average tumor volume of the test substance A1 group was 372mm 3, T / C value was 31%; equimolar amounts of BT8009, BT5528, and Al had a significant growth inhibitory effect on gastric cancer NCI-N87 nude mouse transplanted tumors, and the anti-tumor activities of the three were comparable.

[0451] From Table 16-17 and Figure 22-23 It can be seen that the relative weight loss rate of mice is small, their weight is well maintained, and they have good safety.

[0452] In summary, the dual-target drug of the present invention can significantly inhibit the growth of tumor cells and is dose-dependent. In addition, experiments show that the dual-target drug of the present invention has equivalent efficacy to the single-target drug of equimolar dose. The dual-target PDC drug of the present invention greatly expands the applicable population. A dual-target PDC molecule of the present invention can reach the coverage population of two drugs (Nectin4 and Aph2, Nectin4 and MT1-MMP), and the cost is also lower, which has very good application prospects in clinical practice.

Claims

1. A drug conjugate of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof, in: D is for medicine, L is a cleavable linking group, Y is a linking group formed by coupling an azide functional group with an alkyne functional group, X1 and X2 are each independently a molecular spacer group, and P1 and P2 are each independently selected from cyclic peptide ligands.

2. A drug conjugate according to claim 1, wherein D is selected from cytotoxic agents used in cancer treatment, such as alkylating agents such as cisplatin and carboplatin, and oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites, including purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids, including vinca alkaloids, such as vincristine, vinblastine, vinorelbine and vindesine; podophyllotoxin and its derivatives etoposide and teniposide; Taxanes, including paclitaxel; topoisomerase inhibitors, including camptothecin, irinotecan and topotecan, and type II inhibitors, including acridine, etoposide, etoposide phosphate and teniposide, antitumor antibiotics, including immunosuppressants actinomycin D, doxorubicin, epirubicin, bleomycin, calicheamicin, etc., maytansinoids, such as DM1 or monomethyl auristatins such as MMAE.

3. The drug conjugate according to claim 1, wherein D is selected from DM1 of the following formula Or monomethyl auristatin of the formula 4. The drug conjugate according to any one of claims 1 to 3, wherein L is a linker group comprising one or more amino acid residues selected from alanine (Ala), citrulline (Cit), lysine (Lys), tryptophan (Trp) and valine (Val), such as -Val-Cit-, -Trp-Cit-, -Val-Lys-, -D-Trp-Cit-, -PAB-Cit-Val-glutaryl- or -PAB-cyclobutyl-Ala-Cit-βAla-, wherein PAB represents p-aminobenzyloxycarbonyl; or L is a linker group comprising a cleavable disulfide bond, such as -SS-, -SS(SO3H)-, -SS-(Me)-, -(Me)-SS-(Me)-, -SS-(Me2)- or -SS-(Me)-SO3H-; Preferably, L is -PAB-Cit-Val-glutaryl- or -PAB-cyclobutyl-Ala-Cit-βAla-.

5. The drug conjugate according to any one of claims 1 to 4, wherein the cytotoxic agent is MMAE, and L is selected from -Val-Cit-, -Val-Lys-, -PAB-Cit-Val-glutaryl- or -PAB-cyclobutyl-Ala-Cit-βAla-; preferably, the cytotoxic agent is MMAE, and L is selected from -PAB-Cit-Val-glutaryl-.

6. The drug conjugate according to any one of claims 1 to 5, wherein the DL- portion has the structure of the following formula, the right-hand bond of which is connected to N in formula (I):

7. The drug conjugate according to any one of claims 1 to 4, wherein the cytotoxic agent is DM1 and L is selected from -SS-, -SS(SO3H)-, -SS-(Me)-, -(Me)-SS-(Me)-, -SS-(Me2)- and -SS-(Me)-SO3H-.

8. The drug conjugate according to any one of claims 1 to 7, wherein Y is selected from A mixture of two isomers, R1 is C3-C8 alkylene, R2 is -(C4-C8 alkylene)-CO-NH-, wherein one or more carbon atoms in the C4-C8 alkylene are optionally replaced by an atom or group selected from N, O, S and (C=O), R3 is selected from hydrogen, C1-C6 alkyl, aryl and heteroaryl, or R3 is connected to the carbon atom or optional N atom in -(C4-C8 alkylene)- of R2 to form a 5- to 8-membered cyclic hydrocarbon group or a 5- to 8-membered heterocyclic ring, and the 5- to 8-membered cyclic hydrocarbon group or the 5- to 8-membered heterocyclic ring is optionally condensed with one or two rings independently selected from aromatic rings or heteroaromatic rings.

9. The drug conjugate according to any one of claims 1 to 8, which is a drug conjugate comprising one of the following Y groups or a mixture of two drug conjugates: Wherein n is an integer of 3-6, preferably, the left bond of the group is connected to the carbon atom in formula (I), and the right bond is connected to the X2 part.

10. The drug conjugate according to claim 9, which is a drug conjugate comprising one of the following Y groups or a mixture of two drug conjugates: Its left bond is connected to the carbon atom in formula (I), and its right bond is connected to the X2 part.

11. The drug conjugate according to any one of claims 1 to 10, wherein X1 and X2 are each independently selected from -(CH2) p -(C=O)-(Sar) q -、-(CH2CH2O) k -(Sar) q -、 -(CH2CH2O) k -CH2-(C=O)-β-Ala-(Sar) q -, wherein Sar is sarcosine, k = 1-8, p = 1-4, q = 1-10.

12. The drug conjugate according to claim 11, wherein X1 is selected from X2 is selected from -(CH2CH2O) k -CH2-(C=O) -β-Ala-(Sar) q -, wherein Sar is sarcosine, k = 1-8, q = 1-10.

13. The drug conjugate according to claim 11, wherein X1 and X2 are both selected from -(CH2) p -(C=O)-(Sar) q -,in, p=1-4,q=1-10.

14. The drug conjugate according to any one of claims 1 to 13, wherein P1 and P2 are each independently selected from a bicyclic peptide ligand specific for bindin-4, a bicyclic peptide ligand specific for MT1-MMP or a bicyclic peptide ligand specific for EPHA2, wherein X1 and X2 are linked to the C-terminus or N-terminus of P1 and P2, respectively, provided that P1 and P2 are not the same.

15. The drug conjugate according to claim 14, wherein the binding protein-4 specific bicyclic peptide ligand is selected from 16. The drug conjugate according to claim 14, wherein the MT1-MMP specific bicyclic peptide ligand portion has the following structure 17. The drug conjugate according to claim 14, wherein the EPHA2-specific bicyclic peptide ligand portion has the following structure 18. The drug conjugate according to any one of claims 1 to 17, wherein the drug conjugate is selected from:

19. A drug conjugate selected from the group consisting of:

20. A pharmaceutical composition comprising the drug conjugate according to any one of claims 1 to 19 and one or more pharmaceutically acceptable excipients.

21. Use of the drug conjugate according to any one of claims 1 to 19 in the preparation of a medicament for preventing or treating cancer.

22. The use according to claim 21, wherein the cancer is selected from the group consisting of prostate cancer, lung cancer (e.g. non-small cell lung cancer (NSCLC)), breast cancer (e.g. triple negative breast cancer), gastric cancer, ovarian cancer, esophageal cancer, multiple myeloma and fibrosarcoma.

23. A bicyclic peptide ligand selected from the group consisting of:

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