Drug conjugate for integrin receptor and use thereof

Abstract

A polypeptide-coupled compound having high affinity to an integrin receptor (αVβ3) and a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the polypeptide-coupled compound and the pharmaceutically acceptable salt, and a use of a drug conjugate and the pharmaceutical composition in the preparation of a drug for preventing and / or treating αVβ3 overexpression diseases.

Description

Integrin receptor-coupled drugs and their applications Technical Field

[0001] The present invention belongs to the field of anti-tumor drugs, and specifically relates to a polypeptide-coupled drug targeting integrin receptors, and a preparation method and application thereof. Background Art

[0002] Numerous studies have shown that the molecular expression patterns on the surfaces of tumor cells and tumor vascular endothelial cells differ significantly from those of normal cells, making it possible to distinguish tumor tissue from normal tissue. Using targeting peptides as carriers, PDCs (polypeptide-drug conjugates) are formed through chemical linkage with cytotoxins. These peptides are then used to specifically target tumor cells, releasing the anticancer drug molecules they carry in the specific physiological environment of tumor cells or tissues (e.g., low pH, high expression of proteases, and high reducing properties). Receptor-targeted PDCs obtained based on the above mechanism have demonstrated more effective tumor treatment effects than free chemical drug molecules without peptide conjugation in various tumor models, including breast cancer, prostate cancer, and melanoma.

[0003] Summary of the Invention

[0004] The present invention provides a coupled compound specific for αVβ3, which has high affinity for the target, good selectivity, high stability, excellent pharmacokinetics, and is suitable for injection, inhalation, nasal, ocular, oral or local administration.

[0005] Specifically, the present invention provides a drug conjugate represented by formula (I) or its stereoisomers, pharmaceutically acceptable salts, PLD (I)

[0006] wherein P is selected from RGD peptide, and linear polypeptides and cyclic polypeptides containing RGD peptide sequences;

[0007] In some embodiments, P is selected from iRGD, cRGD;

[0008] In some embodiments, P is selected from the group consisting of iRGD, cRGDyC, cRGDyK, cRGDfC, cRGDfK;

[0009] In some embodiments, P is selected from

[0010] L is a linker; D is a cytotoxic agent;

[0011] In some embodiments, the cytotoxic agent is selected from the following group: alkylating agents, antimetabolites, plant alkaloids, terpenoids, podophyllotoxins and their derivatives, taxanes and their derivatives, topoisomerase inhibitors, tubulin polymerization inhibitors, RNA polymerase inhibitors, antitumor antibiotics; in some embodiments, the cytotoxic agent is selected from the following group: cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide, azathioprine, mercaptopurine, pyrimidine analogs, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, paclitaxel, Camptothecin, irinotecan, isenotecan, topotecan, acridine, etoposide, etoposide phosphate, teniposide, actinomycin D, doxorubicin, epirubicin, eribulin, rubectin, trabectedin, epothilone and its derivatives, bleomycin and its derivatives, dactinomycin and its derivatives, plicamycin and its derivatives, mitomycin C, calicinomycin, maytansine and its derivatives, auristatin and its derivatives; in some embodiments, the cytotoxic agent is selected from the following group: cisplatin, carboplatin , oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide, azathioprine, mercaptopurine, pyrimidine analogs, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, paclitaxel, camptothecin, irinotecan, topotecan, acridine, etoposide, etoposide phosphate, teniposide, actinomycin D, doxorubicin, epirubicin, eribulin, rubectin, trabectedin, epothilone and its derivatives, bleomycin and its derivatives, dactinomycin and its derivatives , plicamycin and its derivatives, mitomycin C, calicheamicin, maytansine and its derivatives, auristatin and its derivatives; in some embodiments, the cytotoxic agent is selected from docetaxel, MMAE, 7-ethylcamptothecin, irinotecan, isotecan, Dxd, eribulin, rubectin or trabectedin; in some embodiments, the cytotoxic agent is selected from docetaxel, MMAE, 7-ethylcamptothecin, irinotecan, Dxd, eribulin, rubectin or trabectedin;

[0012] In some embodiments, the cytotoxic agent is selected from the following structure:

[0013] In some embodiments, the cytotoxic agent is selected from the following structure:

[0014] In some embodiments, the linker is selected from one or more of the following groups: unmodified or modified single amino acids, unmodified or modified amino acid chains of different lengths, alkyl chains containing or not containing O, N heteroatoms,

[0015] m, n, p, q, and u are each independently selected from an integer of 0 to 20;

[0016] v is an integer selected from 1 to 100;

[0017] t is an integer selected from 1 to 150;

[0018] r is an integer selected from 0 to 150;

[0019] In some embodiments, the linker is selected from one or more of the following groups: unmodified or modified single amino acids, unmodified or modified amino acid chains of different lengths, alkyl chains containing or not containing O, N heteroatoms,

[0020] m, n, p, q, and u are each independently selected from an integer of 0 to 20;

[0021] v is an integer selected from 1-100; t is an integer selected from 1-150; r is an integer selected from 0-150;

[0022] In some embodiments, the linker is selected from one or more of the following groups: unmodified or modified single amino acids, unmodified or modified amino acid chains of different lengths, alkyl chains containing or not containing O, N heteroatoms,

[0023] m, n, p, q, and u are each independently selected from an integer of 0 to 20;

[0024] v is an integer selected from 1-100; t is an integer selected from 1-150; r is an integer selected from 0-150;

[0025] In some embodiments, the linker is selected from one or a combination of the following groups:

[0026] Unmodified or modified single amino acids, unmodified or modified amino acid chains of different lengths, alkyl chains with or without O and N heteroatoms,

[0027] m, n, p, q, and u are each independently selected from an integer of 0 to 20;

[0028] v is an integer selected from 1-100; t is an integer selected from 1-150; r is an integer selected from 0-150;

[0029] In some embodiments, the linker is selected from one or a combination of the following groups:

[0030] Unmodified or modified single amino acids, unmodified or modified amino acid chains of varying lengths, wherein the amino acids are selected from the group consisting of valine, citrulline, glycine, aspartic acid, proline, and phenylalanine;

[0031] Alkyl chains with or without O, N heteroatoms,

[0032] n is an integer selected from 1-5; p is an integer selected from 0-5; t is independently an integer selected from 2-100; r is an integer selected from 1-10; in some specific embodiments, the linker is selected from the following structures:

[0033] in, Indicates the site where L and P are linked, and * indicates the site where P and D are linked; in some specific embodiments, the linker is selected from the following structures:

[0034] in, Indicates the site where L and P are linked, and * indicates the site where P and D are linked;

[0035] In some embodiments, the linker is selected from the following structures:

[0036] in, Indicates the site where L and P are linked, and * indicates the site where P and D are linked;

[0037] In some embodiments, the linker is selected from the following structures:

[0038] in, Indicates the site where L and P are linked, and * indicates the site where P and D are linked;

[0039] In some embodiments, the linker is selected from the following structures:

[0040] in, Indicates the site where L and P are linked, and * indicates the site where P and D are linked.

[0041] The first technical solution of the present invention relates to a drug conjugate represented by formula (I) or its stereoisomers or pharmaceutically acceptable salts.

[0042] PLD (I)

[0043] wherein P is selected from RGD peptide, and linear polypeptides and cyclic polypeptides containing RGD peptide sequences;

[0044] L is a linker; D is a cytotoxic agent.

[0045] The second technical solution of the present invention is the drug conjugate of formula (I) or its stereoisomers, pharmaceutically acceptable salts, wherein the cytotoxic agent is selected from the following group: alkylating agents, antimetabolites, plant alkaloids, terpenoids, podophyllotoxins and their derivatives, taxanes and their derivatives, topoisomerase inhibitors, microtubule polymerization inhibitors, RNA polymerase inhibitors, and antitumor antibiotics.

[0046] The third technical solution of the present invention is the drug conjugate of formula (I) or its stereoisomers, pharmaceutically acceptable salts, wherein the cytotoxic agent is selected from the following group: cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide, azathioprine, mercaptopurine, pyrimidine analogs, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, paclitaxel, camptothecin, irinotecan, topotecan, acridine, etoposide, etoposide phosphate, teniposide, actinomycin D, doxorubicin, epirubicin, eribulin, rubectin, trabectedin, epothilones and their derivatives, bleomycins and their derivatives, dactinomycins and their derivatives, plicamycins and their derivatives, mitomycin C, calicheamicin, maytansines and their derivatives, auristatins and their derivatives, or isotecan.

[0047] The fourth technical solution of the present invention is the drug conjugate of formula (I) or its stereoisomers, pharmaceutically acceptable salts, wherein the cytotoxic agent is selected from docetaxel, MMAE, 7-ethylcamptothecin, irinotecan, Dxd, eribulin, rubectin or trabectedin or irinotecan.

[0048] The fifth technical solution of the present invention is the drug conjugate of formula (I) or its stereoisomers, pharmaceutically acceptable salts, wherein the linker is selected from one or a combination of the following groups:

[0049] Unmodified or modified single amino acids, unmodified or modified amino acid chains of different lengths, alkyl chains with or without O and N heteroatoms,

[0050] m, n, p, q, and u are each independently selected from an integer of 0 to 20;

[0051] v is selected from an integer of 1-100; t is selected from an integer of 1-150; and r is selected from an integer of 0-150.

[0052] The sixth technical solution of the present invention is the drug conjugate of formula (I) or its stereoisomers, pharmaceutically acceptable salts, wherein the linker is selected from the following structures:

[0053] in, Indicates the site where L and P are linked, and * indicates the site where P and D are linked.

[0054] As a more specific technical solution of the present invention, a drug conjugate or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the drug conjugate is selected from one of the following structures:

[0055] The present invention also relates to a pharmaceutical composition comprising the drug conjugate described in any of the aforementioned technical solutions, or its stereoisomers, or pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier and / or excipient. Furthermore, the pharmaceutical composition or pharmaceutical preparation comprises 1-1500 mg of the drug conjugate described in any of the aforementioned technical solutions, or its stereoisomers, or pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier and / or excipient.

[0056] The present invention also relates to a use of the drug conjugate described in any of the aforementioned technical solutions or its stereoisomers, pharmaceutically acceptable salts, or compositions thereof in the preparation of a drug for preventing and / or treating diseases that overexpress αVβ3.

[0057] Typically, the disease in which αVβ3 is overexpressed is cancer. Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (adenomas and various types of carcinomas, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, and other cancers), such as bladder and urinary tract cancer, breast cancer, gastrointestinal cancer (including esophageal cancer, stomach (gastric) cancer, small intestine cancer, colon cancer, rectal cancer, and anal cancer), liver cancer (hepatocellular carcinoma), gallbladder and biliary system cancer, exocrine pancreatic cancer, kidney cancer, lung cancer (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck cancer (e.g., tongue cancer, buccal cancer, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, tonsil cancer, salivary gland cancer, nasal cavity cancer, and paranasal sinus cancer), ovarian cancer, fallopian tube cancer, peritoneal cancer, vaginal cancer, vulvar cancer , penile cancer, cervical cancer, uterine fibroids, endometrial cancer, thyroid cancer (e.g., follicular thyroid cancer), renal cancer, prostate cancer, skin and appendage cancer (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthomas, dysplastic nevi); hematologic malignancies (i.e., leukemias, lymphomas) and premalignant hematologic conditions and borderline malignancies, including hematologic malignancies and related conditions of the lymphoid lineage (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer [NK] ] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), and hematologic malignancies and myeloid-related disorders (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocytic leukemia); tumors of mesenchymal origin, such as soft tissue sarcomas, bone or chondrosarcomas, such as osteosarcoma, fibrosarcoma, cartilage Sarcomas, rhabdomyosarcomas, leiomyosarcomas, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiosarcomas, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pinealomas, and schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid carcinoma); tumors of the eye and adnexa (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma);Pediatric and embryonic tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumor); or congenital or other forms of syndrome that predisposes the patient to malignant tumors (e.g., xeroderma pigmentosum). Preferably, the disease in which αVβ3 is overexpressed is selected from cancer. Further, the disease in which αVβ3 is overexpressed is selected from the group consisting of colon cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, brain glioma, and malignant sarcomas, carcinomas, and lymphomas of the breast, ovary, colon, kidney, hepatobiliary, lung, and brain.

[0058] The present invention also provides a method for treating a disease in a mammal or a human, comprising administering to a subject a therapeutically effective amount of the coupled compound according to any one of the aforementioned technical solutions, or its stereoisomers, pharmaceutically acceptable salts, or the pharmaceutical composition, and a pharmaceutically acceptable carrier and / or excipient, wherein the therapeutically effective amount is preferably 1-1500 mg; the diseases preferably include colon cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, glioma, and malignant sarcomas, cancers, and lymphomas of the breast, ovary, colon, kidney, liver and gallbladder, lung, and brain.

[0059] The present invention also provides a method for treating a disease in a mammal or human, comprising administering to the mammal or human a therapeutically effective amount of the conjugated compound of the present invention or a stereoisomer, pharmaceutically acceptable salt, or pharmaceutical composition thereof. In some embodiments, the mammal of the present invention does not include a human.

[0060] As used herein, an "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a conjugated compound disclosed herein to provide some relief from one or more symptoms of the disease or condition being treated. In some embodiments, the result is a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the amount of a conjugated compound disclosed herein required to provide a clinically significant reduction in disease symptoms. Examples of therapeutically effective amounts include, but are not limited to, 1-1500 mg, 1-1400 mg, 1-1300 mg, 1-1200 mg, 1-1000 mg, 1-900 mg, 1-800 mg, 1-700 mg, 1-600 mg, 1-500 mg, 1-400 mg, 1-300 mg, 1-250 mg, 1-200 mg, 1-150 mg, 1-125 mg, 1-100 mg, 1-80 mg, 1-60 mg, 1-50 mg, 1-40 mg, 1-25 mg, 1- 20mg, 5-1500mg, 5-1000mg, 5-900mg, 5-800mg, 5-700mg, 5-600mg, 5-500mg, 5-400mg, 5-300mg, 5-250mg, 5-200mg, 5 -150mg, 5-125mg, 5-100mg, 5-90mg, 5-70mg, 5-80mg, 5-60mg, 5-50mg, 5-40mg, 5-30mg, 5-25mg, 5-20mg, 10-1500mg, 10-1000mg, 10-900mg, 10-800mg, 10-700mg, 10-600mg, 10-500mg, 10-450mg, 10-400mg, 10-300mg, 10-250mg, 10-20 0mg, 10-150mg, 10-125mg, 10-100mg, 10-90mg, 10-80mg, 10-70mg, 10-60mg, 10-50mg, 10-40mg, 10-30mg, 10-20mg; 2 0-1500mg, 20-1000mg, 20-900mg, 20-800mg, 20-700mg, 20-600mg, 20-500mg, 20-400mg, 20-350mg, 20-300mg, 20-25 0mg, 20-200mg, 20-150mg, 20-125mg, 20-100mg, 20-90mg, 20-80mg, 20-70mg, 20-60mg, 20-50mg, 20-40mg, 20-30mg;50-1500mg, 50-1000mg, 50-900mg, 50-800mg, 50-700mg, 50-600mg, 50-500mg, 50-4 00mg, 50-300mg, 50-250mg, 50-200mg, 50-150mg, 50-125mg, 50-100mg; 100-1500mg, 100-1000mg, 100-900mg, 100-800mg, 100-700mg, 100-600mg, 100-500mg, 100-400mg, 100-300mg, 100-250mg, 100-200mg. ;

[0061] The present invention relates to a pharmaceutical composition or pharmaceutical preparation comprising a therapeutically effective amount of the drug conjugate of the present invention or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, and a carrier and / or excipient. The pharmaceutical composition may be in the form of a unit dosage form (the amount of the active ingredient in a unit dosage form is also referred to as the "drug strength"). In some embodiments, the pharmaceutical composition includes but is not limited to 1-1500 mg, 5-1000 mg, 10-800 mg, 20-600 mg, 25-500 mg, 40-200 mg, 50-100 mg, 1 mg, 1.25 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 125 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 0mg, 190mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg, 800mg, 850mg, 900mg, 950mg, 1000mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg of a coupled compound of the present invention or a pharmaceutically acceptable salt thereof.

[0062] A method for treating a disease in a mammal or a human, comprising administering the drug conjugate of the present invention or a stereoisomer, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient to a subject at a daily dose of 1-1500 mg / day. The daily dose can be a single dose or divided doses. In some embodiments, the daily dose includes but is not limited to 10-1500 mg / day, 20-1500 mg / day, 25-1500 mg / day, 50-1500 mg / day, 75-1500 mg / day, 100-1500 mg / day, 200-1500 mg / day, 10-1000 mg / day, 20-1000 mg / day, 25-1000 mg / day, 50-1000 mg / day, 75-1000 mg / day, 10 0-1000 mg / day, 200-1000 mg / day, 25-800 mg / day, 50-800 mg / day, 100-800 mg / day, 200-800 mg / day, 25-400 mg / day, 50-400 mg / day, 100-400 mg / day, 200-400 mg / day, in some embodiments, daily doses include but are not limited to 1 mg / day, 5 mg / day, 10 mg / day, 20 mg / day, 25 mg / day, 50 mg / day, 75 mg / day, 100 mg / day, 125 mg / day, 150 mg / day, 200 mg / day, 400 mg / day, 600 mg / day, 800 mg / day, 1000 mg / day, 1200 mg / day, 1400 mg / day, 1500 mg / day.

[0063] The present invention relates to a kit, which can include a composition in single-dose or multi-dose form, wherein the kit contains the conjugate of the present invention or its stereoisomers, or a pharmaceutically acceptable salt, and the amount of the conjugate of the present invention or the pharmaceutically acceptable salt is the same as that in the above-mentioned pharmaceutical composition.

[0064] In the present invention, the amount of the conjugate of the present invention or its stereoisomers or pharmaceutically acceptable salts is in each case calculated based on the free base form.

[0065] "Preparation specifications" refers to the weight of the main drug contained in each vial, tablet or other unit preparation.

[0066] Synthesis route

[0067] Those skilled in the art can prepare the compounds of the present invention by combining known organic synthesis techniques, using commercially available chemicals and / or compounds described in the chemical literature as starting materials. "Commercially available chemicals" are obtained from reputable commercial sources, including suppliers such as Titan Technology, Anage Chemical, Shanghai Demo, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, Nanjing Yaoshi, WuXi AppTec, and J&K Technology.

[0068] Specific and similar reactants can be selectively identified by indexes of known chemical substances prepared by the American Chemical Society's Chemical Abstracts Service, which are available in most public and university libraries and online. Chemicals that are known but not commercially available in the catalog are optionally prepared by custom chemical synthesis facilities, many of which standard chemical supply facilities (e.g., those listed above) offer custom synthesis services.

[0069] the term

[0070] The peptide sequences of the present invention can be synthesized using standard techniques and then reacted with a cytotoxic agent to which a linker is attached. When doing so, standard chemical methods can be used. This enables rapid, large-scale preparation of soluble materials for further downstream experiments or validation.

[0071] In some embodiments, the drug conjugates of the present invention are synthesized according to the following route:

[0072] For more specific synthesis steps, see Examples.

[0073] the term

[0074] Unless otherwise specified in the present invention, the terms of the present invention have the following meanings:

[0075] A derivative is a product derived from a compound in which the hydrogen atoms or atomic groups in the compound are replaced by other atoms or atomic groups.

[0076] Unless otherwise stated, all amino acids were used in the L-configuration.

[0077] DPhe:D-phenylalanine

[0078] Gln: glutamine

[0079] Ala: alanine

[0080] His:histidine

[0081] Trp: Tryptophan

[0082] Phe: Phenylalanine

[0083] Val: valine

[0084] Gly: glycine

[0085] RGD peptides are a series of polypeptides, including but not limited to iRGD and cRGD. cRGD also includes structures such as cRGDyC, cRGDyK, cRGDfC, and cRGDfK. RGD peptides are generally polypeptides with the following structure or containing the following amino acid sequence: Among them, the iRGD structure is as follows:

[0086] cRGD is a series of compounds, typical compounds include the following structures:

[0087] The preferred cRGD structure is as follows:

[0088] Or cRGDyK, the structure is as follows:

[0089] Common cytotoxic agents and structures:

[0090] DMF: N,N-dimethylacetamide;

[0091] DIC: N,N'-diisopropylcarbodiimide;

[0092] HOBT: 1-hydroxybenzotriazole;

[0093] EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0094] DMA: N,N-dimethylacetamide;

[0095] EEDQ: 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline;

[0096] DIEA: N,N-diisopropylethylamine;

[0097] MTBE: methyl tert-butyl ether;

[0098] TFA: trifluoroacetic acid;

[0099] MeCN: acetonitrile;

[0100] DMAP: 4-dimethylaminopyridine;

[0101] Sub: Resin substitution degree.

[0102] The carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds of the present invention include their isotopes, and the carbon, hydrogen, oxygen, sulfur, nitrogen or halogen involved in the groups and compounds of the present invention are optionally further replaced by one or more of their corresponding isotopes, wherein the isotopes of carbon include 12 C. 13 C and 14 C, hydrogen isotopes include protium (H), deuterium (deuterium, also known as heavy hydrogen), tritium (T, also known as super tritium), oxygen isotopes include 16 O. 17O and 18 O, sulfur isotopes include 32 S. 33 S. 34 S and 36 S, nitrogen isotopes include 14 N and 15 N, an isotope of fluorine 19 F, chlorine isotopes include 35 Cl and 37 Isotopes of Cl, bromine include 79 Br and 81 Br.

[0103] "Pharmaceutically acceptable salts" refer to salts of the compounds of the present invention that retain the biological effectiveness and properties of the free acids or free bases, and the free acids are reacted with non-toxic inorganic or organic bases, or the free bases are reacted with non-toxic inorganic or organic acids.

[0104] A "pharmaceutical composition" refers to a mixture of one or more compounds described herein, or stereoisomers, solvates, or pharmaceutically acceptable salts thereof, with other ingredients, wherein the other ingredients include physiologically / pharmaceutically acceptable carriers and / or excipients.

[0105] "Carrier" refers to a system that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered compound, and can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug and deliver the drug to the target organ. Non-limiting examples include microcapsules and microspheres, nanoparticles, liposomes, etc.

[0106] An "excipient" is a substance that is not itself a therapeutic agent but serves as a diluent, adjuvant, binder, and / or vehicle that is added to a pharmaceutical composition to improve its handling or storage properties or to allow or facilitate the formation of a compound or pharmaceutical composition into a unit dosage form for administration. As known to those skilled in the art, pharmaceutical excipients can serve a variety of functions and can be described as wetting agents, buffers, suspending agents, lubricants, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavoring agents, and sweeteners. Examples of pharmaceutical excipients include, but are not limited to: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, hydroxypropyl methylcellulose, hydroxypropyl cellulose, microcrystalline cellulose, and cross-linked carboxymethylcellulose (e.g., cross-linked sodium carboxymethylcellulose); (4) tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, and maltodextrin. oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffer solutions; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical preparations. DETAILED DESCRIPTION

[0107] The present invention will be described in detail below through examples. Where specific conditions are not specified in the examples, the experimental methods are carried out according to conventional conditions. The examples are provided to better illustrate the present invention, but it should not be understood that the present invention is limited to the examples. Any non-essential improvements and adjustments made by those skilled in the art to the embodiments based on the above invention are still within the scope of protection of the present invention.

[0108] Test Method

[0109] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). NMR shifts (δ) were expressed in 10 -6 The unit of (ppm) is given. NMR measurements were performed using a Bruker Avance III 400 and Bruker Avance 300 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS).

[0110] MS was determined using (Agilent 6120B (ESI) and Agilent 6120B (APCI));

[0111] HPLC determination was performed using an Agilent 1260DAD high pressure liquid chromatograph (Zorbax SB-C 18 100 × 4.6 mm, 3.5 μM);

[0112] Thin layer chromatography silica gel plates used were Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The specifications of the silica gel plates used for thin layer chromatography (TLC) were 0.15 mm to 0.20 mm, and the specifications used for thin layer chromatography separation and purification products were 0.4 mm to 0.5 mm.

[0113] Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

[0114] intermediates

[0115] Preparation of peptide 1

[0116] Step 1: The synthesis of 1a was carried out using standard Fmoc chemistry:

[0117] 1. Add CTC Resin (5.0 mmol, 5.0 g, sub: 1.0 mmol / g) and dichloromethane solvent to the reactor, swell for 30 min, add amino acid solution, react for 1 hour, and add 10 mL of methanol to react for 30 min.

[0118] 2. Drain and rinse three times with DMF.

[0119] 3. Add 20% piperidine / DMF and mix for 30 minutes.

[0120] 4. Drain and rinse five times with DMF.

[0121] 5. Add Fmoc-protected amino acid solution, mix for 30 seconds, then add coupling reagent, bubble nitrogen for 1 hour, and monitor the reaction with ninhydrin.

[0122] 6. Repeat steps 2-5 for the next amino acid coupling.

[0123] 7. In the last step, the product was washed twice with methanol, once with dichloromethane, and twice with methanol, and dried under vacuum to obtain peptide resin 1a (8 g), which was used directly in the next reaction.

[0124] Step 2: Add peptide resin 1a (8 g) to a 150 mL reaction flask, followed by 80 mL of cleavage solution (trifluoroethanol:dichloromethane (v:v) = 3:7), and stir at room temperature for 1 hour. Filter the resin to obtain a filtrate, which is then concentrated under reduced pressure to afford compound 1b (2.5 g).

[0125] Step 3: To a 2 L reaction flask, 1b (2.5 g, 2.4 mmol), dichloromethane (1.2 L), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (687 mg, 3.6 mmol), 1-hydroxybenzotriazole (486 mg, 3.6 mmol), and N-methylmorpholine (0.7 mL) were added sequentially. The mixture was stirred at room temperature for 16 h. The reaction mixture was washed with water (100 mL x 2) and saturated brine (100 mL x 2). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield compound 1c (2 g crude product), which was used directly in the next step.

[0126] Step 4: To a 200 mL reaction flask, trifluoroacetic acid (70 mL), triisopropylsilane (3 mL), and water (2 mL) were added sequentially. After stirring, compound 1c (2 g, 2 mmol) was added and the mixture was stirred at room temperature for 2 h. The reaction solution was added to 500 mL of methyl tert-butyl ether (the methyl tert-butyl ether was cooled to 0°C beforehand). Flocculent material precipitated and the mixture was centrifuged (3 min at 3000 rpm). The precipitate was washed three times with methyl tert-butyl ether and dried under vacuum to obtain the crude peptide, which was then separated by preparative HPLC. Methods: 1. Instrument: Waters 2767 preparative liquid chromatography; chromatographic column: SunFire@PrepC18 (19 mm × 250 mm). 2. The sample was dissolved in acetonitrile and water and filtered through a 0.45 μm filter to prepare a sample solution. 3. Preparative chromatography conditions: a. Mobile phase A, B composition: A: 5 mmol / L ammonium acetate / H2O, B: acetonitrile; b. Gradient elution: mobile phase content 10%-50%; c. Flow rate: 12 mL / min; d. Elution time: 20 min. Retention time: 12 min. Peptide 1 (130 mg) was obtained.

[0127] LCMS (ESI): m / z = 620.3 [M+H] + .

[0128] Preparation of intermediate 1

[0129] Step 1: In a 2L reaction flask, Boc-Gly-OH (2.78 g, 15.9 mmol) and dichloromethane (1.28 L) were added sequentially, followed by N,N'-diisopropylcarbodiimide (2.5 mL, 15.9 mmol), 4-dimethylaminopyridine (1.29 g, 10.6 mmol), and 7-ethylcamptothecin (2 g, 5.3 mmol). The mixture was stirred at room temperature for 16 h. The reaction solution was washed with water (50 mL × 1) and saturated brine (50 mL × 1). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 1a (3 g crude product), which was used directly in the next reaction.

[0130] LCMS (ESI): m / z = 534.3 [M+H] + .

[0131] Step 2: To a 100 mL reaction flask, add dichloromethane (30 mL), trifluoroacetic acid (30 mL), and compound 1a (3 g) in sequence. After complete addition, stir and react at room temperature for 1 hour. Concentrate directly under reduced pressure, and the resulting residue is separated by preparative HPLC. Method: 1. Apparatus: Waters 2767 Preparative HPLC; Column: SunFire@Prep C18 (19 mm × 250 mm). 2. Dissolve the sample in DMF and filter through a 0.45 μm filter to prepare a sample solution. 3. Preparative chromatographic conditions: a. Mobile phase A and B composition: Mobile phase A: acetonitrile; Mobile phase B: water (containing 0.1% trifluoroacetic acid); b. Gradient elution, mobile phase A content ranging from 10% to 60%; c. Flow rate: 12 mL / min; d. Elution time: 20 min. Intermediate 1 (1 g, yield: 41%) was obtained.

[0132] LCMS (ESI): m / z = 434.2 [M+H] + .

[0133] Preparation of intermediate 2

[0134] Step 1: The synthesis of 2a was carried out using standard Fmoc chemistry:

[0135] 1. Add CTC Resin (10.0 mmol, 10.0 g, sub: 1.0 mmol / g) and dichloromethane solvent to the reactor, swell for 30 minutes, add amino acid solution, react for 1 hour, and add 10 mL of methanol to react for 30 minutes.

[0136] 2. Drain and rinse three times with DMF.

[0137] 3. Add 20% piperidine / DMF and mix for 30 minutes.

[0138] 4. Drain and rinse five times with DMF.

[0139] 5. Add Fmoc-protected amino acid solution, mix for 30 seconds, then add coupling reagent, bubble nitrogen for 1 hour, and monitor the reaction with ninhydrin.

[0140] 6. Repeat steps 2-5 for the next amino acid coupling.

[0141] 7. In the last step, the product was washed twice with methanol, once with dichloromethane, and twice with methanol, and dried under vacuum to obtain peptide resin 2a (18 g), which was used directly in the next reaction.

[0142] Step 2: Add peptide resin 2a (18 g) to a 200 mL reaction flask, followed by lysis buffer (trifluoroethanol:dichloromethane (v:v) = 3:7, 150 mL), and stir at room temperature for 1 hour. Filter the resin to obtain the filtrate, which is then concentrated under reduced pressure to afford compound 2b (8 g crude product), which is used directly in the next reaction.

[0143] Step 3: To a 5 L reaction flask, compound 2b (8 g, 7.8 mmol), dichloromethane (4 L), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.2 g, 11.7 mmol), 1-hydroxybenzotriazole (1.6 g, 11.7 mmol), and N-methylmorpholine (2.5 mL) were added sequentially. The mixture was stirred at room temperature for 16 hours. The reaction solution was washed with water (100 mL × 2) and saturated brine (100 mL × 2). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 2c (7 g crude product), which was used directly in the next reaction.

[0144] Step 4: In a 2L reaction flask, methanol (800mL) and compound 2c (7g, 7mmol) were added sequentially. After stirring, 10% palladium-carbon catalyst (1g) was added, hydrogen was introduced, and the reaction was stirred at room temperature for 48 hours. The mixture was filtered directly, the filtrate was collected and concentrated, and the resulting residue was purified by preparative HPLC. Method: 1. Apparatus: Waters 2767 preparative liquid chromatography; chromatographic column: SunFire@PrepC18 (19mm×250mm). 2. The sample was dissolved in acetonitrile and water and filtered through a 0.45μm filter to prepare a sample solution. 3. Preparative chromatography conditions: a. Mobile phase A, B composition: A: 5mmol / L ammonium acetate / H2O, B: acetonitrile; b. Gradient elution: mobile phase content 10%-70%; c. Flow rate: 12mL / min; d. Elution time: 20min, retention time: 17min. Peptide 2 (2g) was obtained.

[0145] LCMS (ESI): m / z = 913.4 [M+H] + .

[0146] Step 5: Peptide 2 (740 mg, 0.81 mmol) was added to DMA (10 mL) and dichloromethane (5 mL), followed by N-hydroxysuccinimide (277 mg, 2.43 mmol) and EDCI (467 mg, 2.43 mmol). The mixture was stirred at room temperature under nitrogen for 18 h. The dichloromethane was removed by concentration in a 25°C water bath, and the resulting residue was purified by reverse phase column chromatography (H2O:MeCN:TFA (v:v:v) = 40:60:0.2) to obtain intermediate 2 (780 mg, yield: 95%).

[0147] LCMS (ESI): m / z = 1010.7 [M+H] + .

[0148] Preparation of peptide 3

[0149] Step 1: Crude peptide 3-1 was synthesized using standard Fmoc chemistry:

[0150] 1. Add Rink Amide-MBHA Resin (2.5 mmol, 5.0 g, sub: 0.5 mmol / g) and dichloromethane solvent to the reactor and allow to swell for 30 minutes. Then add 20% piperidine / N,N-dimethylformamide and mix for 30 minutes. After washing with N,N-dimethylformamide four times, add the amino acid solution and react for 1 hour. Monitor the reaction with ninhydrin.

[0151] 2. Drain and rinse three times with N,N-dimethylformamide.

[0152] 3. Add 20% piperidine / N,N-dimethylformamide and mix for 30 minutes.

[0153] 4. Drain and rinse five times with N,N-dimethylformamide.

[0154] 5. Add Fmoc-protected amino acid solution, mix for 30 seconds, then add coupling reagent, bubble nitrogen for 1 hour, and monitor the reaction with ninhydrin.

[0155] 6. Repeat steps 2 to 5 for the next amino acid coupling.

[0156] 7. In the last step, acetic anhydride and N,N-diisopropylethylamine were added and reacted for 30 minutes. The mixture was then rinsed twice with methanol, once with dichloromethane, and twice with methanol. The mixture was vacuum dried to obtain crude peptide 3-1 (9 g) as a peptide resin, which was directly used in the next reaction.

[0157] Step 2: In a 200 mL reaction flask, trifluoroacetic acid (70 mL), triisopropylsilane (3 mL), and water (2 mL) were added sequentially. After stirring, crude peptide 3-1 (9 g) was added and stirred at room temperature for 2 hours. The reaction solution was added to 500 mL of methyl tert-butyl ether (methyl tert-butyl ether was cooled to 0°C in advance). White flocculent material precipitated and centrifuged (3 minutes at 3000 rpm). The white precipitate was washed three times with methyl tert-butyl ether and dried under vacuum to obtain crude peptide 3-2 (2.3 g).

[0158] Step 3: Add 900 mL of water, 100 mL of acetonitrile, and crude peptide 3-2 (2.3 g) to a 2 L flask, stir to dissolve, and add 0.1 mol / L iodine / acetonitrile solution dropwise until the reaction solution turns yellow. Hold for 10 minutes, then add aqueous ascorbic acid to quench the reaction. Purification method: 1. Apparatus: Waters 2767 preparative liquid chromatography; chromatographic column: SunFire@PrepC18 (19 mm × 250 mm). 2. Dissolve the sample in acetonitrile and water, filter through a 0.45 μm filter to prepare a sample solution. 3. Preparative chromatography conditions: a. Mobile phase A, B composition: A: 0.1% trifluoroacetic acid / H2O, B: acetonitrile; b. Gradient elution: mobile phase content 10%-50%; c. Flow rate: 12 mL / min; d. Elution time: 20 min. Lyophilize to obtain peptide 3 (1 g).

[0159] LCMS (ESI): m / z = 989.4 [M+H] + .

[0160] Preparation of intermediate 3:

[0161] Step 1: Add known compound 3a (2.0 g, 3.9 mmol) and glutaric anhydride (0.89 g, 7.8 mmol) to N,N-dimethylformamide (20 mL), then add N,N-diisopropylethylamine (1.0 g, 7.8 mmol), stir at room temperature for 4 h, and directly purify the reaction solution by C18 reverse phase column chromatography (mobile phase A, B composition: mobile phase A: acetonitrile; mobile phase B: water (containing 0.1% TFA), A / B = 50 / 50) to obtain compound 3b (2.3 g, yield: 94%).

[0162] LCMS (ESI): m / z = 627.4 [M+H] + .

[0163] Step 2: Compound 3b (2.3 g, 3.6 mmol) was added to a mixed solution of TFA / DCM = 1:1 (20 mL), stirred at room temperature for 4 h, concentrated to dryness, and the crude product was purified by C18 reverse phase column chromatography (mobile phase A, B composition: mobile phase A: acetonitrile; mobile phase B: water (containing 0.1% TFA), A / B = 45 / 55) to obtain intermediate 3 (1.8 g, yield: 93%).

[0164] LCMS (ESI): m / z = 527.4 [M+H] + .

[0165] Preparation of intermediate 4:

[0166] Step 1: Compound 2A (1.5 g, 2.3 mmol), exitecan mesylate (1.5 g, 2.3 mmol), HOBT (0.31 g, 2.3 mmol) and DIEA (1.1 g, 6.9 mmol) were added to N,N-dimethylacetamide (15 mL), stirred at room temperature for 18 h, added with water (100 mL), extracted with ethyl acetate (100 mL × 2), the organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (methanol: dichloromethane (v:v) = 0-10%) to give compound 4a (1.3 g, 59.6%).

[0167] LCMS (ESI): m / z = 941.4 [M+H] + .

[0168] Step 2: Compound 4a (1.3 g, 1.38 mmol) was added to a mixed solution of TFA / DCM = 1:10 (26 mL), stirred at room temperature for 1 h, concentrated to dryness, and the crude product was purified by C18 reverse phase column chromatography (mobile phase A, B composition: mobile phase A: acetonitrile; mobile phase B: water (containing 0.1% TFA), A / B = 45 / 55) to obtain intermediate 4 (0.8 g, yield: 60.6%).

[0169] LCMS (ESI): m / z = 841.8 [M+H] + .

[0170] Preparation of intermediate 5:

[0171] Step 1: Compound cRGDyK (100 mg, 0.16 mmol), disuccinimidyl glutarate (115 mg, 0.48 mmol), and DIEA (92 mg, 0.96 mmol) were added to N,N-dimethylformamide (2 mL) and stirred at room temperature for 18 h. The reaction solution was purified by preparative HPLC. Method: A C18 reverse-phase preparative column was used with a mobile phase consisting of deionized water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% TFA (B). Gradient elution (B content = 5% to 70%) was performed. The elution time was 15 min, the flow rate was 12 mL / min, the column temperature was 30°C, and the retention time was 5.01 min. Intermediate 5 (30 mg, yield: 22.38%) was obtained.

[0172] LCMS (ESI): m / z = 831.3 [M+H] + .

[0173] Example 1: Preparation of Compound 1

[0174] Steps 1 to 4: CTC resin (100 g, 1.08 mmol / g) and Fmoc-L-citrulline (40.0 g, 100 mmol) were added to dichloromethane (600 mL), followed by the addition of N,N-diisopropylethylamine (25.8 g, 200 mmol). The mixture was purged with nitrogen for 2 hours. The mixture was filtered and the resin was washed three times with DMF. A 20% piperidine / DMF solution was added to the resin mixture and the mixture was purged with nitrogen for 30 minutes. The mixture was filtered and the resin was washed five times with DMF. DMF (600 mL), Boc-L-valine (44.0 g, 200 mmol), HOBT (27.0 g, 200 mmol), and DIC (25.2 g, 200 mmol) were then added to the resin mixture. The mixture was purged with nitrogen for 2 hours. Color development with ninhydrin and phenol indicated the reaction was complete. The resin was filtered, washed three times with DMF, twice with methanol, once with dichloromethane, and twice more with methanol, and then dried. A 30% trifluoroethanol / dichloromethane solution was added to the resin mixture, and nitrogen was bubbled through the reaction for 1 hour. The mixture was filtered, and the mother liquor was collected and concentrated to yield compound 1D (32 g, 86% yield over four steps).

[0175] LCMS (ESI): m / z = 375.1 [M+H] + .

[0176] Step 5: Compound 1D (32 g, 0.085 mol) was added to dichloromethane (320 mL) and methanol (160 mL), followed by p-aminobenzyl alcohol (12.6 g, 0.1 mol) and EEDQ (42 g, 1.7 mol). The mixture was stirred at room temperature for 16 h. The mixture was concentrated directly, and the resulting residue was purified by column chromatography (dichloromethane:methanol (v:v) = 5:1) to afford compound 1E (27 g, 66% yield).

[0177] LCMS (ESI): m / z = 480.1 [M+H] + .

[0178] Step 6: 7-Ethylcamptothecin (392 mg, 1.0 mmol) was added to dry dichloromethane (10 mL), followed by DMAP (127 mg, 1.0 mmol) and DIEA (403 mg, 3.0 mol). Under nitrogen, the temperature was lowered to 0°C, triphosgene (155 mg, 0.5 mmol) was added, and the mixture was stirred at 0°C for 30 minutes. Compound 1E (500 mg, 1.0 mmol) was then added, and the mixture was stirred at room temperature for 16 hours. The mixture was concentrated directly, and the resulting residue was purified by column chromatography (dichloromethane:methanol (v:v) = 10:1) to afford compound 1F (700 mg, 76% yield).

[0179] LCMS(ESI): m / z=782.3[M-Boc+H] + ,882.3[M+H] + .

[0180] Step 7: Compound 1F (660 mg, 0.74 mmol) was added to a mixture of trifluoroacetic acid and dichloromethane (v:v = 1:9, 10 mL) and stirred at room temperature for 4 h. The mixture was concentrated in a 30°C water bath, and the resulting residue was purified by reverse-phase column chromatography (HO:MeCN:TFA (v:v:v) = 50:50:0.2) to afford compound 1G (400 mg, 68% yield).

[0181] LCMS (ESI): m / z = 782.3 [M+H] + .

[0182] Step 8: Compound 1G (150 mg, 0.19 mmol) was added to DMA (1 mL), followed by glutaric anhydride (32 mg, 0.38 mmol) and DIEA (37 mg, 0.38 mmol). The mixture was stirred at room temperature for 4 h. The product was purified by reverse-phase column chromatography (HO:MeCN:TFA (v:v:v) = 50:50:0.2) to afford compound 1H (150 mg, 87% yield).

[0183] LCMS (ESI): m / z = 896.3 [M+H] + .

[0184] Step 9: Compound 1H (150 mg, 0.16 mmol) was added to DMA (1 mL) and dichloromethane (0.5 mL), followed by N-hydroxysuccinimide (57 mg, 0.48 mmol) and EDCI (96 mg, 0.48 mmol). The mixture was stirred at room temperature under nitrogen for 18 h. The dichloromethane was removed by concentration in a 25°C water bath, and the resulting residue was purified by reverse-phase column chromatography (HO:MeCN:TFA (v:v:v) = 40:60:0.2) to afford compound 1I (135 mg, 81% yield).

[0185] LCMS(ESI): m / z=497.4[M / 2+H] + .

[0186] Step 10: Compound 1I (12 mg, 12 μmol) and peptide 1 (9 mg, 14.4 μmol) were added to DMF (1 mL), followed by DIEA (10 mg, 84 μmol), and the mixture was allowed to react at room temperature for 4 hours. Liquid phase preparative separation and purification (liquid phase preparative conditions: C18 reverse phase preparative column, mobile phase: deionized water containing 0.1% trifluoroacetic acid (A), acetonitrile containing 0.1% trifluoroacetic acid (B), gradient elution, B content = 5% to 70%, elution time 15 min, flow rate 12 mL / min, column temperature: 30°C, retention time: 5.07 min) was performed to obtain compound 1 (6 mg, yield: 33%).

[0187] LCMS(ESI): m / z=749.5[M / 2+H] + ,1497.6[M+H] + .

[0188] Example 2: Preparation of Compound 2

[0189] Step 1: Compound 1E (0.92 g, 1.92 mmol) and di(p-nitrobenzene) carbonate (1.17 g, 3.85 mmol) were added to N,N-dimethylformamide (10 mL), followed by N,N-diisopropylethylamine (0.37 g, 2.90 mmol). The mixture was reacted at room temperature under nitrogen for 16 h. Water (50 mL) was added to the reaction solution, which was then extracted with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography (methanol:dichloromethane (v:v) = 0-10%) to afford compound 2A (0.8 g, yield: 64.63%).

[0190] Step 2: Compound 2A (0.5 g, 0.78 mmol) and intermediate 1 (0.47 g, 0.86 mmol) were added to DMF (5 mL), followed by DIEA (0.25 g, 1.93 mmol). The mixture was reacted at room temperature for 16 h. Water (30 mL) was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography (methanol:dichloromethane (v:v) = 0-10%) to afford 2B (0.6 g, 81.92%).

[0191] LCMS (ESI): m / z = 939.3 [M+H] + .

[0192] Step 3: Compound 2B (0.5 g, 0.53 mmol) was added to dichloromethane (10 mL), followed by TFA (1 mL). The mixture was allowed to react at room temperature for 2 h. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography (H₂O:MeCN:TFA (v:v:v) = 50:50:0.1) to afford compound 2C (0.35 g, 69.30% yield).

[0193] LCMS (ESI): m / z = 839.5 [M+H] + .

[0194] Step 4: Compound 2C (0.11 g, 0.12 mmol) and intermediate 2 (0.12 g, 0.12 mmol) were added to DMF (3 mL), followed by DIEA (0.047 g, 0.36 mmol). The mixture was allowed to react at room temperature for 16 h. The resulting solution was purified by reverse-phase column chromatography (HO:MeCN:TFA (v:v:v) = 55:45:0.1) to afford compound 2D (0.06 g, 28.84% yield).

[0195] LCMS (ESI): m / z = 867.8 [M / 2+H] + .

[0196] Step 5: Compound 2D (0.06 g, 0.035 mmol) was added to a mixture of TFA / TIS / H2O (v:v:v = 93:5:2, 3 mL) and allowed to react at room temperature for 15 min. The reaction solution was directly poured into MTBE (40 mL) and centrifuged. The precipitate was again centrifuged with MTBE (40 mL x 2). The resulting precipitate was purified by preparative HPLC. Method: A C18 reverse-phase preparative column was used with a mobile phase consisting of deionized water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% TFA (B). Gradient elution was performed with a B content of 5% to 70%. The elution time was 15 min, the flow rate was 12 mL / min, the column temperature was 30°C, and the retention time was 6.56 min. Compound 2 (5 mg, yield: 10%) was obtained.

[0197] LCMS (ESI): m / z = 713.5 [M / 2+H] + .

[0198] Example 3

[0199] Step 1: Compound 2C (0.11 g, 0.12 mmol) and glutaric anhydride (0.027 g, 0.24 mmol) were added to N,N-dimethylformamide (2 mL), followed by N,N-diisopropylethylamine (0.047 g, 0.36 mmol). The mixture was allowed to react at room temperature for 16 h. The reaction solution was directly purified by C18 reverse-phase column chromatography (mobile phase A, B composition: mobile phase A: acetonitrile; mobile phase B: water (containing 0.1% TFA), A / B = 50 / 50) to obtain compound 3A (0.08 g, yield: 70%).

[0200] LCMS (ESI): m / z = 953.6 [M+H] + .

[0201] Step 2: Compound 3A (0.08 g, 0.084 mmol), N-hydroxysuccinimide (0.029 g, 0.25 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.048 g, 0.25 mmol) were added to N,N-dimethylformamide (2 mL) and reacted at room temperature for 16 h. The reaction solution was directly purified by C18 reverse-phase column chromatography (mobile phase A, B composition: mobile phase A: acetonitrile; mobile phase B: water (containing 0.1% TFA), A / B = 60 / 40) to obtain compound 3B (0.06 g, yield: 68%).

[0202] LCMS (ESI): m / z=526 [M / 2+H] + .

[0203] Step 3: Compound 3B (0.03 g, 0.029 mmol) and peptide 1 (0.036 g, 0.058 mmol) were added to N,N-dimethylformamide (2 mL), followed by N,N-diisopropylethylamine (0.011 g, 0.085 mmol). The mixture was allowed to react at room temperature for 5 h. The reaction solution was directly purified by preparative HPLC. Purification conditions: C18 reverse-phase preparative column, mobile phase: deionized water containing 0.1% trifluoroacetic acid (A), acetonitrile containing 0.1% trifluoroacetic acid (B), gradient elution, B content = 5% to 70%, elution time 15 min, flow rate 12 mL / min, column temperature: 30°C. Compound 3 (9 mg, yield: 20%) was obtained.

[0204] LCMS (ESI): m / z=776 [M / 2+H] + .

[0205] Example 4

[0206] Step 1: Compound 3B (0.05 g, 0.047 mmol) and peptide 3 (0.045 g, 0.047 mmol) were added to N,N-dimethylformamide (2 mL), followed by N,N-diisopropylethylamine (0.012 g, 0.094 mmol). The mixture was allowed to react at room temperature for 5 h. The reaction solution was directly purified by preparative HPLC. Purification conditions: C18 reverse-phase preparative column, mobile phase: deionized water containing 0.1% trifluoroacetic acid (A), acetonitrile containing 0.1% trifluoroacetic acid (B), gradient elution, B content = 5% to 70%, elution time 15 min, flow rate 12 mL / min, column temperature: 30°C, retention time: 6.74 min. Compound 4 (15 mg, yield: 16%) was obtained.

[0207] LCMS (ESI): m / z = 962.9 [M / 2+H] + .

[0208] Example 5

[0209] Step 1: Compound 3B (0.35 g, 0.33 mmol) and intermediate 3 (0.176 g, 0.33 mmol) were added to N,N-dimethylformamide (4 mL), followed by N,N-diisopropylethylamine (0.086 g, 0.66 mmol). The mixture was allowed to react at room temperature for 5 h. The resulting solution was purified by C18 reverse-phase column chromatography (mobile phase A, B composition: mobile phase A: acetonitrile; mobile phase B: water (containing 0.1% TFA), A / B = 50 / 50) to afford compound 5A (420 mg, yield: 86%).

[0210] LCMS (ESI): m / z = 731.7 [M / 2+H]+ .

[0211] Step 2: Compound 5A (0.42 g, 0.287 mmol), N-hydroxysuccinimide (0.024 g, 0.86 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.039 g, 0.86 mmol) were added to N,N-dimethylformamide (2 mL) and dichloromethane (1 mL) under nitrogen atmosphere and reacted at room temperature for 16 h. The reaction solution was directly purified by C18 reverse-phase column chromatography (mobile phase A, B composition: mobile phase A: acetonitrile; mobile phase B: water (containing 0.1% TFA), A / B = 60 / 40) to obtain compound 5B (0.38 g, yield: 85%).

[0212] LCMS (ESI): m / z = 780.4 [M / 2+H] + .

[0213] Step 3: Compound 5B (0.05 g, 0.032 mmol) and peptide 3 (0.032 g, 0.032 mmol) were added to N,N-dimethylformamide (2 mL), followed by N,N-diisopropylethylamine (0.009 g, 0.064 mmol). The mixture was allowed to react at room temperature for 5 h. The reaction solution was directly purified by preparative HPLC. Purification conditions: C18 reverse-phase preparative column, mobile phase: deionized water containing 0.1% trifluoroacetic acid (A), acetonitrile containing 0.1% trifluoroacetic acid (B), gradient elution, B content = 5% to 70%, elution time 15 min, flow rate 12 mL / min, column temperature: 30°C. Compound 5 (30 mg, yield: 39%) was obtained.

[0214] LCMS (ESI): m / z = 962.9 [M / 2+H] + .

[0215] Example 6

[0216] Step 1: Compound 6A (3.0 g, 3.5 mmol) and N-hydroxysuccinimide (0.82 g, 7.0 mmol) were added to dichloromethane (36 mL), followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.37 g, 7.0 mmol). The mixture was reacted at room temperature under nitrogen for 16 h. Water (100 mL) was added to the reaction solution, which was then extracted with dichloromethane (100 mL). The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography (methanol:dichloromethane (v:v) = 0-5%) to afford compound 6B (3.0 g, yield: 89.82%).

[0217] Step 2: Compound 6B (3.0 g, 3.2 mmol) and Boc-L-lysine (0.79 g, 3.2 mmol) were added to N,N-dimethylacetamide (30 mL), followed by DIEA (1.24 g, 9.6 mmol). The mixture was reacted at room temperature for 16 h. Water (30 mL) was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography (methanol:dichloromethane (v:v) = 0-10%) to afford compound 6C (3.2 g, 94.12%).

[0218] LCMS (ESI): m / z = 1068.7 [M+H] + .

[0219] Step 3: Compound 6C (3.2 g, 3.0 mmol) was added to a 20% piperidine / DMF mixture (30 mL) and allowed to react at room temperature for 2 h. The reaction solution was purified by reverse-phase column chromatography (H₂O:MeCN:TFA (v:v:v) = 50:50:0.1) to afford compound 6D (2.6 g, 90.59% yield).

[0220] LCMS (ESI): m / z = 847.0 [M+H] + .

[0221] Step 4: Compound 6D (2.6 g, 0.27 mmol), D-glucose (0.12 g, 1.62 mmol), and acetic acid (0.02 g, 0.027 mmol) were added to methanol (30 mL) under nitrogen atmosphere at 50°C for 1 h. Sodium cyanoborohydride (1.02 g, 1.62 mmol) was then added and the mixture was allowed to react at 50°C for 18 h. The resulting solution was purified by reverse-phase column chromatography (H₂O:MeCN:TFA (v:v:v) = 60:40:0.1) to afford compound 6E (2.3 g, 72.33% yield).

[0222] LCMS (ESI): m / z = 1175.0 [M / 2+H] + .

[0223] Step 5: Compound 6E (0.37 g, 0.315 mmol), intermediate 4 (0.3 g, 0.315 mmol), HATU (0.18 g, 0.47 mmol), and DIEA (0.13 g, 0.95 mmol) were added to DMF (4 mL) and reacted at room temperature for 18 h. The reaction solution was purified by reverse-phase column chromatography (HO:MeCN:TFA (v:v:v) = 50:50:0.1) to provide compound 6F (0.2 g, yield: 31.80%).

[0224] LCMS (ESI): m / z = 999.3 [M / 2+H] + .

[0225] Step 6: Compound 6F (0.2 g, 0.1 mmol) was added to a mixture of trifluoroacetic acid and dichloromethane (1:10) (2 mL) and allowed to react at room temperature for 1 h. The reaction solution was concentrated, and the resulting residue was purified by preparative HPLC. Method: A C18 reverse-phase preparative column was used with a mobile phase consisting of deionized water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% TFA (B). Gradient elution (B content = 5% to 70%) was performed, with an elution time of 15 min, a flow rate of 12 mL / min, and a column temperature of 30°C. This afforded 6G (60 mg, yield: 29.85%).

[0226] LCMS (ESI): m / z = 949.4 [M / 2+H] + .

[0227] Step 7: Compound 6G (30 mg, 0.014 mmol), intermediate 5 (13 mg, 0.014 mmol), and DIEA (6 mg, 0.052 mmol) were added to DMF (1 mL) and reacted at room temperature for 2 h. The reaction solution was purified by preparative HPLC. Method: C18 reverse-phase preparative column, mobile phase: deionized water containing 0.1% trifluoroacetic acid (A), acetonitrile containing 0.1% TFA (B), gradient elution, B content = 5% to 70%, elution time 15 min, flow rate 12 mL / min, column temperature: 30°C. Compound 6 (20 mg, yield: 54.05%) was obtained.

[0228] LCMS (ESI): m / z = 871.6 [M / 3+H] + ,1307.1[M / 2+H] + .

[0229] Example 7

[0230] Step 1: Intermediate 4 (100 mg, 0.118 mmol), disuccinimidyl glutarate (32 mg, 0.099 mmol), and DIPEA (45 mg, 0.354 mmol) were added to DMF (2 mL) and reacted at room temperature for 1 h. Peptide 1 (73 mg, 0.118 mmol) and DIPEA (45 mg, 0.354 mmol) were then added and reacted at room temperature for 2 h. The reaction solution was purified by preparative HPLC. Method: A C18 reverse-phase preparative column was used with a mobile phase consisting of deionized water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% TFA (B). Gradient elution was performed with a B content of 5% to 70%. The elution time was 15 min, the flow rate was 12 mL / min, and the column temperature was 30°C. Compound 7 (10 mg, yield: 5.40%) was obtained.

[0231] LCMS (ESI): m / z = 779.0 [M / 2+H] + .

[0232] Example 8

[0233] Step 1: Intermediate 4 (530 mg, 0.55 mmol), N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine (260 mg, 0.55 mmol), and HATU (316 mg, 0.83 mmol) were added to DMF (5 mL), followed by DIPEA (215 mg, 1.66 mmol). The mixture was reacted at room temperature for 16 h. The reaction solution was purified by reverse phase column chromatography (H2O:MeCN:NH4OAc (v:v:v) = 40:60:0.5) to afford compound 8A (580 mg, 81.00% yield).

[0234] LCMS (ESI): m / z = 1291.6 [M+H] + .

[0235] Step 2: Compound 8A (580 mg, 0.45 mmol) was added to a 20% piperidine / DMF solution (5 mL), and the reaction was carried out at room temperature for 1 min. The reaction solution was purified by reverse phase column chromatography (H2O:MeCN:NH4OAc (v:v:v) = 50:50:0.5) to obtain compound 8B (460 mg, yield: 95.83%).

[0236] LCMS (ESI): m / z = 1069.5 [M+H] + .

[0237] Step 3: Compound 8B (165 mg, 0.154 mmol), disuccinimidyl glutarate (50 mg, 0.154 mmol), and DIPEA (59 mg, 0.463 mmol) were added to DMF (2 mL) and reacted at room temperature for 1 h. Peptide 1 (131 mg, 0.154 mmol) and DIPEA (59 mg, 0.463 mmol) were then added and reacted at room temperature for 2 h. The reaction solution was purified by reverse-phase column chromatography (H₂O:MeCN:NH₄OAc (v:v:v) = 50:50:0.5) to afford compound 8C (180 mg, yield: 65.45%).

[0238] LCMS (ESI): m / z = 893.0 [M / 2+H] + .

[0239] Step 4: Compound 8C (180 mg, 0.100 mmol) was added to dichloromethane (2 mL), followed by TFA (0.2 mL). The mixture was allowed to react at room temperature for 1 h. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography (H₂O:MeCN:TFA (v:v:v) = 55:45:0.1) to afford compound 8D (140 mg, yield: 82.84%).

[0240] LCMS (ESI): m / z = 843.0 [M / 2+H] + .

[0241] Step 5: Compound 8D (20 mg, 0.011 mmol), methyl-tetracosyl polyethylene glycol succinimidyl acrylate (13 mg, 0.011 mmol), and DIPEA (5 mg, 0.033 mmol) were added to DMF (1 mL) and reacted at room temperature for 2 h. The reaction solution was purified by preparative HPLC. Method: A C18 reverse-phase preparative column was used with a mobile phase consisting of deionized water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% TFA (B). Gradient elution was performed with a B content of 5% to 70%. The elution time was 15 min, the flow rate was 12 mL / min, and the column temperature was 30°C. Compound 8 (7 mg, yield: 22.15%) was obtained.

[0242] LCMS (ESI): m / z = 708.0 [M / 4+H] + ,943.5[M / 3+H] + ,1414.9[M / 2+H] + .

[0243] Example 9

[0244] Step 1: Compound 8D (30 mg, 0.018 mmol), methyl nonapolyethylene glycol succinimidyl acrylate (10 mg, 0.018 mmol), and DIPEA (7 mg, 0.054 mmol) were added to DMF (1 mL) and reacted at room temperature for 2 h. The reaction solution was purified by preparative HPLC. Method: A C18 reverse-phase preparative column was used with a mobile phase consisting of deionized water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% TFA (B). Gradient elution (B content = 5% to 70%) was performed. The elution time was 15 min, the flow rate was 12 mL / min, and the column temperature was 30°C. Compound 9 (6 mg, yield: 15.58%) was obtained.

[0245] LCMS (ESI): m / z = 723.3 [M / 3+H] + ,1084.7[M / 2+H] + .

[0246] Example 10

[0247] Step 1: Compound 2A (2.1 g, 3.2 mmol) and DIPEA (3.6 g, 32 mmol) were added to DMF (40 mL) under nitrogen atmosphere. Stir at room temperature for 10 minutes, then cool to 0°C. MMAE (2.0 g, 3.2 mmol) and HOBT (0.38 g, 3.2 mmol) were added, and the mixture was stirred for 30 minutes. The mixture was then returned to room temperature and stirred for 18 hours. The reaction mixture was purified by reverse-phase column chromatography (HO:MeCN:TFA (v:v:v) = 45:55:0.1) to afford compound 10A (2.7 g, 67.84% yield).

[0248] LCMS (ESI): m / z = 1223.4 [M+H] + .

[0249] Step 2: Compound 10A (2.0 g, 1.6 mmol) was added to a 1:10 mixture of TFA / DCM (34 mL) and stirred at room temperature for 2 h. The solvent was concentrated to dryness at 25°C, and THF (110 mL) and potassium carbonate (2.26 g, 16 mmol) were added, followed by stirring at room temperature for 3 h. The product was concentrated to dryness and purified by reverse-phase column chromatography (H₂O:MeCN:TFA (v:v:v) = 50:50:0.1) to afford compound 10B (1.4 g, yield: 76.50%).

[0250] LCMS (ESI): m / z = 1123.4 [M+H] + .

[0251] Step 3: Compound 10B (100 mg, 0.08 mmol), disuccinimidyl glutarate (26 mg, 0.08 mmol), and DIPEA (30 mg, 0.24 mmol) were added to DMF (2 mL) and reacted at room temperature for 1 h. Peptide 1 (59 mg, 0.08 mmol) and DIPEA (30 mg, 0.24 mmol) were then added and reacted at room temperature for 2 h. The reaction solution was purified by preparative HPLC. Method: A C18 reverse-phase preparative column was used with a mobile phase consisting of deionized water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% TFA (B). Gradient elution was performed with a B content of 5% to 70%. The elution time was 15 min, the flow rate was 12 mL / min, and the column temperature was 30°C. Compound 10 (24 mg, yield: 16.21%) was obtained.

[0252] LCMS (ESI): m / z = 920.1 [M / 2+H] + .

[0253] Example 11

[0254] Step 1: Intermediate 1 (227 mg, 0.27 mmol), N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine (127 mg, 0.27 mmol), and HATU (155 mg, 0.27 mmol) were added to DMF (4 mL), followed by DIPEA (110 mg, 0.81 mmol). The mixture was reacted at room temperature for 16 h. The reaction solution was purified by reverse-phase column chromatography (H2O:MeCN:NH4OAc (v:v:v) = 40:60:0.5) to afford compound 11A (240 mg, 68.77% yield).

[0255] LCMS (ESI): m / z = 1289.6 [M+H] + .

[0256] Step 2: Compound 11A (240 mg, 0.18 mmol) was added to a 20% piperidine / DMF solution (5 mL), and the mixture was reacted at room temperature for 1 min. The reaction solution was purified by reverse phase column chromatography (H2O:MeCN:NH4OAc (v:v:v) = 50:50:0.5) to obtain compound 11B (160 mg, yield: 80.80%).

[0257] LCMS (ESI): m / z = 1067.5 [M+H] + .

[0258] Step 3: Compound 11B (160 mg, 0.15 mmol), disuccinimidyl glutarate (49 mg, 0.15 mmol), and DIPEA (58 mg, 0.45 mmol) were added to DMF (2 mL) and reacted at room temperature for 1 h. Peptide 1 (127 mg, 0.15 mmol) and DIPEA (58 mg, 0.45 mmol) were then added and reacted at room temperature for 2 h. The reaction solution was purified by reverse-phase column chromatography (H₂O:MeCN:NH₄OAc (v:v:v) = 50:50:0.5) to afford compound 11C (85 mg, yield: 31.83%).

[0259] LCMS(ESI):m / z=842.3[(M-Boc) / 2+H] + ,892.3[M / 2+H] + .

[0260] Step 4: Compound 11C (85 mg, 0.047 mmol) was added to dichloromethane (2 mL), followed by TFA (0.2 mL). The mixture was allowed to react at room temperature for 1 h. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase column chromatography (H₂O:MeCN:TFA (v:v:v) = 55:45:0.1) to afford compound 11D (80 mg, 99.75% yield).

[0261] LCMS (ESI): m / z = 842.4 [M / 2+H] + .

[0262] Step 5: Compound 11D (30 mg, 0.018 mmol), methyl nonapolyethylene glycol succinimidyl acrylate (9 mg, 0.018 mmol), and DIPEA (6 mg, 0.054 mmol) were added to DMF (1 mL) and reacted at room temperature for 2 h. The reaction solution was purified by preparative HPLC. Method: A C18 reverse-phase preparative column was used with a mobile phase consisting of deionized water containing 0.1% trifluoroacetic acid (A) and acetonitrile containing 0.1% TFA (B). Gradient elution (B content = 5% to 70%) was performed. The elution time was 15 min, the flow rate was 12 mL / min, and the column temperature was 30°C. Compound 11 (15 mg, yield: 38.86%) was obtained.

[0263] LCMS (ESI): m / z = 722.6 [M / 3+H] + ,1083.6[M / 2+H] + .

[0264] Example 12

[0265] Step 1: 1-Maleimido-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-aza-triacontane-31-oic acid (Compound 12A) (400 mg, 0.67 mmol) was added to dichloromethane (5 mL) solvent. N-hydroxysuccinimide (85 mg, 0.74 mmol) and N,N'-dicyclohexylcarbodiimide (150 mg, 0.74 mmol) were added to the reaction solution and reacted at room temperature for 4 hours. After completion of the reaction, the mixture was dried under reduced pressure at 30°C. The resulting residue was dissolved in methanol (5 mL) and purified by reverse phase column chromatography (column model: C18 spherical 20-35um100A 40g; acetonitrile:water (0.1% trifluoroacetic acid) (v:v) = 5%-80%) to obtain Compound 12B (362 mg, yield: 77.76%).

[0266] LC-MS (ESI): m / z = 690.2 [M+H] + .

[0267] Step 2: Compound 12B (100 mg, 0.087 mmol) was added to N,N-dimethylformamide (3 mL) solvent, followed by the addition of N,N-diisopropylethylamine (12 mg, 0.091 mmol) and mercapto-β-cyclodextrin (60 mg, 0.087 mmol). The mixture was reacted at room temperature for 4 hours. After the reaction was completed, compound 12C (5.6% N,N-dimethylformamide solution) was obtained, which was directly used in the next step.

[0268] Step 3: The solution of compound 12C from the previous step, compound 8D (5 mg, 0.003 mmol), and N,N-diisopropylethylamine (1.9 mg, 0.015 mmol) were added sequentially to N,N-dimethylformamide (2 mL) and allowed to react at room temperature for 2 hours. After completion, the reaction solution was filtered through a filter head and the resulting solution was separated and purified using a preparative liquid chromatography system. Preparation method: Instrument: Octopus Plus high pressure preparative; Column: Durashell C18 (A) (21.2 mm × 250 mm, 10 μm); Mobile phase: A, 0.1% trifluoroacetic acid; B, acetonitrile; Gradient: 5%-80% B; Flow rate: 40 mL / min; Wavelength: 210 nm; The solvent was then dried in a lyophilizer at -80°C to obtain compound 12 (4.02 mg, yield: 39.72%).

[0269] LC-MS(ESI):m / z=1706.2[M / 2+H] + .

[0270] Biological testing

[0271] 1. Recombinant human αVβ3 ELISA test

[0272] Rhvitronectin was diluted to a final concentration of 1 μg / ml in TBS buffer, and 50 μl was transferred to a 96-well plate for coating overnight at 4°C. The plate was washed three times with TBS buffer, followed by the addition of 150 μl of blocking buffer (TBS buffer containing 1% BSA) and blocking at 37°C for 1 hour. The plate was washed three times with TBS buffer, and human αVβ3 integrin protein was diluted to 1 μg / ml in TBS buffer containing 0.1% BSA. 50 μl of integrin protein was transferred to a 96-well plate, followed by the addition of 1 μl of various compound concentrations or DMSO, and incubation at room temperature for 2 hours. Biotinylated anti-αv antibody was diluted to 0.5 μg / ml in TBS buffer containing 0.1% BSA. The plate was washed three times with TBS buffer, and 50 μl of antibody was added and incubated at room temperature for 1 hour. The plate was washed three times with TBS buffer, and 50 μl of Streptavidin-HRP was added and incubated at room temperature for 20 minutes. Add 50 μl of TMB substrate and incubate at room temperature for 20 min. Finally, add 25 μl of stop buffer (2 M H2SO4) and read the OD value of the plate at 450 nm on a microplate reader. Calculate the IC using GraphPad Prism 6 software. 50 value.

[0273] Table 1. IC values ​​of compounds against human αVβ3 protein 50 value

[0274] AA stands for IC 50 ≤5nM, A means 5nM<IC 50 ≤30nM, B means 30nM<IC 50 ≤50nM, C means 50nM<IC 50 ≤100nM.

[0275] Conclusion: The compounds of the present invention, such as the compounds in the examples, showed high binding activity to human αVβ3 protein.

[0276] 2. Detection of proliferation inhibitory activity in WM-2664 and SW620 cells

[0277] WM-2664 cells (MINGZHOUBIO, MZ-1627) were cultured in a 37°C, 5% CO2 incubator. At the beginning of the experiment, the cells were resuspended into a cell suspension of a certain concentration and transferred to a 96-well plate (Fisher Scientific, 655090), with 4000 cells / well and 95 μL per well, and placed in an incubator to adhere overnight. Then 5 μL of the test compound at different concentrations was added to each well and the cells were cultured in a 37°C, 5% CO2 incubator for 72 hours. After 72 hours, the detection solution was added according to the instructions of the CellCounting-Lite 2.0Luminescent Cell Viability Assay (Vazyme, DD1101-03), and the fluorescence signal value was measured using a BMG (PHERAstar FSX) microplate reader. GraphPad Prism8.0 software was used to calculate the IC 50 value.

[0278] SW620 cells were cultured in a 37°C, 5% CO2 incubator. At the beginning of the experiment, the cells were resuspended into a cell suspension of a certain concentration and transferred to a 96-well plate (Greiner, 655090), with 1000 cells / well and 90 μL per well, and placed in an incubator to adhere overnight. Then, 10 μL of the test compound at different concentrations was added to each well and the cells were cultured in a 37°C, 5% CO2 incubator for 72 hours. After 72 hours, the detection solution was added according to the instructions of Celltiter-Glo (Promega, G7558), and the fluorescence signal value was measured using a BMG (PHERAstar FSX) microplate reader. GraphPad Prism8.0 software was used to calculate the IC 50 value.

[0279] Selectivity fold = (IC of compound in SW620 cells) 50 / SW620 cell IC corresponding to toxin 50 ) / (IC of compound in WM-2664 cells 50 / WM-2664 cell IC corresponding to toxin 50 ).

[0280] Table 2. IC values ​​of compounds on WM-2664 cells 50 Value and selectivity

[0281] A stands for IC 50 ≤10nM, B means 10nM<IC 50 ≤50nM, C means 50nM<IC 50 ≤100nM, D means 100nM<IC 50 ≤1000nM.

[0282] Conclusion: The compounds of the present invention, such as the example compounds, showed high inhibitory activity against WM-2664 cells, while their inhibitory activity against SW620 cells was relatively weak.

[0283] 3. Detection of MKN74 cell proliferation inhibitory activity

[0284] MKN74 cells (COBIOER, CBP60490) were cultured in a 37°C, 5% CO2 incubator. At the beginning of the experiment, the cells were resuspended into a cell suspension of a certain concentration and transferred to a 96-well plate (Fisher Scientific, 655090), with 2000 cells / well and 95 μL per well, and placed in an incubator to adhere overnight. Then, 5 μL of the test compound at different concentrations was added to each well and the cells were cultured in a 37°C, 5% CO2 incubator for 72 hours. After 72 hours, the detection solution was added according to the instructions of the CellCounting-Lite 2.0Luminescent Cell Viability Assay (Vazyme, DD1101-03), and the fluorescence signal value was measured using a BMG (PHERAstar FSX) microplate reader. GraphPad Prism8.0 software was used to calculate the IC 50 value.

[0285] Table 3. IC of compounds against MKN74 cells 50 value

[0286] A stands for IC 50 ≤100nM, B means 100nM<IC 50 ≤500nM, C means 500nM<IC 50 ≤1000nM, D represents IC 50 >1000nM.

[0287] Conclusion: The compounds of the present invention, such as the compounds in the examples, have relatively weak inhibitory activity against MKN74 cells.

[0288] 4. In vitro plasma stability test

[0289] Add the prepared compound solution to a predetermined amount of biological matrix to prepare stability samples at two concentration levels (a low-concentration quality control sample and a high-concentration quality control sample), with two aliquots for each concentration level. Under the required experimental conditions, the compound stability test result is calculated as the peak area ratio of the samples at a predetermined time / the peak area ratio of the sample at zero time multiplied by 100%.

[0290] Experimental results:

[0291] Table 4.1 Residual rate (%) of test compound at 40 ng / mL after 24 h at 37°C

[0292] Table 4.2 Residual rate (%) of test compound at 4000 ng / mL after 24 h at 37°C

[0293] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good in vitro plasma stability.

[0294] 5. Pharmacokinetic test in rats

[0295] Experimental animals: Male SD rats, about 220 g, 6 to 8 weeks old, purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0296] Experimental design: On the day of the experiment, SD rats were randomly divided into groups according to body weight. They were fasted but not watered for 12-14 hours before administration and fed 4 hours after administration.

[0297] Before and after drug administration, 0.15 mL of blood was collected intraorbitally under isoflurane anesthesia. The blood was placed in an EDTAK2 centrifuge tube and centrifuged at 5000 rpm at 4°C for 10 minutes to collect plasma. Blood was collected at 2, 5, 15, 30 minutes, and 1, 2, 3, 4, and 6 hours. All samples were stored at -80°C prior to analysis and quantitative analysis was performed using LC-MS / MS.

[0298] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good pharmacokinetic properties in rats.

[0299] 6. Pharmacokinetic test in mice

[0300] Experimental animals: Male Balb / c mice, 22-25 g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. or Beijing Huafukang Biotechnology Co., Ltd.

[0301] Experimental Design: On the day of the experiment, Balb / c mice were randomly divided into groups based on body weight. They were fasted (but not water) for 12–14 hours prior to dosing and fed 4 hours after dosing. Dosing was performed according to Table 6.1.

[0302] Table 6.1 Dosage Information Note: Intravenous administration solvent: 5% DMA + 5% HS15 + 90% Saline

[0303] (DMA: dimethylacetamide; HS15: polyethylene glycol-15-hydroxystearate; Saline: normal saline)

[0304] Before and after drug administration, 0.06 mL of blood was collected via the orbital cavity under isoflurane anesthesia. The blood was placed in an EDTAK2 centrifuge tube and centrifuged at 5000 rpm at 4°C for 10 minutes to collect plasma. Blood was collected at 0, 5, 15, 30 minutes, and 1, 2, 4, 7, and 24 hours. All samples were stored at -80°C prior to analysis and quantitative analysis was performed using LC-MS / MS.

[0305] Table 6.2 Pharmacokinetic parameters of test compounds in mouse plasma

[0306] *: The blood collection time points are 2, 5, 15, 30 min, 1, 2, 3, 4, and 6 h.

[0307] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good pharmacokinetic properties in mice.

[0308] 7. Beagle dog pharmacokinetic test

[0309] Experimental animals: Male beagle dogs, weighing approximately 8-11 kg, were purchased from Beijing Mas Biotechnology Co., Ltd.

[0310] Experimental method: On the day of the experiment, beagle dogs were randomly divided into groups according to body weight. They were fasted but not watered for 12-14 hours before administration and fed 4 hours after administration.

[0311] Before and after dosing, 1 mL of blood was collected from the jugular vein or limb vein and placed in an EDTAK2 centrifuge tube. The tubes were centrifuged at 5000 rpm at 4°C for 10 minutes, and plasma was collected. Blood was collected at the following time points: 0, 5, 15, 30 minutes, and 1, 2, 4, 6, 8, 10, 12, 24, and 48 hours. All samples were stored at -80°C prior to analysis and quantitative analysis was performed using LC-MS / MS.

[0312] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good pharmacokinetic properties in beagle dogs.

[0313] 8. Monkey pharmacokinetic test

[0314] Experimental animals: Male cynomolgus monkeys, 3-5 kg, 3-6 years old, purchased from Suzhou Xishan Biotechnology Co., Ltd.

[0315] Experimental method: On the day of the experiment, monkeys were randomly divided into groups according to body weight. They were fasted but not watered for 14-18 hours before administration and fed 4 hours after administration.

[0316] Before and after dosing, 1.0 mL of blood was collected from a limb vein and placed in an EDTAK2 centrifuge tube. The tubes were centrifuged at 5000 rpm at 4°C for 10 minutes, and plasma was collected. Blood was collected at the following time points: 0, 5 minutes, 15 minutes, 30 minutes, and 1, 2, 4, 6, 8, 10, 12, and 24 hours. All samples were stored at -80°C prior to analysis and quantitative analysis was performed using LC-MS / MS.

[0317] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good pharmacokinetic properties in monkeys.

[0318] 9. hERG potassium channel effect test

[0319] Experimental platform: electrophysiology manual patch clamp system

[0320] Cell line: Chinese hamster ovary (CHO) cell line stably expressing hERG potassium channel

[0321] Experimental methods: CHO (Chinese Hamster Ovary) cells stably expressing hERG potassium channels were used to record hERG potassium channel currents using the whole-cell patch clamp technique at room temperature. Glass microelectrodes were pulled from glass electrode blanks (BF150-86-10, Sutter) using a puller. The tip resistance after perfusing the electrode liquid was about 2-5 MΩ. The glass microelectrode was inserted into the amplifier probe to connect to the patch clamp amplifier. The clamping voltage and data recording were controlled and recorded by a computer using pClamp 10 software, with a sampling frequency of 10 kHz and a filter frequency of 2 kHz. After obtaining the whole-cell recording, the cell was clamped at -80 mV to induce the hERG potassium current (I hERG ) was administered with a 2-second depolarization step from -80 mV to +20 mV, followed by repolarization to -50 mV for 1 second before returning to -80 mV. This voltage stimulus was administered every 10 seconds, and administration began after confirming that the hERG potassium current was stable (for at least 1 minute). Compounds were administered for at least 1 minute at each test concentration, and at least two cells (n ≥ 2) were tested for each concentration.

[0322] Data processing: Data analysis was performed using pClamp 10, GraphPad Prism 5, and Excel. The degree of inhibition of hERG potassium current (peak hERG tail current induced at -50 mV) by different compound concentrations was calculated using the following formula: Inhibition% = [1-(I / Io)] × 100%

[0323] Wherein, Inhibition% represents the inhibition percentage of the compound on hERG potassium current, and I and Io represent the amplitude of hERG potassium current before and after drug addition, respectively.

[0324] Compound IC 50 Calculated using GraphPad Prism 5 software by fitting the following equation: Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC50-X)*HillSlope))

[0325] Where X is the Log value of the test sample concentration, Y is the inhibition percentage at the corresponding concentration, and Bottom and Top are the minimum and maximum inhibition percentages, respectively.

[0326] Conclusion: The compounds of the present invention, such as the compounds in the examples, have no inhibitory effect on hERG potassium channel current.

[0327] 10. Liver microsome stability test

[0328] In this study, liver microsomes from five species, including humans, dogs, rats and mice, were used as in vitro models to evaluate the metabolic stability of the test substances.

[0329] At 37°C, 1 μM of the test substance was incubated with microsomal proteins and coenzyme NADPH. The reaction was terminated by adding ice-cold acetonitrile containing an internal standard after a certain time (5, 10, 20, 30, 60 min). The concentration of the test substance in the sample was detected by LC-MS / MS. The T value was calculated based on the ln value of the drug residual rate in the incubation system and the incubation time. 1 / 2 , and further calculated the liver microsomal intrinsic clearance CL int(mic) and hepatic intrinsic clearance CL int(Liver) .

[0330] Conclusion: The compounds of the present invention, such as the compounds in the examples, have good liver microsomal stability.

[0331] 11. CYP450 enzyme inhibition test

[0332] The purpose of this study was to evaluate the effects of test substances on the activities of five isoenzymes of cytochrome P450 (CYP) in human liver microsomes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) using an in vitro test system. Specific probe substrates for the CYP450 isoenzymes were incubated with human liver microsomes and varying concentrations of the test substances. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was added to initiate the reaction. After the reaction, the samples were processed and the metabolites produced by the specific substrates were quantitatively detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Changes in CYP enzyme activity were measured, and the IC values ​​were calculated. 50 The inhibitory potential of the test substance on each CYP enzyme isoform was evaluated.

[0333] Conclusion: The compounds of the present invention, such as the compounds in the examples, have no inhibitory activity on the five isoenzymes of human liver microsomal cytochrome P450 (CYP).

[0334] 12. In vivo efficacy experiments in mice

[0335] 12.1. Antitumor efficacy test in WM-2664 subcutaneous xenograft tumor model

[0336] WM-2664 cells were cultured in a 37°C, 5% CO2 incubator. WM-2664 cells resuspended in L15 medium were cultured at a concentration of 1×10 7 0.1 mL was inoculated subcutaneously on the right side of BALB / c nude mice. The tumor grew to approximately 150-200 mm. 3 The animals were randomly divided into groups at about 1 hour. After grouping, the animals were given drugs and the long and short diameters of the tumors were measured with a vernier caliper to calculate the tumor volume. The volume calculation formula is: volume (TV) = 0.5 × long diameter × short diameter 2 During the experiment, the tumor volume was measured twice a week. The tumor volume was used to calculate the tumor growth inhibition rate TGI (anti-tumor activity evaluation index): TGI (%) = 100% - (TV Treatment D n -TV Treatment-D0) / (TV Control-D n -TV Control-D0)×100%. TV Treatment D n : tumor volume of the treatment group on day n; TV Treatment D0: tumor volume of the treatment group at the time of grouping; TV Control D n : tumor volume of the control group on day n; TV Control D0: tumor volume of the control group at the time of grouping.

[0337] Conclusion: The compounds of the present invention, such as the compounds in the examples, showed good anti-tumor activity in the WM-2664 subcutaneous xenograft tumor model.

[0338] 12.2. Antitumor efficacy test in SW620 subcutaneous xenograft tumor model

[0339] SW620 cells were cultured in a 37°C, 5% CO2 incubator. SW620 cells resuspended in L15 medium were plated at a density of 5×10 6 0.1 mL was inoculated subcutaneously on the right side of BALB / c nude mice. The tumor grew to approximately 150-200 mm. 3The animals were randomly divided into groups at about 1 hour. After grouping, the animals were given drugs and the long and short diameters of the tumors were measured with a vernier caliper to calculate the tumor volume. The volume calculation formula is: volume (TV) = 0.5 × long diameter × short diameter 2 During the experiment, the tumor volume was measured twice a week. The tumor volume was used to calculate the tumor growth inhibition rate TGI (anti-tumor activity evaluation index): TGI (%) = 100% - (TV Treatment D n -TV Treatment-D0) / (TV Control-D n -TV Control-D0)×100%. TV Treatment D n : tumor volume of the treatment group on day n; TV Treatment D0: tumor volume of the treatment group at the time of grouping; TV Control D n : tumor volume of the control group on day n; TV Control D0: tumor volume of the control group at the time of grouping.

[0340] Conclusion: The compounds of the present invention, such as the compounds in the examples, showed relatively weak tumor inhibitory effects in the SW620 subcutaneous xenograft tumor model.

[0341] 13. Tissue distribution test of WM2664 subcutaneous xenograft tumor model

[0342] WM-2664 cells were cultured in a 37°C, 5% CO2 incubator. WM-2664 cells resuspended in L15 medium were cultured at a concentration of 1×10 7 0.1 mL was inoculated subcutaneously on the right side of BALB / c nude mice. The tumor grew to approximately 150-200 mm. 3 Animals were randomly divided into groups at approximately 1 hour. After grouping, the animals were administered medication. After administration, 0.06 mL of blood was collected from the eye sockets under isoflurane anesthesia. The blood was placed in an EDTAK2 centrifuge tube and centrifuged at 5000 rpm at 4°C for 10 minutes to collect plasma. Blood was collected at 0, 15, 30 minutes, and 1 and 2 hours. The animals were euthanized, and tumor, bone marrow, duodenum, ileum, colon, heart, liver, spleen, lung, kidney, brain, fat, stomach, and skeletal muscle tissue were collected after dissection. Tissue collection was performed at 1 hour and 2 hours. All samples were stored at -80°C prior to analysis and testing. LC-MS / MS was used to quantify the compound and toxin concentrations in the samples.

[0343] Conclusion: The compounds of the present invention, such as the compounds in the examples, showed that the toxins were highly enriched in the tumor tissue in the WM-2664 subcutaneous xenograft tumor model.

Claims

1. A drug conjugate represented by formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof, PLD (I) in, P is selected from RGD peptide, and linear polypeptide and cyclic polypeptide containing RGD peptide sequence; L is a linker; D is a cytotoxic agent.

2. The drug conjugate according to claim 1 or its stereoisomers, pharmaceutically acceptable salts, wherein the cytotoxic agent is selected from the following group: alkylating agents, antimetabolites, plant alkaloids, terpenoids, podophyllotoxins and their derivatives, taxanes and their derivatives, topoisomerase inhibitors, microtubule polymerization inhibitors, RNA polymerase inhibitors, and antitumor antibiotics.

3. The drug conjugate according to claim 2, or a stereoisomer or pharmaceutically acceptable salt thereof, wherein the cytotoxic agent is selected from the following group: cisplatin, carboplatin, oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide, azathioprine, mercaptopurine, pyrimidine analogs, vincristine, vinblastine, vinorelbine, vindesine, etoposide, teniposide, paclitaxel, camptothecin, irinotecan, isitecan, topotecan, anacrine, etoposide, etoposide phosphate, teniposide, actinomycin D, doxorubicin, epirubicin, eribulin, rubitidine, trabectedin, epothilone and its derivatives, bleomycin and its derivatives, dactinomycin and its derivatives, plicamycin and its derivatives, mitomycin C, calicheamicin, maytansine and its derivatives, auristatin and its derivatives.

4. The drug conjugate according to claim 3, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the cytotoxic agent is selected from docetaxel, MMAE, 7-ethylcamptothecin, irinotecan, isotecan, Dxd, eribulin, rubectin or trabectedin.

5. The drug conjugate or its stereoisomers or pharmaceutically acceptable salt according to claim 1, wherein the linker is selected from one or a combination of the following groups: Unmodified or modified single amino acids, unmodified or modified amino acid chains of different lengths, alkyl chains with or without O and N heteroatoms, m, n, p, q, and u are each independently selected from an integer of 0 to 20; v is an integer selected from 1-100; t is selected from an integer of 1-150; r is selected from an integer of 0-150.

6. The drug conjugate according to claim 5, or its stereoisomers, or pharmaceutically acceptable salts, wherein the linker is selected from the following structures: in, indicates the site where L and P are linked, and * indicates the site where P and D are linked.

7. A drug conjugate or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the drug conjugate is selected from one of the following structures:

8. A pharmaceutical composition or pharmaceutical preparation comprising the coupled compound according to any one of claims 1 to 7, or a stereoisomer thereof, or a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier and / or excipient.

9. The pharmaceutical composition or pharmaceutical preparation according to claim 8, comprising 1-1500 mg of the coupled compound according to any one of claims 1-7 or its stereoisomer, a pharmaceutically acceptable salt and a carrier and / or excipient.

10. Use of the drug conjugate according to any one of claims 1 to 7 or its stereoisomers, pharmaceutically acceptable salts, or the pharmaceutical composition according to claim 8 or 9 in the preparation of a drug for preventing and / or treating a disease in which αVβ3 is overexpressed, wherein the disease in which αVβ3 is overexpressed is preferably selected from cancer.

11. A method for treating a disease in a mammal or a human, the method comprising administering to a subject a therapeutically effective amount of the coupled compound according to any one of claims 1 to 7, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 8 or 9, wherein the therapeutically effective amount is preferably 1-1500 mg, and the disease is preferably cancer, more preferably colon cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, brain glioma, and malignant sarcomas, cancers, and lymphomas of the breast, ovary, colon, kidney, liver and gallbladder, lung, and brain.

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