Dual-ligand drug complexes and their uses

Abstract

This application relates to a complex compound or a pharmaceutically acceptable salt thereof, said complex compound or pharmaceutically acceptable salt comprising an effective load and two target molecules. This application also relates to a pharmaceutical composition comprising the complex compound of this application or a pharmaceutically acceptable salt thereof. This application further relates to a method for delivering an effective load to a subject in need and a method for treating a disease, both methods comprising administering to the subject a therapeutically effective amount of the complex compound of this application or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of this application.

Description

Technical Field

[0001] This application relates to the field of biomedical chemistry. More specifically, this application relates to dual-ligand drug complexes, pharmaceutical compositions comprising said dual-ligand drug complexes, and methods for delivering a payload to a subject in need using said dual-ligand drug complexes and methods for treating diseases using said dual-ligand drug complexes. Prior Technology

[0002] Typically, the pathological and physiological characteristics of diseased cells differ significantly from those of normal cells. One manifestation of this is the presence of specific or overexpressed substances (e.g., antigens, chemical signals, receptors) on the surface of diseased cells, which are absent or poorly expressed in normal cells. Based on this principle, antibody-drug conjugates (ADCs) and peptide-drug conjugates (PDCs) have been developed to treat diseases. Currently, although some ADCs and PDCs are on the market or in clinical trials, their clinical application is significantly limited due to the design principles of these drugs.

[0003] Due to their complexity and large molecular weight, ADCs face numerous challenges in development, including a lack of suitable targets, production difficulties, and poor drug stability. Currently, ADCs are primarily used in cancer therapy. In some cases, the affinity of targeting antibodies for cancer cell surface antigens can reach as high as 10⁻⁹ to 10⁻¹² (Kd, moles / L), thus exhibiting high specificity for both target cells and normal cells with the same target receptors. However, because ADCs have a long metabolic time in vivo (1 to 3 weeks), they continuously kill normal cells during this period, significantly increasing their toxic side effects. Therefore, the ideal indication for ADCs should be diseases characterized by a significant difference in the amount of cell surface antigens between tumor and normal cells. However, very few diseases currently meet this stringent requirement.

[0004] PDCs are used to treat a variety of diseases in clinical or preclinical studies, but these are simply linking chemotherapy drugs with peptides, or adding peptides to nanoparticles or polymeric materials that encapsulate chemotherapy drugs. This makes it difficult for most peptides to enter cells due to their large molecular weight and charged nature. Therefore, most of these PDCs are currently only suitable for extracellular therapy, which severely limits the application scope and efficacy of PDCs.

[0005] Drug complexes can also be ligand-drug complexes (LDCs), where the ligand can be a peptide or a small molecule. However, the application of LDCs faces many challenges in terms of bioavailability, stability, efficacy, and toxicity. For example, many ligands cannot enter cells due to their large molecular weight, lipophilicity, or other properties, which limits their therapeutic applications. Furthermore, if the ligand is combined with routine chemotherapy drugs (e.g., doxorubicin, paclitaxel, etc.), the efficacy is usually low, while if it is combined with highly potent drug molecules (e.g., MMAE, DM1), the toxicity is high, and may even lead to death in animals before reaching the therapeutically effective dose for tumor treatment.

[0006] Therefore, there is an urgent need in the field to obtain improved LDCs that can act on highly expressed receptors that are widely present on the surface of diseased cells, broaden the target range and therapeutic window, and enhance drug efficacy while avoiding drug side effects. Summary of the Invention

[0007] One aspect of this application discloses a complex compound or a pharmaceutically acceptable salt thereof comprising an effective load and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a prostate-specific membrane antigen ligand portion, respectively.

[0008] Another aspect of this application discloses a complex compound or a pharmaceutically acceptable salt thereof, the complex compound or a pharmaceutically acceptable salt thereof comprising an effective load and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a ligand portion having the formula (I): (I).

[0009] Another aspect of this application discloses a complex compound or a pharmaceutically acceptable salt thereof comprising an effective load and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and P10, and the effective load is camptothecin or any derivative thereof.

[0010] In some embodiments, the prostate-specific membrane antigen ligand contained in the complex compound or a pharmaceutically acceptable salt thereof comprises the following structure: .

[0011] In some embodiments, the prostate-specific membrane antigen ligand contained in the complex compound or a pharmaceutically acceptable salt thereof comprises the following structure: .

[0012] In some embodiments, the prostate-specific membrane antigen ligand contained in the complex compound or a pharmaceutically acceptable salt thereof comprises the following structure: .

[0013] In some embodiments, the prostate-specific membrane antigen ligand contained in the complex compound or a pharmaceutically acceptable salt thereof comprises the following structure: .

[0014] In some embodiments, the two target molecules contained in the complex compound or its pharmaceutically acceptable salt are different.

[0015] In some embodiments, the synergistic molecules contained in the complex compound or its pharmaceutically acceptable salt are cell-interacting molecules.

[0016] In some embodiments, the complex compound or its pharmaceutically acceptable salt comprises two targeting molecules that interact with different cellular molecules.

[0017] In some embodiments, the synergistic molecule contained in the complex compound or its pharmaceutically acceptable salt is an endocytic molecule capable of mediating endocytosis.

[0018] In some embodiments, the synergistic molecule contained in the complex compound or its pharmaceutically acceptable salt binds to a molecule selected from the group consisting of: FORR1, TRPV6, FOLH1 (PMSA), LHRH, Her2, Trop2, Her3, NECTIN4, LRP1, GLUT1, EGFR1, AXL, CA9, CD44, Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR family, CD40, CD40L, OX40, OX40L, GITRL, GITR, 4-BBL, 4- 1BB, CD70, CD27, ICOSL, ICOS, HHLA2, CD28, CD86 / 80, CD28, MHCII antigen, TCR, CTLA-4, CD155, CD122, CD113, IGIT, PD-L1, PD1, Galectin-9, TIM-3, HVEM, BTLA, CD160, VISTA, B7-H4, B7-H3, phosphatidylinosermine, HHLA2, LAG3, Galectin-3, LILRB4, SIGLEC15, NKG2A, NKG2D, SLAMF7, KIR2DL1, KIR2DL2, KIR2DL3, FGFR1, FGFR2, FGFR4, NeuGcGM3, and CXCR4.

[0019] In some embodiments, the complex compound or a pharmaceutically acceptable salt thereof comprises a prostate-specific membrane antigen ligand portion and a synergistic molecule portion, the synergistic molecule portion being bound to molecules selected from the group consisting of: FORL1, TRPV6, FOLH1 (PMSA), SSTR2, and LHRH.

[0020] In some embodiments, the complex compound or a pharmaceutically acceptable salt thereof comprises a ligand portion of formula (I) and a synergistic molecule portion, said synergistic molecule portion being bound to a molecule selected from the group consisting of: FORL1, TRPV6, SSTR2, and LHRH.

[0021] In some other embodiments, the complex compound or a pharmaceutically acceptable salt thereof comprises P10 and a synergistic molecule portion, said synergistic molecule being bound to a molecule selected from the group consisting of: FORL1, TRPV6, FOLH1 (PMSA), and LHRH.

[0022] In some embodiments, the synergistic molecule contained in the complex compound or a pharmaceutically acceptable salt thereof is folic acid or an analogue thereof. In some embodiments, the folic acid analogue is selected from the group consisting of 5-methyltetrahydrofolate, 5-methoxytetrahydrofolate, methotrexate, and 5,10-methylenetetrahydrofolate.

[0023] In some embodiments, the complex compound or a pharmaceutically acceptable salt thereof comprises one, two, three, four, or more effective loads. In some embodiments, the effective load is selected from the group consisting of small molecule compounds, nucleotides, peptides, and proteins. In some embodiments, the effective load is a small molecule compound. In some embodiments, the small molecule compound is selected from the group consisting of camptothecin and any derivative thereof, auristatin and any derivative thereof, maytansine and any derivative thereof, radioisotope complexes, cyclooxygenase-2 inhibitors, paclitaxel and any derivative thereof, epothilone and any derivative thereof, bleomycin and any derivative thereof, dextrin and any derivative thereof, purcamycin and any derivative thereof, and mitomycin C. In some embodiments, the small molecule compound is camptothecin and any derivative thereof, auristatin and any derivative thereof, maytansine and any derivative thereof, radioisotope complexes, or cyclooxygenase-2 inhibitors.

[0024] In some embodiments, the payload contained in the complex compound of this application or its pharmaceutically acceptable salt is linked to at least one of the target molecules via a linker.

[0025] In some embodiments, the linkers contained in the complex compound of this application or its pharmaceutically acceptable salt are peptide linkers, disulfide linkers, pH-dependent linkers, or combinations thereof.

[0026] In some embodiments, the peptide linker can be cleaved by protease or reduction under specific physiological conditions. In some embodiments, the peptide linker is selected from the group consisting of: cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-lysine, cysteine-glutamic acid, aspartic acid-aspartic acid, and aspartic acid-aspartic acid-lysine. Optionally, the carboxylic acid in the above amino acids is acetylated.

[0027] In some embodiments, the disulfide linker is selected from the group consisting of DMDS, MDS, DSDM, and NDMDS.

[0028] In some implementations, the pH-dependent linker is aconitine.

[0029] In some embodiments, the linker of the complex compound of this application or a pharmaceutically acceptable salt thereof comprises the following structure: , , , , , , , , , , and , Alternatively, the linker may be a combination of the above structure and a peptide linker.

[0030] In some embodiments, the two target molecules contained in the complex compound of this application or a pharmaceutically acceptable salt thereof are linked by a spacer region. In some embodiments, the spacer region of this application contains an amino acid sequence selected from the group consisting of: SEQ ID NO:1-14, Arg-Arg, Ala-Ser-Asn, Ala-Ala-Ala, Ser-Ser-Arg, Pro-Arg, and Pro-Leu-Gly.

[0031] In some embodiments, the composite compound of this application is CB-20B, and its structural formula is as follows:

[0032] In some embodiments, the composite compound of this application is CB-20BK, and its structural formula is as follows:

[0033] In some embodiments, the composite compound of this application is CB-60S, and its structural formula is as follows:

[0034] In some embodiments, the composite compound of this application is CB-60SK, and its structural formula is as follows:

[0035] In some embodiments, the composite compound of this application is CB-20C, and its structural formula is as follows:

[0036] In some embodiments, the composite compound of this application is CB-1020, and its structural formula is as follows:

[0037] In some embodiments, the composite compound of this application is CB-1320, and its structural formula is as follows:

[0038] In some embodiments, the composite compound of this application is CB-1820, and its structural formula is as follows:

[0039] In some embodiments, the composite compound of this application is CR19428, and its structural formula is as follows:

[0040] In some embodiments, the composite compound of this application is 20R-SM09, and its structural formula is as follows:

[0041] In some embodiments, the composite compound of this application is CB-20R, and its structural formula is as follows: , where M is a radioactive isotope.

[0042] In some embodiments, the composite compound of this application is CB-18G, and its structural formula is as follows:

[0043] In some embodiments, the composite compound of this application is CR19426, and its structural formula is as follows:

[0044] In some embodiments, the composite compound of this application is CB-10S, and its structural formula is as follows:

[0045] In some embodiments, the composite compound of this application is CR19425, and its structural formula is as follows:

[0046] In some embodiments, the composite compound of this application is CB-50S, and its structural formula is as follows:

[0047] Another aspect of this application discloses a pharmaceutical composition comprising the complex compound of this application or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0048] In some embodiments, the composition is used for intravenous, subcutaneous, oral, intramuscular, or intraventricular administration.

[0049] Another aspect of this application discloses a method for delivering a payload to a subject in need, comprising administering to the subject a therapeutically effective amount of the complex compound described in this application or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in this application.

[0050] Another aspect of this application discloses a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the complex compound described in this application or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in this application.

[0051] In some embodiments, the method of treating a disease in the subject of this application further includes administering one or more therapeutic agents in combination with the complex compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

[0052] Another aspect of this application discloses the use of a complex compound described in this application or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in this application, in the preparation of a medicament for treating a disease in the target.

[0053] Another aspect of this application discloses a complex compound or a pharmaceutically acceptable salt thereof described in this application for use in treating a disease in the subject of this application, or a pharmaceutical composition described in this application.

[0054] In some implementations, the disease is selected from the group consisting of: cancer, immune diseases, cardiovascular diseases, metabolic diseases, and neurological diseases.

[0055] In some implementations, the cancer is selected from the group consisting of: prostate cancer, breast cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma, and multiple myeloma.

[0056] In some embodiments, the immune disease is an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of connective tissue diseases, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

[0057] In some implementations, cardiovascular diseases are selected from the group consisting of: angina pectoris, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia, and congenital heart disease.

[0058] In some implementations, metabolic diseases are selected from the group consisting of diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.

[0059] In some implementations, the neurological disease is selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma, and epilepsy. Simple Explanation of the Diagram

[0060] Figure 1 shows the chemical structural formulas of the complex compounds CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09, CB-20R, CB-18G, CR19426, CB-10S, CR19425 and CB-50S.

[0061] Figure 2A shows the time-varying curves of Cy5-pep-20BK binding and endocytosis with different cells (from top to bottom: LNCaP cells, KB cells, DU145 cells, and NCI-H460 cells). Figure 2B shows the time-varying curves of Cy5-pep-20AK binding and endocytosis with different cells (from top to bottom: LNCaP cells, DU145 cells, NCI-H460 cells, and KB cells).

[0062] Figure 3 shows the fluorescence images of Cy5-FA binding to and endocytosis in different cells over time. Complete circular patterns represent the fluorescence of the cell nucleus, while dotted patterns represent the fluorescence of Cy5-FA.

[0063] Figure 4A shows the inhibitory activity of the complex compound CB-20BK on the proliferation of the illustrated tumor cells. Figure 4B shows the inhibitory activity of the complex compound CB-20B on the proliferation of the illustrated tumor cells. Figure 4C shows the inhibitory activity of the complex compound CB-10S on the proliferation of the illustrated tumor cells. Figure 4D shows the inhibitory activity of the complex compound CB-60S on the proliferation of the illustrated tumor cells. Figure 4E shows the inhibitory activity of the complex compound CB-60SK on the proliferation of the illustrated tumor cells. Figure 4F shows the inhibitory activity of the complex compound CB-18G on the proliferation of the illustrated tumor cells. Figure 4G shows the inhibitory activity of the complex compound CB-50S on the proliferation of the illustrated tumor cells.

[0064] Figures 5A-5E show the tumor-inhibiting effect of the complex compound CB-20BK in mice.

[0065] Figures 6A-6C show the tumor-inhibiting effect of the complex compound CB-20B in mice.

[0066] Figures 7A-7E show the tumor-inhibiting effect of the complex compound CB-18G in mice.

[0067] Figures 8A-8B show the effect of injectable CBP-1018 on tumor volume in lung cancer models LU2505 and LU1206. Implementation

[0068] Although this application discloses various aspects and embodiments, it will be apparent to those skilled in the art that various equivalent changes and modifications can be made to these aspects and embodiments without departing from the spirit and scope of this application. The various aspects and embodiments disclosed in this application are for illustrative purposes only and are not intended to be limiting; the true scope is indicated by the claims in the appended patent applications. All publications, patents, or patent applications referenced in this application are incorporated herein by reference in their entirety. Unless otherwise stated, all technical terms used in this application have the same meaning as commonly understood by those skilled in the art to which this application pertains.

[0069] As used herein and in the claims of the appended patent applications, the singular forms “a,” “an,” “one,” and “the” include plural objects unless the context clearly indicates otherwise. The terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably in this application. It should also be noted that the terms “comprising,” “including,” and “having” are used interchangeably.

[0070] As used herein and in the appended claims, the term "analyte" includes structural analogs and functional analogs. Structural analogs are compounds having similar chemical structures, which may contain one or more different atoms or one or more different functional groups. Functional analogs are compounds having the same or similar chemical, biological, or pharmacological effects. For example, folic acid analogs include 5-methyltetrahydrofolate, 5-methoxytetrahydrofolate, methotrexate, and 5,10-methylenetetrahydrofolate.

[0071] As used herein and in the claims of the appended patent applications, the term "derivative" refers to a class of more complex compounds derived from a parent compound molecule by substituting one or more atoms or groups of atoms with other atoms or groups of atoms. For example, camptothecin derivatives include irinotecan, SN-38, Dxd, topotecan, GI-147211C, topotecan, 9-aminocamptothecin, 7-hydroxymethylcamptothecin, 7-aminomethylcamptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, 9-nitrocamptothecin, gemmatothecin, karenitecin, silatecan, letopotecan, ezaranotecan, difluorotecan, belototecan, letopotecan, and S39625.

[0072] One aspect of this application discloses a complex compound or a pharmaceutically acceptable salt thereof comprising an effective load and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a prostate-specific membrane antigen ligand portion, respectively.

[0073] Another aspect of this application discloses a complex compound or a pharmaceutically acceptable salt thereof, the complex compound or a pharmaceutically acceptable salt thereof comprising an effective load and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a ligand portion having the formula (I): (I).

[0074] Another aspect of this application discloses a complex compound or a pharmaceutically acceptable salt thereof comprising an effective load and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and P10, and the effective load is camptothecin or any derivative thereof.

[0075] As used in this application, the term "payload" refers to a molecule or substance intended to be delivered to target cells or tissues. Without limitation, a payload can be any molecule or substance intended for the diagnosis, treatment, or prevention of a disease in a subject. In some embodiments, the payload has a molecular weight of less than or equal to about 5 kDa. In some embodiments, the payload has a molecular weight of less than or equal to about 1.5 kDa. In some embodiments, the payload is a drug or diagnostic reagent that has been deemed safe and effective for use by an appropriate drug review and registration agency (e.g., FDA, EMEA, or NMPA).

[0076] In some embodiments, the payload of this application is a small molecule compound, a nucleotide (e.g., DNA, plasmid DNA, RNA, siRNA, antisense oligonucleotides, or nucleic acid aptamers), a peptide, or a protein (e.g., an enzyme). In some embodiments, the payload is a small molecule compound.

[0077] In some embodiments, the payload of this application includes, but is not limited to: anticancer drugs, radioactive substances, vitamins, anti-AIDS drugs, antibiotics, immunosuppressants, antiviral drugs, enzyme inhibitors, neurotoxins, opioids, modulators of cell-extracellular matrix interactions, vasodilators, antihypertensive drugs, hypnotics, antihistamines, anticonvulsants, muscle relaxants, anti-Parkinsonian substances, anticonvulsants and muscle contraction agents, antiparasitic and / or antiprotozoan drugs, analgesics, antipyretics, steroid and nonsteroidal anti-inflammatory drugs, anti-angiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, prostaglandins, antidepressants, antipsychotics, antiemetics, or imaging agents.

[0078] In some embodiments, the active ingredient of this application has free amino or carboxyl groups before being linked to the composite compound of this application. The active ingredient is incorporated into the composite compound through a ylated reaction between the aforementioned amino or carboxyl groups and the corresponding groups of the composite compound (e.g., linkers). In some embodiments, modification of the aforementioned free amino or carboxyl groups (e.g., by incorporating them into the composite compound of this application) significantly reduces the activity of the active ingredient (e.g., by at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%).

[0079] As used in this application, "small molecule compound" refers to a compound having a molecular weight of less than or equal to about 2 kDa. In some embodiments, the small molecule compound has a molecular weight of less than or equal to about 1.5 kDa. In some preferred embodiments, the small molecule compound has a molecular weight of less than or equal to about 1 kDa, 800 Da, 700 Da, 600 Da, or 500 Da. In some embodiments, the small molecule compound of this application is selected from the group consisting of: camptothecin and any derivative thereof (e.g., SN38 or Dxd), auristatin and any derivative thereof (e.g., MMAE and MMAF), maytansine and any derivative thereof, cyclooxygenase-2 inhibitors (e.g., celecoxib), radioisotope complexes, paclitaxel and any derivative thereof, epothilone and any derivative thereof, bleomycin and any derivative thereof, dextrin and any derivative thereof, purcamycin and any derivative thereof, and mitomycin C. In some embodiments, the small molecule compound is camptothecin and any derivative thereof, auristatin and any derivative thereof, a radioisotope complex, or a cyclooxygenase-2 inhibitor. In some embodiments, the small molecule compounds described in this application are drugs for alleviating or treating cancer. In some embodiments, the small molecule compounds described in this application are drugs for alleviating or treating autoimmune diseases.

[0080] The term "camptothecin" as used in this application refers to a cytotoxic alkaloid, primarily derived from the camptothecin plant (Campsis grandiflora) of the Davidiaceae family, which exhibits strong antitumor activity. The camptothecin and its derivatives in this application include camptothecin and its derivatives that are currently existing or will be developed in the future. The camptothecin and its derivatives in this application include, but are not limited to: camptothecin, irinotecan, SN-38, Dxd, topotecan, GI-147211C, topotecan, 9-aminocamptothecin, 7-hydroxymethylcamptothecin, 7-aminomethylcamptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, 9-nitrocamptothecin, gemmatothecin, karenitecin, silatecan, letopotecan, esaxatecan, diflutecan, belototecan, letopotecan, and S39625.

[0081] As used in this application, the term "aurestatin and any derivative thereof" refers to the natural antitumor product salipodin 10 and a series of its derivatives. These compounds induce cell arrest during mitosis by interfering with microscopic self-assembly, exhibiting strong cellular cytotoxicity. Aurestatin and any derivative thereof in this application include both currently existing and subsequently developed aurestatin and any derivative thereof. Aurestatin and its derivatives in this application include, but are not limited to, aurestatin, monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), monomethylaurestatin D (MMAD), AFP, and AFHPA.

[0082] The term "cyclooxygenase-2 inhibitor" as used in this application refers to a specific class of cyclooxygenase-2 inhibitors. Cyclooxygenase-2 participates in the development and invasion of malignant tumors through multiple mechanisms. Cyclooxygenase-2 inhibitors can inhibit tumor cell migration and adhesion, as well as intravascular invasion, thereby inhibiting the occurrence and development of malignant tumors. The cyclooxygenase-2 inhibitors in this application include existing or future cyclooxygenase-2 inhibitors. Cyclooxygenase-2 inhibitors include, but are not limited to, celecoxib, rofecoxib, parecoxib, vortexib, and etoxib.

[0083] As used in this application, the term "radioisotope complex" refers to a special type of complex containing a radioisotope, wherein the chelating agent in the complex can chelate with the radioisotope and provide a more stable binding link to the target material. As used in this application, the term "radioisotope" refers to an element that spontaneously emits radiation (e.g., alpha rays, beta rays, or gamma rays). The radioisotopes in this application include all currently existing or future radioisotopes that can be used for treatment and diagnosis. The radioactive isotopes in this application include, but are not limited to, 67Cu, 64Cu, 90Y, 109Pd, 111Ag, 149Pm, 153Sm, 165Ho, 166Ho, 177Lu, 186Re, 188Re, 99mTc, 67Ga, 68Ga, 111In, 90Y, 177Lu, 186Re, 188Re, 197Au, 198Au, 199Au, 105Rh, 161Tb, 149Pm, 44Sc, 47Sc, 70As, 71As, 72As, 73As, 74As, 76As, 77As, 212Pb, 212Bi, 213Bi, 225Ac, 117mSn, 67Ga, 201Tl, 123I, 131I, 160Gd, 148Nd, 89Sr and 211 At. In some implementations,The active ingredients of H2dedpa, H4octapa, H2azapa, DTPA, CHX-A''-DTPA, and DTPA-bis anhydride、Maleimide-DTPA、DTPA(tBu)4、DiamSar CB-TE2A、Cyclam、DO2A、DOTA、OTA-GA(tBu)4 Maleimide-DOTA-GA、p-NCS-Bz-DOTA-GA、NH2-DOTA-GA、DOTA-GA anhydride、DOTA-tris(tBu) ester、Propargyl-DOTA-tris(tBu) ester、DO3AM-acetic acid、DO3AM-N-(2-aminoethyl)ethanamide、DO3AtBu-N-(2-aminoethyl)ethanamide、DOTA-di(tBu) ester、DOTA-tris(tBu) ester NHS ester、DOTA-NHS ester、Propargyl-DOTA-tris(tBu) ester、DOTADOTA-GA anhydride、DOTA-GA(tBu)4 、p-NCS-Bz-DOTA-GA、NH2-DOTA-GA、Maleimide-DOTA-GA、AGuIX、Gado-H、CYCLEN、DO2AtBu、DO3AtBu、DO3AEt、DO3AM、DOTAEt、DOTPrEt、cis-Gly oxal-Cyclene、Mono-N-Benzyl-Cyclene、Trans-N-Dibenyl-Cyclene、TriBOC-Cyclene、Mono-N-Benzyl-TACN、DiBOC-TACN、Cross-bridge-Cyclam (CB-Cyclam)、(13)aneN4、TACN、TACN·3HCl、TACD、Mono-N-benzyl-TACD、DiBOC-TACD、1,7-Dioxa-4,10-diazacyclododecane, C-Methyl-Ester-Cyclam, C-Carboxylic-Acid-Cyclam, trans-N-Dimethyl-Cyclam, TETRAM, TETAEt, TETAMEt2, TETAMMe2, TETAM, CPTA, CB-Cyclam derivatives, CB-TE2A, Methylamino-(13)aneN4, Bis-(13)aneN4, Oxo-(13)aneN4, Mono-N-Benzyl-(13)aneN4, TriBOC-(13)aneN4, TRITRAM, TRI3AEt, TRI3AtBu, TRITAM, TRITA, Mono-N-Benzyl-Cy clam, Formaldehyde-Cyclam, cis-Glyoxal-Cyclam, Dioxocyclam, Oxocyclam, trans-N-Dibenzyl-Cyclam, TriBOC-Cyclam, DOTP, DOTMA, TETA, DOTAM, DiAmSar, CB-Cyclam, CB-TE2A, NOTA, NOTAM, NH2 -NODA-GA, Iodo-NODA-GA, NCS-MP-NODA, NH2-MPAA-NODA, NODA-GA(tBu)3 , NODA-GA-NHS ester, Maleimide-NODA-GA, NOTA-NHS ester, Maleimide-NOTA, Propargyl-NOTA(tBu)2, p-NCS-benzyl-NODA-GA, NOTA(tBu)2, NCS-MP-NODA, NH2-MPAA-NODA, NH2-NODA-GA, Iodo-NODA-GA and TACN. ,

[0084] In some embodiments, the complex compound of this application or its pharmaceutically acceptable salt comprises one effective load. In some embodiments, the complex compound of this application or its pharmaceutically acceptable salt comprises two or more effective loads. For example, the complex compound of this application or its pharmaceutically acceptable salt comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more effective loads. In complex compounds containing multiple effective loads, each effective load may be the same or different from each other. In some embodiments, at least two effective loads are different from each other.

[0085] As used in this application, the term "targeting molecule" refers to any molecule or portion capable of targeting the complex compound of this application to a target site, target tissue, target organ, target cell, or intracellular region of a target cell. In some embodiments, the targeting molecule causes the complex compound of this application to be distributed in greater quantities at the target site, target tissue, target organ, target cell, or intracellular region of a target cell compared to non-target sites, non-target tissues, non-target organs, non-target cells, or intracellular regions of a target cell, for example, by at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500%, or higher. In some embodiments, the targeting molecule causes the complex compound with the targeting molecule to be distributed in greater quantities at the target site, target tissue, target organ, target cell, or intracellular region of a target cell compared to a complex compound without the targeting molecule, for example, by at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500%, or higher. In some implementations, the targeting molecule can trigger or promote the specific binding of a complex compound containing such a targeting molecule to the target molecule, trigger or promote the endocytosis of the complex compound by the target cell, or trigger or promote the enrichment of the complex compound around the target cell and / or its entry into the target cell.

[0086] In some embodiments, the complex compound of this application comprises at least two targeting molecules. In some embodiments, the two or more targeting molecules comprised in the complex compound of this application are the same or different. In some embodiments, at least two of the two or more targeting molecules comprised in the complex compound of this application are different. In some embodiments, the two or more targeting molecules comprised in the complex compound of this application are all different from each other. In some embodiments, at least two of the two or more targeting molecules comprised in the complex compound of this application are capable of specifically binding to different cell surface proteins or markers. In some embodiments, the two or more targeting molecules comprised in the complex compound of this application are capable of specifically binding to different cell surface proteins or markers.

[0087] In some embodiments, the complex compound of this application contains at least two targeting molecules, at least one of which is a synergistic molecule.

[0088] As used in this application, the term "synergistic molecule" refers to any molecule or portion capable of synergizing with other targeting molecules contained in the complex compound of this application to better trigger or promote the specific binding of the complex compound to the target molecule, trigger or promote the endocytosis of the complex compound by the target cell, trigger or promote the enrichment of the complex compound around and / or entry into the target cell, and / or otherwise cause the specific binding and retention of the complex compound to the target cell. In some embodiments, the synergistic molecule causes the complex compound of this application to be distributed in greater quantities, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500%, or higher, at the target site, target tissue, target organ, target cell, or target cell region compared to non-target sites, non-target tissues, non-target organs, non-target cells, or regions within non-target cells. In some embodiments, the synergistic molecule causes the complex compound containing the synergistic molecule to be distributed in greater quantities at the target site, target tissue, target organ, target cell, or intracellular region of the target cell compared to the complex compound without the synergistic molecule, for example, by at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500%, or higher. In some embodiments, the synergistic molecule causes the complex compound containing the synergistic molecule to have higher activity against the target cells compared to the complex compound without the synergistic molecule, for example, by at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500%, or more.

[0089] In some embodiments, the synergistic molecules of this application are cell-interaction molecules.

[0090] As used in this application, the term "cell-interacting molecule" refers to a compound that can interact with cell surface material of a target cell to trigger or promote the specific binding of a complex containing such a cell-interacting molecule to the cell, trigger or promote the endocytosis of the complex by the target cell, and / or trigger or promote the enrichment of the complex around the target cell and / or entry into the target cell.

[0091] Cell-interacting molecules can be small chemical molecules or large biomolecules. In some embodiments, cell-interacting molecules are small molecule compounds or peptides. In some embodiments, cell-interacting molecules are small molecule compounds or peptides comprising 2-50, 2-40, 2-30, 2-25, 2-22, 2-20, 2-18, 2-15, 2-12, 2-10, 2-8, 4-50, 5-50, 5-40, 5-30, 5-25, 5-22, 5-20, 5-18, 5-15, 5-12, 5-10, 6, 7, 8, or 9 amino acids.

[0092] In some embodiments, the targeting molecule is a ligand capable of binding to cell surface receptors or other molecules. In some embodiments, at least one targeting molecule is a ligand capable of binding to cell surface receptors or other molecules.

[0093] The ligands of this application can include a wide variety of chemical or biological molecules that can have specific binding affinity to selected targets, such as cell surface receptors, cell surface antigens, cells, tissues, organs, etc. In some embodiments, the ligands can specifically bind to proteins or markers expressed on the surface of target cells. In some embodiments, the ligands of this application bind to cell surface proteins or markers with an affinity of 10⁻⁶ to 10⁻¹¹ M (Kd value). In some embodiments, the ligands of this application bind to cell surface proteins or markers with an affinity of at least 10⁻⁷, at least 10⁻⁸, or at least 10⁻⁹ M (Kd value). In some embodiments, the ligands of this application bind to cell surface proteins or markers with an affinity of less than 10⁻⁶, less than 10⁻⁷, or less than 10⁻⁸ M (Kd value). In some embodiments, the ligands of this application bind to cell surface proteins or markers with a certain affinity, wherein the affinity of the ligand for the target cell surface protein or marker is at least two, three, four, five, six, eight, ten, twenty, fifty, one hundred, or more times higher than its affinity for non-target cell surface proteins or markers. In some embodiments, the cell surface proteins or markers of this application exhibit significantly higher performance in target cells (e.g., cancer cells) than in normal cells. The term "significant" as used in this application refers to a statistically significant difference, or a significant difference that can be recognized by those skilled in the art.

[0094] In some embodiments, the expression levels of the cell surface proteins or markers of this application in target cells (e.g., cancer cells) are 2 to 1,000,000 times higher than their expression levels in normal cells. For example, the expression levels in target cells (e.g., cancer cells) are 2 to 10 times, 2 to 100 times, 2 to 1,000 times, 2 to 10,000 times, 2 to 100,000 times, or 2 to 1,000,000 times higher than their expression levels in normal cells (which can be any value within the above range, including the endpoints of the range). In some embodiments, the expression levels of cell surface receptors in target cells (e.g., cancer cells) are at least 10 times, at least 100 times, at least 1,000 times, at least 10,000 times, or at least 100,000 times higher than their expression levels in normal cells. In some embodiments, the expression levels of cell surface receptors on normal cells are reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% when compared to the expression levels of cell surface proteins or markers on target cells (e.g., cancer cells). In some embodiments, the cell surface proteins or markers described herein are undetectable in normal cells.

[0095] In some embodiments, the cell surface proteins or markers of this application are cell surface receptors.

[0096] In some embodiments, the cell surface receptors of this application are selected from the group consisting of: transferrin receptor (TFR), low-density lipoprotein receptor (LDLR), folate receptor (FR), auxin-inhibiting hormone receptor, urate kinase receptor, tumor necrosis factor receptor (TNFR), integrin receptor (LFA-1), SST-14 receptor (SSTR2), GNRH receptor (GNRHR), TRPV6, and integrin α receptor.

[0097] In some embodiments, the cell surface proteins or markers of this application are cell surface antigens.

[0098] In some embodiments, the cell surface antigens of this application are selected from the group consisting of: prostate-specific membrane antigen, MUC1 mucin, acute lymphoblastic leukocyte antigen, Thy-1 cell surface antigen, Melan-A protein, squamous cell carcinoma antigen, galactoglobulin 3, and human leukocyte antigen.

[0099] In some embodiments, the cell-interacting molecules of this application can bind to molecules selected from the group consisting of: FOLR1, TRPV6, FOLH1 (PMSA), LHRH, Her2, Trop2, Her3, NECTIN4, LRP1, GLUT1, EGFR1, AXL, CA9, CD44, Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR family, CD40, CD40L, OX40, OX40L, GITRL, GITR, 4-BBL, 4-1BB, CD 70, CD27, ICOSL, ICOS, HHLA2, CD28, CD86 / 80, CD28, MHCII antigen, TCR, CTLA-4, CD155, CD122, CD113, IGIT, PD-L1, PD1, Galectin-9, TIM-3, HVEM, BTLA, CD160, VISTA, B7-H4, B7-H3, phosphatidylinosermine, HHLA2, LAG3, Galectin-3, LILRB4, SIGLEC15, NKG2A, NKG2D, SLAMF7, KIR2DL1, KIR2DL2, KIR2DL3, FGFR1, FGFR2, FGFR4, NeuGcGM3, and CXCR4.

[0100] In some embodiments, the complex compound of this application or a pharmaceutically acceptable salt thereof comprises a prostate-specific membrane antigen ligand portion and a synergistic molecule portion, said synergistic molecule portion binding to molecules selected from the group consisting of: FORL1, TRPV6, FOLH1 (PMSA), SSTR2, and LHRH.

[0101] In some embodiments, the complex compound of this application or a pharmaceutically acceptable salt thereof comprises a ligand portion of formula (I) and a synergistic molecule portion, said synergistic molecule portion being bound to molecules selected from the group consisting of: FORL1, TRPV6, SSTR2 and LHRH.

[0102] In some other embodiments, the complex compound of this application or a pharmaceutically acceptable salt thereof comprises P10 and a synergistic molecule moiety, said synergistic molecule being bound to a molecule selected from the group consisting of: FORL1, TRPV6, FOLH1 (PMSA) and LHRH.

[0103] In some embodiments, a cooperating molecule in the complex compound of this application or its pharmaceutically acceptable salt is an endocytic molecule capable of mediating endocytosis. As used herein, the term "endocytosis" refers to the ability of the complex compound or its pharmaceutically acceptable salt to mediate its own endocytosis, internalization, or uptake into the target cell after interaction with the target cell. As used herein, the term "endocytic molecule" refers to a molecule that, after interaction with the target cell, is capable of mediating the endocytosis, internalization, or uptake of the complex compound of this application or its pharmaceutically acceptable salt into the target cell.

[0104] In some embodiments, the endocytic molecules are selected from the group consisting of folic acid and its analogues, peptides that can mediate endocytosis, and membrane-penetrating peptides.

[0105] In some embodiments, the endocytic molecule of this application is folic acid or an analogue thereof.

[0106] Folic acid, due to its small molecular weight, lack of immunogenicity, and good stability, is conducive to forming chemical bonds with other groups. Folic acid can bind with high affinity to folate receptors expressed on the cell surface to mediate cellular uptake of folate. Although the expression level of folate receptors is very low in most normal cells, it is expressed in high levels in a large number of cancer cells to meet the high demand for folate from rapidly dividing cells under low folate conditions (see Kelemen LE, Int J Cancer, 2006; 119: 243-50; Kane MA et al., J Clin Invest. 1988; 81: 1398-406; Matsue H et al., Proc Natl Acad Sci USA. 1992; 89: 6006-9; Zhao R et al., Annu Rev Nutr. 2011; 31: 177-201). Folic acid can specifically bind to folic acid receptors on the cell surface, and it can also mediate the endocytosis of complex compounds or their pharmaceutically acceptable salts into target cells.

[0107] In some embodiments, the folic acid analogues are selected from the group consisting of 5-methyltetrahydrofolate, 5-methoxytetrahydrofolate, methotrexate, and 5,10-methylenetetrahydrofolate.

[0108] In some implementations, the endocytic molecule is a peptide capable of mediating endocytosis.

[0109] In some embodiments, the peptide capable of mediating endocytosis comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, Arg-Gly-Asp (referred to as RGD), and a homologous peptide having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence homology with any one of SEQ ID NO: 16-18, wherein the homologous peptide is a functional equivalent of the peptide represented by SEQ ID NO: 16-18.

[0110] In some embodiments, the peptides capable of mediating endocytosis as described in this application have a conserved amino acid substitution at only one amino acid site compared to the sequences of SEQ ID NO: 16-20 and RGD. In some embodiments, the peptides capable of mediating endocytosis as described in this application have conserved amino acid substitutions at 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid sites compared to the sequences of SEQ ID NO: 16-20.

[0111] Without affecting their biological activity, the peptides that mediate endocytosis as described in this application may also contain non-naturally occurring amino acids, including, for example, β-fluoroalanine, 1-methylhistidine, γ-methylene glutamine, α-methylleucine, 4,5-dehydrolysine, hydroxyproline, 3-fluorophenylalanine, 3-aminotyramine, 4-methyltryptophan, etc.

[0112] The percentage of homology can be determined using a variety of methods well known in the art. For example, sequences can be compared using the following publicly available tools: BLASTp software (available from the National Center for Biotechnology Information (NCBI) website: http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, see also Altschul SF et al., J. Mol. Biol. , 215:403–410 (1990); Stephen F. et al., Nucleic Acids Res. , 25:3389–3402 (1997)), ClustalW2 (available from the European Institute for Bioinformatics website: http: / / www.ebi.ac.uk / Tools / msa / clustalw2 / , see also Higgins DG et al., Methods in Enzymology , 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)) and Tcoffee (available from the website of the Swedish Institute of Bioinformatics, see also Poirot O. et al., Nucleic Acids Res., 31(13): 3503-6 (2003); Notredame C. et al., J. Mol. Boil., 302(1): 205-17 (2000)). If software is used for sequence alignment, the default parameters provided in the software can be used, or the parameters can be customized in other ways to suit the alignment purpose. All of these are within the knowledge of those usually skilled in the art.

[0113] As used in this application, the term "functional equivalent" refers to a derived peptide that retains biological activity substantially similar to that of the original peptide from which the derived peptide originates. Functional equivalents can be natural derivatives or synthetically prepared. Exemplary functional equivalents include amino acid sequences having one or more amino acid substitutions, deletions, or additions, provided that the peptide's biological activity is preserved. The substituted amino acid ideally possesses similar chemical-physical properties to the substituted amino acid. Ideally, similar chemical-physical properties include similarity in charge, bulkiness, hydrophobicity, hydrophilicity, etc.

[0114] In some embodiments, the functional equivalent includes conservative substitutions of amino acid residues. Conservative substitutions of amino acid residues refer to substitutions between amino acids with similar properties, such as substitutions between polar amino acids (e.g., substitution between glutamine and aspartamine), substitutions between hydrophobic amino acids (e.g., substitution between leucine, isoleucine, methionine, and valine), and substitutions between amino acids with the same charge (e.g., substitution between arginine, lysine, and histidine, or substitution between glutamine and aspartic acid), etc.

[0115] In some embodiments, the endocytic molecule is a membrane-penetrating peptide. Membrane-penetrating peptides ( [C]ell- [P] enetrating [P]eptides (CPP), also known as protein transduction domains (PTDs), are short peptides (typically fewer than 40 amino acids) capable of entering the cell interior in a receptor-independent manner. When complexed with a payload, the transmembrane peptide mediates the transmembrane transport of the payload and possesses protein transduction activity. In some embodiments, the transmembrane peptides described herein are selected from the group consisting of tumor homing peptides, mitochondrial penetrating peptides, cell-activating transmembrane peptides, and antimicrobial peptides. In some embodiments, the transmembrane peptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 19 (RRRRRRRRR, referred to as R9) and SEQ ID NO: 20 (GRKKRRQRRRPPQ, which is a Tat peptide, i.e., a transmembrane peptide of the HIV transcription protein transactivator).

[0116] In some embodiments, one of the target molecules in the complex compound of this application or a pharmaceutically acceptable salt thereof is a prostate-specific membrane antigen ligand portion.

[0117] As used in this application, "prostate-specific membrane antigen" refers to a type II transmembrane glycoprotein present in the membrane of prostate epithelial cells, composed of 750 amino acids, including 19 intracellular amino acids, 24 transmembrane amino acids, and 707 extracellular amino acids. Prostate-specific membrane antigen is expressed in normal prostate epithelial cells, but its expression level is much higher in prostate cancer cells. Compared to traditional prostate-specific antigens used in clinical testing, prostate-specific membrane antigen is a more sensitive and specific tumor marker for prostate cancer, especially showing high expression in hormone-refractory prostate cancer and metastatic prostate cancer lesions, demonstrating high sensitivity and specificity in differentiating prostate cancer from other types of malignant tumors. Furthermore, prostate-specific membrane antigen is also highly specifically expressed on tumor vascular endothelial cells in various non-prostate-derived solid tumors (such as lung cancer, bladder cancer, gastric cancer, pancreatic cancer, kidney cancer, and colorectal cancer).

[0118] As used in this application, the term "prostate-specific membrane antigen ligand" refers to antibodies, nucleic acid aptamers, and small molecules that can specifically recognize and bind to prostate-specific membrane antigens. The prostate-specific membrane antigen ligands in this application include existing or future prostate-specific membrane antigen ligands, as well as fragments of the aforementioned ligands, provided that these fragments retain the ability to bind to prostate-specific membrane antigens. Antibody ligands are the most common prostate-specific membrane antigen ligands, including but not limited to monoclonal antibodies J591, J533, J415, and E99 (e.g., see Liu H, Rajasekaran AK, Moy P et al., Constitutive and antibody-induced internalization of prostate-specific memberane antigen [J]. Cancer Res, 1998, 58 (18): 4055-4060). Nucleic acid aptamers are single-stranded DNA or RNA molecules that bind with high affinity and high specificity to prostate-specific membrane antigens (PSA), obtained through index-enrichment ligand systems. These PSA ligands include, but are not limited to, xPSM-A10 aptamers and their derivatives, and xPSM-A9 aptamers and their derivatives (e.g., see Lupoid SE et al., Identification and Characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen, Cncer Res, 2002, 62(14):4029-4033). Compared to antibody-based and nucleic acid aptamer-based ligands, small molecule PSA ligands have advantages such as small molecular weight, high permeability, low immunogenicity, and ease of synthesis. These include, but are not limited to, glutamate-based and aminophosphate-based small molecule ligands.

[0119] In some embodiments, the prostate-specific membrane antigen small molecule ligand of this application may be selected from the group consisting of: 2-[[methylphosphino]methyl]glutaric acid; 2-[[ethylphosphino]methyl]glutaric acid; 2-[[propylphosphino]methyl]glutaric acid; 2-[[butylphosphino]methyl]glutaric acid; 2-[[cyclohexylphosphino]methyl]glutaric acid; 2-[[phenylphosphino]methyl]glutaric acid; 2-[[2-(tetrahydrofuranyl)phosphino]methyl]glutaric acid; 2-[[(2-tetrahydropyranyl)phosphino]methyl]glutaric acid; 2-[[((4-pyridyl)methyl)phosphino]methyl]glutaric acid; 2-[[((2-pyridyl)methyl]methyl]glutaric acid; 2-[[(phenylmethyl)hydroxyphosphono]methyl]glutaric acid; 2-[[((2-phenylethyl)methyl)hydroxyphosphono]methyl]glutaric acid; 2-[[((3-phenylpropyl)methyl)hydroxyphosphono]methyl]glutaric acid; 2-[[((3-phenylbutyl)methyl)hydroxyphosphono]methyl]glutaric acid; 2-[[((2-phenylbutyl)methyl)hydroxyphosphono]methyl]glutaric acid; 2-[[(4-phenylbutyl)hydroxyphosphono]methyl]glutaric acid; and 2-[[(aminomethyl)hydroxyphosphono]methyl]glutaric acid; 2-[[methylhydroxyphosphono]oxy]glutaric acid; 2-[[ethylhydroxyphosphono]oxy]glutaric acid; 2-[[propyl] 2-[[Butylhydroxyphosphono]oxy]glutaric acid; 2-[[phenylhydroxyphosphono]oxy]glutaric acid; 2-[[((4-pyridyl)methyl)hydroxyphosphono]oxy]glutaric acid; 2-[[((2-pyridyl)methyl)hydroxyphosphono]oxy]glutaric acid; 2-[[(phenylmethyl)hydroxyphosphono]oxy]glutaric acid; and 2-[[((2-phenylethyl)methyl)hydroxyphosphono]oxy]glutaric acid; 2-[[(N-hydroxy)aminomethane]methyl]glutaric acid; 2-[[(N-hydroxy-N-methyl)aminomethane]methyl]glutaric acid; 2-[[(N-butyl-N-hydroxy)aminomethane]methyl]glutaric acid; 2-[[(N-benzyl-N-hydroxy)aminomethane]methyl]glutaric acid; 2-[[(N-benzyl-N-hydroxy)aminomethane]methyl]glutaric acid; 2-[[(N-benzyl-N-hydroxy)aminomethane]methyl]glutaric acid; 2-[[(N-hydroxy-N-phenyl)aminomethane]methyl]glutaric acid; 2-[[(N-hydroxy-N-2-phenylethyl)aminomethane]methyl]glutaric acid; 2-[[(N-ethyl-N-hydroxy)aminomethane]methyl]glutaric acid; 2-[[(N-hydroxy-N-propyl)aminomethane]methyl]glutaric acid; 2-[[(N-hydroxy-N-3-phenylpropyl)aminomethane]methyl]glutaric acid; 2-[[(N-hydroxy-N-4-pyridyl)aminomethane]methyl]glutaric acid; 2-[[(N-hydroxy)aminoamine]methyl]glutaric acid; 2-[[N-hydroxy(methyl)aminoamine]methyl]glutaric acid; 2-[[N-hydroxy(benzyl)aminoamine]methyl]glutaric acid;2-[[N-hydroxy(phenyl)amino]methyl]glutaric acid; 2-[[N-hydroxy(2-phenylethyl)amino]methyl]glutaric acid; 2-[[N-hydroxy(ethyl)amino]methyl]glutaric acid; 2-[[N-hydroxy(propyl)amino]methyl]glutaric acid; 2-[[N-hydroxy(3-phenylpropyl)amino]methyl]glutaric acid; and 2-[[N-hydroxy(4-pyridyl)amino]methyl]glutaric acid; 2-[(thionyl)methyl]glutaric acid; 2-[(methylthionyl)methyl]glutaric acid; 2-[(ethylthionyl)methyl]glutaric acid; 2-[(propylthionyl)methyl]glutaric acid; 2-[(butylthionyl)methyl]glutaric acid; 2-[(phenylthionyl)methyl]glutaric acid; 2-[[(2-phenylethyl)thionyl]methyl]glutaric acid; 2-[[(3-phenylpropyl)thionyl]methyl]glutaric acid; 2-[[(4-pyridyl)thionyl]methyl]glutaric acid; 2-[(benzylthionyl)methyl]glutaric acid; 2-[(sulfonylurea)methyl]glutaric acid; 2-[(methanesulfonyl) 2-[(ethanesulfonyl)methyl]glutaric acid; 2-[(propanesulfonyl)methyl]glutaric acid; 2-[(butyryl)methyl]glutaric acid; 2-[(phenylsulfonyl)methyl]glutaric acid; 2-[[(2-phenylethyl)sulfonyl]methyl]glutaric acid; 2-[[(3-phenylpropyl)sulfonyl]methyl]glutaric acid; 2-[[ (4-pyridyl)sulfonyl]methyl]glutaric acid; 2-[(benzylsulfonyl)methyl]glutaric acid; 2-[(sulfoximinyl))methyl]glutaric acid; 2-[(methylimino)methyl]glutaric acid; 2-[(ethylimino)methyl]glutaric acid; 2-[(propylimino)methyl]glutaric acid; 2-[(butylimino)methyl]glutaric acid; 2-[(phenylimino]methyl]glutaric acid; 2-[[(2-phenylethyl)imino]methyl]glutaric acid; 2-[[(3-phenylpropyl)imino]methyl]glutaric acid; 2-[[(4-pyridyl)imino]methyl]glutaric acid; and 2-[(benzyl]methyl]methyl]glutaric acid [(2-phenylethyl)methyl]glutaric acid; N-[methylhydroxyphosphono]glutamic acid; N-[ethylhydroxyphosphono]glutamic acid; N-[propylhydroxyphosphono]glutamic acid; N-[butylhydroxyphosphono]glutamic acid; N-[phenylhydroxyphosphono]glutamic acid; N-[(phenylmethyl)hydroxyphosphono]glutamic acid; N-[((2-phenylethyl)methyl)hydroxyphosphono]glutamic acid; and N-methyl-N-[phenylhydroxyphosphono]glutamic acid. The prostate-specific membrane antigen ligands of this application also include all prostate-specific membrane antigen small molecule ligands disclosed in PCT applications WO2010 / 108125 and WO2006 / 093991, the entire contents of which are incorporated herein by reference.

[0120] In some embodiments, the prostate-specific membrane antigen small molecule ligand of this application is a glutaric acid derivative. In some embodiments, the prostate-specific membrane antigen small molecule ligand of this application is an aminocarbonyl derivative of glutaric acid.

[0121] In some embodiments, the prostate-specific membrane antigen small molecule ligand of this application comprises the following structure:

[0122] In some embodiments, the prostate-specific membrane antigen ligand contained in the complex compound or a pharmaceutically acceptable salt thereof comprises the following structure: .

[0123] In some embodiments, the prostate-specific membrane antigen ligand contained in the complex compound or a pharmaceutically acceptable salt thereof comprises the following structure:

[0124] In some embodiments, the prostate-specific membrane antigen ligand contained in the complex compound or a pharmaceutically acceptable salt thereof comprises the following structure: .

[0125] In some embodiments, the prostate-specific membrane antigen ligand contained in the complex compound or a pharmaceutically acceptable salt thereof comprises the following structure:

[0126] In some embodiments, a target molecule in the complex compound of this application or a pharmaceutically acceptable salt thereof has a ligand moiety shown in formula (I): (I),

[0127] Alternatively, it may be a ligand portion that is at least 70%, at least 80%, at least 85%, or at least 90% homologous to its amino acid sequence, or it may be a ligand portion that has at most 3, 2, or 1 amino acid substitutions (e.g., conservative substitutions).

[0128] In some embodiments, a target molecule in the complex compound of this application or its pharmaceutically acceptable salt is P10 or a ligand moiety having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, or at least 93% amino acid sequence homology with it or having at most 3, 2, or 1 amino acid substitutions (e.g., conservative substitutions).

[0129] As used in this application, the term "P10" refers to a peptide having the amino acid sequence Cys-Lys-Glu-Phe-Leu-His-Pro-Ser-Lys-Val-Asp-Leu-Pro-Arg.

[0130] In some embodiments, the two target molecules of the complex compound or its pharmaceutically acceptable salt provided in this application are a synergistic molecule portion and a prostate-specific membrane antigen ligand portion, respectively. In some embodiments, the synergistic molecule is capable of mediating endocytosis. In some embodiments, the two target molecules of the complex compound or its pharmaceutically acceptable salt provided in this application are folic acid or an analogue thereof and a prostate-specific membrane antigen ligand portion, respectively.

[0131] In some embodiments, the two target molecules of the complex compound or its pharmaceutically acceptable salt provided in this application are a synergistic molecule portion and a ligand portion having the formula (I). In some embodiments, the synergistic molecule is capable of mediating endocytosis. In some embodiments, the two target molecules of the complex compound or its pharmaceutically acceptable salt provided in this application are folic acid or an analogue thereof and a ligand portion having the formula (I).

[0132] In some embodiments, the two target molecules of the complex compound or its pharmaceutically acceptable salt provided in this application are a synergistic molecule and P10, respectively. In some embodiments, the synergistic molecule is capable of mediating endocytosis. In some embodiments, the two target molecules of the complex compound or its pharmaceutically acceptable salt provided in this application are folic acid or an analogue and P10, respectively.

[0133] In some embodiments, the composite compound provided in this application comprises only a single effective load coupled to two target molecules. In some embodiments, the composite compound provided in this application comprises multiple effective loads coupled to two target molecules.

[0134] As used in this application, the term "composite" refers to the connection of two chemical groups by covalent bonds, which can be either a direct covalent bond between the two chemical groups or an indirect connection of the two chemical groups by a linker.

[0135] In some embodiments, the complex compound or a pharmaceutically acceptable salt thereof comprises an effective load (e.g., one) and two target molecules, wherein the effective load is directly covalently linked to at least one target molecule. In some embodiments, the effective load is directly covalently linked to both target molecules.

[0136] In some embodiments, the complex compound or a pharmaceutically acceptable salt thereof comprises an effective load (e.g., one) and two target molecules, wherein the effective load is covalently linked to at least one target molecule via a linker. In some embodiments, the effective load is covalently linked to both target molecules via linkers.

[0137] As used in this application, the term "linker" refers to a molecule or portion that covalently links a payload to a target molecule. A linker includes a functional group for linking the payload to at least one target molecule. In some embodiments, the functional group may contain two reactive moieties, one for linking to the payload and the other for linking to the target molecule. In some embodiments, the functional groups are different from each other. In some embodiments, the functional group comprises a group containing a thiol reactive moiety and an amine reactive moiety. In some embodiments, the functional groups are the same as each other. In some embodiments, the functional group is a maleimine group. In some embodiments, the linker contains an amino acid. In some embodiments, the carboxylic acid in the amino acid contained in the linker is amide-treated. In some embodiments, the linker contains a short-chain polyethylene glycol (e.g., comprising 2-10, 2-8, 3-8, 4-8, 4-7, 4-6, or 5 repeating units).

[0138] In some embodiments, the linker of this application is a multivalent linker capable of binding at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload and at least one target molecule. The payload bound to the multivalent linker may be the same or different, and the target molecule bound to the multivalent linker may be the same or different.

[0139] In one aspect, the linker should be sufficiently stable to avoid accidental release of the payload during blood circulation, thereby increasing the effective amount of payload delivered to the target cells or tissues and avoiding toxicity. In another aspect, the linker should be capable of releasing the payload around or inside the target cells to effectively kill the target cells or block their function. In some embodiments, the linker comprises at least one cleavable functional group. Preferably, the cleavable functional group is sufficiently stable outside the target cells, but cleaves upon entering the target cells to release the payload. In some embodiments, the cleavable functional group exhibits a cleavage efficiency in target cells that is at least 10, 20, 30, 50, 100, or more times higher than its cleavage efficiency in blood or serum.

[0140] The cleavable linker can be cleaved by hydrolysis, enzymatic reactions, reduction reactions, or by pH changes. In some embodiments, the linker is cleavable under specific physiological conditions (e.g., at a suitable pH). In some embodiments, the linker can be cleaved in an acidic environment at pH about 6.5 or lower, or by reagents such as enzymes. In some embodiments, the linker is sensitive to cleavage agents, such as pH, redox potential, or the presence of degrading molecules.

[0141] In some embodiments, the linker is non-lytic. As used in this application, a non-lytic linker refers to a linker that remains substantially intact during intracellular metabolism.

[0142] In some embodiments, the linker is a peptide linker, which consists of straight-chain or branched-chain amino acids linked by peptide bonds. In some embodiments, the peptide linker can be cleaved by proteases that are highly or specifically expressed in or around target cells, such as cathepsin B in lysosomes or endosomes. The peptide linkers used in this application can have various lengths. Typically, the peptide linkers of this application are 1 to 50 amino acids long. In some embodiments, the length of the peptide linker is 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid. In some embodiments, the length of the peptide linker is 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2 amino acids. The number of amino acids in the peptide linker described in this application can be any integer value within the above range, including the endpoints of the range. In some embodiments, the length of the peptide linker is preferably 1, 2, 3, 4, or 5 amino acids. In some embodiments, the peptide linker is cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-lysine, cysteine-glutamic acid-aspartic acid-aspartic acid, and aspartic acid-aspartic acid-lysine. Optionally, the carboxylic acid in the above amino acids is acetylated.

[0143] In some embodiments, the linker is a disulfide linker containing disulfide bonds. The disulfide bonds can cleave under reducing conditions within cells, while remaining stable in the circulatory system. The disulfide linkers of this application can be DSDM, DMDS, MDS, or NDMDS. The structures of these disulfide linkers are shown in Table 1 below. Table 1: Structure of DSDM, DMDS, MDS and NDMDS [name] [structure] DSDM DMDS MDS NDMDS

[0144] In some embodiments, the linker is a pH-dependent linker. The pH-dependent linker described in this application can cleave at a specific pH environment. In some embodiments, the pH-dependent linker is stable under alkaline conditions but cleaves under acidic conditions (e.g., at pH 6.5 or lower). In some embodiments, the pH-dependent linker is maleic aconitine.

[0145] In some embodiments, the linker of the complex compound or its pharmaceutically acceptable salt is ,

[0146] Alternatively, it could be a combination of the above structure and a peptide linker (e.g., a peptide linker containing 1-3 amino acids linked to a target molecule).

[0147] In some embodiments, the linker of the complex compound or its pharmaceutically acceptable salt has the following structure: ,

[0148] Alternatively, it could be a combination of the above structure and a peptide linker (e.g., a peptide linker containing 1-3 amino acids linked to a target molecule).

[0149] In some embodiments, the linker of the complex compound or its pharmaceutically acceptable salt has the following structure: ,

[0150] Alternatively, it could be a combination of the above structure and a peptide linker (e.g., a peptide linker containing 1-3 amino acids linked to a target molecule).

[0151] In some embodiments, the linker of the complex compound or its pharmaceutically acceptable salt has the following structure: ,

[0152] Alternatively, it could be a combination of the above structure and a peptide linker (e.g., a peptide linker containing 1-3 amino acids linked to a target molecule).

[0153] In some embodiments, the linker of the complex compound or its pharmaceutically acceptable salt has the following structure: ,

[0154] Alternatively, it could be a combination of the above structure and a peptide linker (e.g., a peptide linker containing 1-3 amino acids linked to a target molecule).

[0155] In some embodiments, the linker of the complex compound or its pharmaceutically acceptable salt has the following structure: ,

[0156] Alternatively, it could be a combination of the above structure and a peptide linker (e.g., a peptide linker containing 1-3 amino acids linked to a target molecule).

[0157] In some embodiments, the linker of the complex compound or its pharmaceutically acceptable salt has the following structure: ,

[0158] Alternatively, it could be a combination of the above structure and a peptide linker (e.g., a peptide linker containing 1-3 amino acids linked to a target molecule).

[0159] In some embodiments, the linker of the complex compound or its pharmaceutically acceptable salt has the following structure: ,

[0160] Alternatively, it could be a combination of the above structure and a peptide linker (e.g., a peptide linker containing 1-3 amino acids linked to a target molecule).

[0161] In some embodiments, the linker of the complex compound or its pharmaceutically acceptable salt has the following structure: ,

[0162] Alternatively, it could be a combination of the above structure and a peptide linker (e.g., a peptide linker containing 1-3 amino acids linked to a target molecule).

[0163] In some embodiments, the linker of the complex compound or its pharmaceutically acceptable salt has the following structure: ,

[0164] Alternatively, it could be a combination of the above structure and a peptide linker (e.g., a peptide linker containing 1-3 amino acids linked to a target molecule).

[0165] In some embodiments, the linker of the complex compound or its pharmaceutically acceptable salt has the following structure:

[0166] Alternatively, it could be a combination of the above structure and a peptide linker (e.g., a peptide linker containing 1-3 amino acids linked to a target molecule).

[0167] In some embodiments, the linker of the complex compound or its pharmaceutically acceptable salt has the following structure: ,

[0168] Alternatively, it could be a combination of the above structure and a peptide linker (e.g., a peptide linker containing 1-3 amino acids linked to a target molecule).

[0169] In some embodiments, the connectors of this application may include any one or a combination thereof as described above.

[0170] In some embodiments, the payload is directly or indirectly complexed with a first target molecule, and the first target molecule is directly or indirectly complexed with a second target molecule. In some embodiments, the payload is directly complexed with both the first and second target molecules. In some embodiments, the payload is indirectly complexed with both the first and second target molecules. In some embodiments, the payload is indirectly complexed with the first target molecule (e.g., via a linker), and the first target molecule is directly or indirectly complexed with the second target molecule. In some embodiments, the payload is complexed with the first target molecule via a first linker, and the payload is complexed with the second target molecule via a second linker. In some embodiments, the linker is a multivalent linker that binds to at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the payload and two target molecules.

[0171] In some embodiments, the two target molecules are connected to each other by a spacer region. In some embodiments, the spacer region may be cleaved by a protease specifically expressed by the target cell or by a protease expressed by the target cell. Such proteases include, for example, the proteases listed in Table 2 below. In some embodiments, the spacer region contains an amino acid sequence selected from any one of the amino acid sequences listed in Table 2 below. Table 2: List of Enzymatically Cleavable Sequences [Protein] [Amino acid sequence of the recognition site] [SEQ ID NO.] cathepsin B RR - Bean pod protein ASN - Matripase KSRAEDE SEQ ID NO: 1 MMP-2 PLGLAG SEQ ID NO: 2 Prostate-specific antigen SSLY SEQ ID NO: 3 Matrix Dissolving Agent-3 AAA - TMPRSS2 LLRSLIG SEQ ID NO: 4 Urokinase plasminogen activator SSR - Activated protein C LVKR SEQ ID NO: 5 Factor Ixa LVVR SEQ ID NO: 6 Factor VIIa QLTR SEQ ID NO: 7 Factor Xa LEGR SEQ ID NO: 8 thrombin PR - Calpain-a PLFAEP SEQ ID NO: 9 Calpain-2 GLGSEP SEQ ID NO: 10 Intestinal peptidase DDDDK SEQ ID NO: 11 MMP-8 GPSG SEQ ID NO: 12 cathepsin L PLG - Convertase 5 RSKR SEQ ID NO: 13 Calpain-3 VGVF SEQ ID NO: 14

[0172] As used in this application, the terms "cleavable" or "cleavable" refer to metabolic or reaction processes occurring on the complex compounds provided in this application, thereby disrupting the linker between the payload and the target molecule, or the spacer region between the target molecules, to release the free payload or target molecule. The linker or spacer region is cleaved by a protease or under specific physiological conditions (e.g., pH environment).

[0173] In some embodiments, the complex compound has a structure represented by formulas I, II, III, or IV, wherein n, m, p, and q are independently 0 or 1, representing the independent presence or absence of a linker or spacer region. The term "molecule" in the following formulas is short for "targeting molecule".

[0174] In some embodiments, the complex compound or its pharmaceutically acceptable salt provided in this application comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) effective load as provided in this application, two targeting molecules as provided in this application, and optionally a linker or spacer region as provided in this application. In some embodiments, the complex compound or its pharmaceutically acceptable salt provided in this application comprises one effective load as provided in this application, one ligand that specifically binds to a cell surface protein or marker as provided in this application, one synergistic molecule as provided in this application, and a linker or spacer region as provided in this application.

[0175] In some embodiments, the complex compound has a structure of formula V, VI, VII or VIII as shown below, wherein n, m, p, q and s are independently 0 or 1, which independently represent the presence or absence of linkers, multivalent linkers and spacer regions.

[0176] In some embodiments, the complex compound provided in this application or its pharmaceutically acceptable salt comprises an effective load and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a prostate-specific membrane antigen ligand portion, such as CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09, and CB-20R.

[0177] In some embodiments, the complex compound provided in this application or its pharmaceutically acceptable salt comprises one or more active loadings and two target molecules, wherein the two target molecules are a synergistic molecule portion and a ligand portion having the formula (I), such as CB-18G, CB-1820 and CR19426.

[0178] In some embodiments, the complex compound provided in this application or its pharmaceutically acceptable salt comprises an effective load and two target molecules, wherein the two target molecules are a synergistic molecule moiety and P10, and the effective load is camptothecin and any derivative thereof, such as CB-10S, CR19425 and CB-50S.

[0179] In some embodiments, the complex compounds of this application are selected from the group consisting of the following compounds: CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09, CB-20R, CB-18G, CR19426, CB-10S, CR19425, and CB-50S (the specific structures of each complex compound are shown in Figure 1). In some embodiments, the complex compounds of this application are formed by covalently linking a linker-drug moiety and a ligand moiety. The linker-drug portion of this application includes an effective load and a linker, and the ligand portion of this application includes two target molecules and an optional spacer region or linker. The two portions react to form a covalent bond to form the complex compound of this application. The covalent bond can be formed between the linker in the linker-drug portion and the ligand molecule in the ligand portion, or it can be formed between the linker in the linker-drug portion and the spacer region or linker of the ligand portion.

[0180] The complex compound CB-20B of this application is formed by covalently linking the linker-drug moiety LT1002 and the ligand moiety 20B-SM09. The complex compound CB-20BK of this application is formed by covalently linking the linker-drug moiety LT1002 and the ligand moiety 20BK-SM09. The complex compound CB-60S of this application is formed by covalently linking the linker-drug moiety LT2000C and the ligand moiety 60S-SM09. The complex compound CB-60SK of this application is formed by covalently linking the linker-drug moiety LT2000C and the ligand moiety 60SK-SM09. The complex compound CB-20C of this application is formed by covalently linking the linker-drug moiety LD1001 and the ligand moiety 20BK-SM09. The complex compound CB-1020 of this application is formed by covalently linking the linker-drug moiety LT1002 and the ligand moiety 1020BK-SM09. The complex compound CB-1320 of this application is formed by covalently linking the linker-drug moiety LT1002 and the ligand moiety 1320BK-SM09. The complex compound CB-1820 of this application is formed by covalently linking the linker-drug moiety LT1002 and the ligand moiety 1820BK-SM09. The complex compound CR19428 of this application is formed by covalently linking the linker-drug moiety CR19423 and the ligand moiety 20BK-SM09. The complex compound CB-20R of this application is formed by complexing 20R-SM09 with the radioactive isotope ion M. The complex compound CB-18G of this application is formed by covalently linking the linker-drug moiety LT1002 and the ligand moiety 18G-SM09. The complex compound CR19426 of this application is formed by covalently linking the linker-drug moiety CR19423 and the ligand moiety 18G-SM09. The complex compound CB-10S of this application is formed by covalently linking the linker-drug moiety LT1000 and the ligand moiety CBSM09. The complex compound CR19425 of this application is formed by covalently linking the linker-drug moiety CR19423 and the ligand moiety CBSM09. The complex compound CB-50S of this application is formed by covalently linking the linker-drug moiety LT1000N3 and the ligand moiety 50S-SM09. The structures are shown in Table 3 below. Table 3: Structure of linker-drug and ligand moieties [Abbreviation] [structure] LD1001 LT1000 LT1000N3 LT1002 LT2000C CBSM09 18G-SM09 20B-SM09 20BK-SM09 20R-SM09 50S-SM09 60S-SM09 60SK-SM09 1020BK-SM09 1320BK-SM09 1820BK-SM09 CR19423

[0181] In some embodiments, the complex compound provided in this application, or its pharmaceutically acceptable salt, enters the bloodstream and extracellular space (intercellular matrix). Because the linker is very stable in the extracellular environment and cannot release drug molecules, the toxicity of the drug molecules is blocked. This complex is a non-cytotoxic or low-toxic drug that does not have a toxic effect on normal cells.

[0182] In some embodiments, the complex compound provided in this application, or its pharmaceutically acceptable salt, binds to multiple receptors or antigens that are simultaneously highly expressed on diseased cells. This synergistic effect significantly increases the affinity of the complex compound for target cells and reduces the possibility of binding to normal cells. This allows for the delivery of highly effective toxic drugs such as MMAE / Dxd / SN38 / radioisotope complexes, enhancing efficacy, broadening the therapeutic window, and avoiding drug side effects.

[0183] In some embodiments, after the complex compound provided in this application or its pharmaceutically acceptable salt enters the target cell, the linker can be cleaved and released to release drug molecules (equivalent to removing the modifying groups of the drug molecule) through changes in the intracellular environment (specific enzymatic cleavage, pH change, disulfide bond reduction, etc.), thereby producing a therapeutic effect on tumor cells.

[0184] In some embodiments, the complex compound of this application or a pharmaceutically acceptable salt thereof can be used to specifically deliver a payload to target cells in a target tissue environment. Generally, the two targeting molecules of the complex compound or a pharmaceutically acceptable salt thereof offer three advantages. First, the two targeting molecules can act in multiple ways (often synergistically), thereby improving therapeutic efficacy while reducing side effects. Second, the combination of the two targeting molecules increases the affinity or affinity of the complex compound or a pharmaceutically acceptable salt thereof for the target receptor or target cells, thereby enhancing its specificity and avoiding off-target toxicity. Finally, when properly designed, the combination of two targeting molecules can meet the multifunctional requirements typically required for drug complexes.

[0185] The complex compound or its pharmaceutically acceptable salt of this application achieves unexpected technical effects, including but not limited to: (1) the combination of a ligand capable of binding to cell surface receptors and a synergistic molecule capable of mediating endocytosis enables the complex compound to specifically enter target cells; (2) the complex compound or its pharmaceutically acceptable salt enhances the affinity and targeting specificity of the drug compound, thereby delivering highly effective chemotherapeutic agents (such as MMAE) to patients, broadening the therapeutic window of such agents and avoiding side effects; (3) the linker prevents the release of the payload outside the target cells (e.g., the circulatory system, intercellular matrix, etc.), ensuring the stability of the complex compound in the bloodstream and reducing drug toxicity. After entering the target cells, the linker is cleaved, releasing the payload, thereby exerting the drug's effect, while avoiding multidrug resistance (MDR); (4) a wide variety of drugs can be delivered in the form of the complex compound of this application, thus expanding the application range of related drugs. Therefore, the complex compound or its pharmaceutically acceptable salt of this application not only broadens the target range and therapeutic window of LDC drugs, but also reduces the toxicity and side effects of some drugs.

[0186] As used in this application, the terms "peptide," "protein," and "peptide" can refer to a single amino acid or a polymer of amino acids. Peptides, proteins, or peptides as described in this application may contain naturally occurring amino acids, as well as non-naturally occurring amino acids, or analogues and analytes of amino acids. Peptides, proteins, or peptides can be obtained by any method well known in the art, such as, but not limited to, isolation and purification from natural substances, recombinant expression, chemical synthesis, etc.

[0187] Another aspect of this application discloses pharmaceutical compositions containing the complex compound provided in this application or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0188] As used in this application, the term "pharmaceutically acceptable" means, to the extent of reasonable medical judgment, that it is suitable for contact with human and other animal cells without undue toxicity, irritation, allergic reactions, etc., and is commensurate with a reasonable benefit / risk ratio.

[0189] As used in this application, the term "pharmaceutically acceptable salt" refers to the relatively non-toxic inorganic and organic acid addition salts and base addition salts of the complex compounds of this application. Representative acid addition salts include hydrobromide, hydrochloride, sulfate, hydrogen sulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthate, methanesulfonate, glucono-p-heptanoate, lactobionate, aminosulfonate, malonate, salicylate, propionate, methylene-bis-β-hydroxynaphthylcarboxylate, gentianate, hydroxyethylsulfonate, di-p-toluenemethyltartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylaminosulfonate, and quinic acid laurylsulfonate, etc. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. In some embodiments, sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases, including, for example, sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, and zinc hydroxide. Suitable amine-base addition salts are prepared from amines of sufficient basicity to form stable salts, and preferably include the following amines commonly used in medicinal chemistry because they have low toxicity and are acceptable for medical use: ammonia, ethylenediamine, N-methylglucosamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, guanidine, tris(hydroxymethyl)aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, diphenylhydroxymethylamine, dehydroabiidine, N-ethylguanidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids (e.g., lysine and arginine), and dicyclohexylamine, etc.

[0190] As used in this application, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable solvent, suspension, or any other pharmaceutically inert carrier used to deliver the complex compound provided in this application to a subject, without interfering with the structure and properties of the complex compound. Certain such carriers can formulate the complex compound into, for example, tablets, pills, capsules, liquids, gels, syrups, pastes, suspensions, and soft lozenges for oral ingestion by a subject. Certain such carriers can formulate the complex compound into formulations for injection, infusion, or topical application.

[0191] Pharmaceutically acceptable carriers used in the pharmaceutical compositions provided in this application include, but are not limited to, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous carriers (e.g., sodium chloride injection, Ringer's injection, isotonic dextran injection, sterile water injection, or dextran and lactated Ringer's injection), non-aqueous carriers (e.g., plant-derived fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents, isotonic agents (e.g., sodium chloride or dextran), buffers (e.g., phosphoric acid or citrate buffers), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspensions / dispersants (e.g., sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween-80)), diluents, adjuvants, excipients, or non-toxic excipients, other ingredients known in the art, or various combinations thereof. Suitable ingredients may include, for example, fillers, binders, buffers, preservatives, lubricants, flavorings, thickeners, colorants, or emulsifiers.

[0192] In some embodiments, the pharmaceutical composition is an injectable formulation. Injectable formulations include sterile aqueous solutions or dispersants, suspensions, or emulsions. In all cases, injectable formulations should be sterile and fluid for easy injection. Injectable formulations should remain stable under production and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof, and / or vegetable oils. Injectable formulations should maintain appropriate fluidity. For example, appropriate fluidity can be maintained by using a coating such as lecithin, by using surfactants, etc. Microbial inhibition can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.

[0193] In some embodiments, the pharmaceutical composition is an oral formulation. Oral formulations include, but are not limited to, capsules, flat capsules, pills, tablets, lozenges (using a flavoring base, typically sucrose and gum arabic or tragacanth), powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as soft lozenges (using an inert base, such as gelatin and glycerin, or sucrose and gum arabic), and / or as mouthwashes, etc.

[0194] In solid dosage forms intended for oral administration (e.g., capsules, tablets, pills, sugar-coated pills, powders, granules, etc.), the complex compound is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium hydrogen phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silica; (2) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants, For example, glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or cassava starch, alginic acid, certain silicates and sodium carbonate; (5) solution retardants, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, such as acetyl alcohol and glyceryl monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; and (10) colorants.

[0195] In liquid dosage forms intended for oral administration, the complex compound is mixed with any of the following: pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the complex compound, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, isopropanol, 1,3-butanediol, oils (especially cottonseed oil, peanut oil, corn oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters and mixtures thereof. In addition to inert diluents, the oral composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, aroma enhancers, and preservatives.

[0196] In some embodiments, the pharmaceutical composition is an oral spray or nasal spray formulation. Spray formulations include, but are not limited to, aqueous aerosols, non-aqueous suspensions, liposome formulations, or solid particulate formulations. Aqueous aerosols are prepared by mixing an aqueous solution or suspension of the pharmaceutical agent with a routinely pharmaceutically acceptable carrier and stabilizer. The carrier and stabilizer vary depending on the specific compound, but typically include nonionic surfactants (Tween or polyethylene glycol), oleic acid, lecithin, amino acids such as glycine, buffer solutions, salts, sugars, or sugar alcohols. Aerosols are typically prepared from isotonic solutions and are capable of being delivered by spraying.

[0197] In some embodiments, the pharmaceutical composition may be used in combination with one or more other drugs. In some embodiments, the pharmaceutical composition comprises at least one other drug. In some embodiments, the other drugs are antitumor drugs, cardiovascular drugs, anti-inflammatory drugs, antiviral drugs, digestive system drugs, nervous system drugs, respiratory system drugs, immune system drugs, dermatological drugs, metabolic drugs, etc.

[0198] In some embodiments, the pharmaceutical composition may be administered to a subject in need via suitable routes, including but not limited to oral, injectable (e.g., intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural, intraperitoneal, etc.), mucosal (e.g., intranasal, intraoral, etc.), sublingual, rectal, transdermal, intraocular, and pulmonary administration. In some embodiments, the pharmaceutical composition may be administered intravenously, subcutaneously, orally, intramuscularly, or intracardiacly.

[0199] Due to the properties of some payloads, such as high toxicity or high hydrophilicity, it is desirable to deliver the payload more specifically and efficiently to the recipient. For example, in cancer treatment, it is desirable to deliver chemotherapeutic agents specifically to cancer cells without causing toxicity to normal cells. Therefore, another aspect of this application discloses a method for delivering a payload to a recipient, the method comprising administering to the recipient a therapeutically effective amount of the complex compound provided in this application, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided in this application. The payload described in this application can be any agent that researchers, veterinarians, doctors, or other physicians are seeking to elicit a biological or medical response in tissues, systems, individual animals, or humans to prevent, inhibit, improve, or treat disease.

[0200] As used in this application, the term "object" refers to humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. Objects can also be livestock, such as cattle, pigs, sheep, poultry, and horses, or domesticated animals, such as dogs and cats. Objects can be male (e.g., male) or female (e.g., female), and can be elderly, adult, adolescent, child, or infant. Humans can be Caucasian, African, Asian, Semitic, or of other ethnic backgrounds, or a mixture of these ethnic backgrounds.

[0201] As used in this application, the term "therapeuticly effective amount" refers to an amount by which a complex compound or its pharmaceutically acceptable salt or pharmaceutical composition alleviates, to a certain extent, one or more symptoms of a disease or condition in a subject; an amount that partially or completely restores one or more physiological or biochemical parameters associated with or causing a disease or condition to normal; and / or reduces the likelihood of developing a disease or condition. This amount typically varies depending on a variety of factors that can be determined and described by someone of ordinary skill in the art based on the scope of the description provided in this application. These include, but are not limited to: the specific subject and their age, weight, height, general physical condition and medical history; the specific compound used, and the carrier and chosen route of administration of its formulation; and the nature and severity of the condition being treated.

[0202] In some embodiments, the amount of the complex compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is sufficient to inhibit the disease or condition in the subject, or to preventively inhibit or prevent the onset of the disease or condition. Although the therapeutically effective amount may vary in different subjects, it typically ranges from 0.01 to 100 mg / kg, for example 0.01 to 90 mg / kg, 0.01 to 80 mg / kg, 0.01 to 70 mg / kg, 0.01 to 60 mg / kg, 0.01 to 50 mg / kg, 0.01 to 40 mg / kg, 0.01 to 30 mg / kg, 0.01 to 20 mg / kg, 0.01 to 10 mg / kg, 0.01 to 5 mg / kg, 0.01 to 4 mg / kg, 0.01 to 3 mg / kg, 0.01 to 2 mg / kg, 0.01 to 1 mg / kg, and 0.01 to 0.1 mg / kg. The therapeutic effective amount described in this application can be equal to any value within the above-mentioned numerical range, including the endpoints of that range.

[0203] Another aspect of this application discloses a method for delivering a payload to a subject in need, the method comprising administering to the subject a therapeutically effective amount of the complex compound provided in this application or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided in this application.

[0204] Another aspect of this application discloses a method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the complex compound provided in this application or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided in this application.

[0205] In some implementations, the disease is cancer, including but not limited to prostate cancer, breast cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, thyroid cancer, pancreatic cancer, colon cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma, and multiple myeloma.

[0206] In some embodiments, cancer cells of the cancer exhibit expression of the cell surface receptors or antigens mentioned in this application. In some embodiments, cancer cells of the cancer exhibit high expression of the cell surface receptors or antigens mentioned in this application (e.g., according to Depmap data (see https: / / depmap.org / portal / ), the corresponding gene expression is at least 0, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10). In some embodiments, cancer cells of the cancer exhibit high expression of FOLR1 and FOLH1, TRPV6 and FOLH1, GNRHR and FOLH1, SSTR2 and FOLH1, FOLR1 and SSTR2, or TRPV6 and FOLR1. In some embodiments, the disease is an immune disease, such as an autoimmune disease, including but not limited to connective tissue diseases, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

[0207] In some implementations, the disease is a cardiovascular disease, including but not limited to angina, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia, and congenital heart disease.

[0208] In some implementations, the disease is a metabolic disease, including but not limited to diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.

[0209] In some implementations, the disease is a neurological disease, including but not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma, and epilepsy.

[0210] In some embodiments, the method provided in this application further includes administering one or more therapeutic agents in combination with a complex compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition. In some embodiments, the therapeutic agent targets an anticancer therapeutic target, induces or enhances an immune response against cancer, or is a chemotherapeutic agent.

[0211] The present application will be described in more detail below through specific embodiments. The following embodiments are provided for illustrative purposes only and are not intended to limit the invention in any way. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same results. Example

[0212] The following embodiments are intended to further illustrate this application. Through this description, the advantages and features of this application will become clear. However, these descriptions are merely exemplary and should not be construed as limiting the scope of this application.

[0213] [Example] [1] Preparation of complex compounds

[0214] Synthesis of complex compounds CB-20BK, CB-18G, CB-20B, CB-10S, CB-20C, FA-MMAE, CB-20AK, CB-1020, CB-1320 and CB-1820

[0215] 1. Weigh 10g of Rink amide-am resin (hereinafter referred to as "Rink Resin", Xi'an Lanxiao Technology New Material Co., Ltd., item number 183599-10-2) with a degree of substitution of 0.45 mmol / g, load it into a solid-phase reaction column, add DCM, and bubble nitrogen into the solvent to allow the resin to swell for 30 minutes; remove the solvent, remove the Fmoc protecting group on the resin with DBLK, and then wash it 5 times with DMF. Weigh 4.79g (9mmol) of Fmoc-Lys(Dde)-OH and 1.47g (10.8mmol) of HOBt, dissolve them in DMF, add 1.67ml (10.8mmol) of DIC to the above solution under an ice-water bath at 0℃, mix and activate for 5 minutes, add the solution to the above reaction column, react for 3 hours, remove the solvent, and wash the resin in the reaction column 3 times. Then remove the Fmoc protecting group with DBLK.

[0216] 2. Repeat the above operation, sequentially compounding Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH and intermediate 108 according to their structures. (Intermediate 108)

[0217] 3. Remove the Dde protecting group twice with 2% hydrazine hydrate / DMF, 10 minutes each time, then wash the resin five times with DMF. Then, sequentially compound Fmoc-Glu-OtBu and pteroic acid. Finally, shrink the resin twice with methanol, remove the solvent, and obtain 17.4 g of protected peptide resin.

[0218] 4. Add 17.4 g of the peptide resin obtained in the previous step to a 250 ml single-necked flask. Prepare 139 ml of lysis buffer with a TFA:H₂O:TIS ratio of 95:3:2 (volume ratio) and weigh 2.1 g of DTT into the lysis buffer. Add the lysis buffer to the flask and react at room temperature for 2.5 hours. Filter the solution, wash the resin with 30 ml of TFA, combine the filtrates, and add them to 834 ml of anhydrous diethyl ether. A yellow solid precipitates out. Centrifuge to obtain the solid, wash the solid with anhydrous diethyl ether, and dry under vacuum to obtain 6.4 g of yellow solid. The crude product yield is 93.4%. The HPLC purity is 82.3%. The product was prepared and separated by HPLC (preparation conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile, elution gradient (15-25)%B, time 60 minutes, and fraction collection). The fraction containing qualified product was freeze-dried to obtain 4.73g of 20BK-SM09 with a purity of 98.8%.

[0219] 5. Weigh 4.09 g (3.11 mmol) of Mc-Val-Cit-PAB-MMAE (LT1002) into a 1000 mL single-necked flask, add 500 mL of phosphate buffer and 100 mL of acetonitrile, stir, maintain pH=7.2 until clear, add 4.73 g (3.11 mmol) of intermediate 20BK-SM09, react at room temperature for 2 hours, and monitor the reaction by HPLC. After the reaction is complete, filter, and separate the filtrate by HPLC (preparation conditions: C18 column, mobile phase A: ammonium bicarbonate solution (pH=7.2), B: acetonitrile, elution gradient (25-35)% B, time 60 min, collect the fraction), and freeze-dry the fraction containing qualified product to obtain 6.96 g of CB-20BK product, with a purity of 98.8% and a yield of 78.8%.

[0220] Similarly, the complex compounds CB-18G, CB-20B, CB-10S, CB-20C, FA-MMAE (structure shown below), CB-20AK (structure shown below), CB-1020, CB-1320 and CB-1820 can be obtained through steps similar to those described above. (FA-MMAE) (CB-20AK)

[0221] Synthesis of complex compounds CB-50S, CB-60S and CB-60SK

[0222] 1. Weigh 10g of Wang Resin (hereinafter referred to as "Wang Resin", Xi'an Lanxiao New Material Technology Co., Ltd., item number 1365700-43-1) with a degree of substitution of 1.1 mmol / g, load it into a solid-phase reaction column, add DMF, bubble nitrogen to the solvent, and allow it to swell for 30 minutes; weigh 14.3g (22 mmol) of Fmoc-Arg(pbf)-OH, 3.56g (26.4 mmol) of HOBt, and 0.27g (2.2 mmol) of DMAP, dissolve them in DMF, add 4.1 ml of DIC (26.4 mmol) under an ice-water bath at 0℃, mix and activate for 5 minutes, add the solution to the reaction column, react for 3 hours, remove the solvent and wash 3 times.

[0223] 2. Dissolve 10.4 ml of acetic anhydride and 8.9 ml of pyridine in 50 ml of DMF, mix and add to the washed resin, block at room temperature for 5 hours, wash three times with DMF, shrink with methanol and dry the resin to obtain Fmoc-Arg(pbf)-Wang Resin, with a degree of substitution of 0.53 mmol / g.

[0224] 3. Weigh 3.8 g (2 mmol) of Fmoc-Arg(pbf)-Wang Resin (Sub=0.53 mmol / g) into the reaction column, wash three times with DMF, and then add DMF to swell the resin for 30 minutes. Then remove the Fmoc protecting group with DBLK, and wash six times with DMF. Weigh 2.0 g (6 mmol) of Fmoc-Pro-OH and 0.97 g (7.2 mmol) of HOBt, dissolve them in DMF, add 1.1 ml of DIC (7.2 mmol) under an ice-water bath at 0°C, mix and activate for 5 minutes, add to the reaction column, react for 2 hours, and then remove the Fmoc protecting group with DBLK.

[0225] 4. Repeat the above steps, sequentially compounding Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-propargyl-Gly-OH, Fmoc-Glu-OtBu, and pteroic acid according to their structures. The mixture is condensed twice with methanol, and the solvent is removed by vacuum extraction to obtain 8.4 g of peptide resin.

[0226] 5. Add 8.4g of the peptide resin obtained in the previous step to a 250ml single-necked flask. Prepare 67ml of lysis buffer with a TFA:H2O:TIS ratio of 95:3:2 (volume ratio) and weigh 0.92g of DTT into the lysis buffer. Add the lysis buffer to the flask and react at room temperature for 2.5 hours. Filter off the resin, wash the resin with 20ml of TFA, combine the filtrates, and add them to 402ml of anhydrous diethyl ether to precipitate a yellow solid. Centrifuge to obtain the solid, wash the solid with anhydrous diethyl ether, and vacuum dry to obtain 4.06g of yellow solid. The crude product yield is 97.3%. The HPLC purity is 84.6%. HPLC preparative separation (preparation conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile, elution gradient (20-29)%B, time 60 minutes, collect the fraction), and freeze-dry the fraction containing the qualified product to obtain 2.86g of 50S-SM09 with a purity of 97.6%.

[0227] 6. Weigh 1.29 g (1.37 mmol) of LT1000N3 into a 500 mL single-necked flask, add 270 mL of mixed solvent (ACN:H2O=1:4), and 393 mg (2.74 mmol) of CuBr, and stir. Add 2.86 g (1.37 mmol) of intermediate 50S-SM09, and react at room temperature for 2-3 hours, monitoring the reaction with HPLC during the reaction. After the reaction is complete, filter and separate by HPLC (preparation conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile, elution gradient (22-40)% B, time 60 minutes, collect the fraction). Freeze-dry the fraction containing qualified product to obtain 3.17 g of CB-50S with a purity of 98.6% and a yield of 76.4%.

[0228] Similarly, the complex compounds CB-60S and CB-60SK can be obtained through steps similar to those described above.

[0229] Synthesis of CB-20R

[0230] 1. Weigh 5 g of Rink Resin with a degree of substitution of 0.45 mmol / g and load it into a solid-phase reaction column. Add DCM and bubble with nitrogen until the solvent is absorbed, allowing the resin to swell for 30 minutes. Remove the solvent, remove the Fmoc protecting group from the resin using DBLK, and then wash five times with DMF. Weigh 2.4 g (4.5 mmol) of Fmoc-Lys(Dde)-OH and 0.74 g (5.4 mmol) of HOBt, dissolve them in DMF, add 0.84 ml (5.4 mmol) of DIC under an ice-water bath at 0°C, mix and activate for 5 minutes, add to the reaction column, react for 3 hours, then dry and wash three times. Then remove the Fmoc protecting group using DBLK.

[0231] 2. Repeat the above operation, sequentially compounding Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH and intermediate 108 according to their structures.

[0232] 3. Remove the Dde protecting group twice with 2% hydrazine hydrate / DMF, 10 minutes each time, then wash 5 times with DMF. Compound DOTA-tris(tBu) ester. Remove the Dde protecting group twice more with 2% hydrazine hydrate / DMF, 10 minutes each time, then wash 5 times with DMF. Compound Fmoc-Glu-OtBu and pteroic acid sequentially, then shrink twice with methanol, dry, and obtain 9.2g of protected peptide resin.

[0233] 4. Add 9.2 g of the peptide resin obtained in the previous step to a 250 ml single-necked flask. Prepare 74 ml of lysis buffer with a TFA:H₂O:TIS ratio of 95:3:2 (volume ratio) and weigh 1.05 g of DTT into the lysis buffer. Add the lysis buffer to the flask and react at room temperature for 2.5 hours. Filter off the resin, wash the resin with 20 ml of TFA, combine the filtrates, and add them to 560 ml of anhydrous diethyl ether to precipitate a yellow solid. Centrifuge, wash the solid with anhydrous diethyl ether, and vacuum dry to obtain 3.8 g of yellow solid, with a crude product yield of 87.4% and an HPLC purity of 81.2%. Separate the product by HPLC (preparation conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile, elution gradient (15-25)%B, time 60 minutes, collect the fraction). Freeze-dry the fraction containing the synthesized product to obtain 2.9 g of 20R-SM09 with a purity of 97.8%.

[0234] 5. CB-20R was obtained by complexing 20R-SM09 with the radioactive isotope ion M. Specifically, the radiolabeled 177 Lu (approximately 50 MBq) was mixed with 100 μl of 0.5 M sodium acetate buffer (pH = 5). 40 μl of a 1 mM CB-20R solution dissolved in 10% DMSO aqueous solution, 2 μl of saturated ascorbic acid solution, and 100 μl of a solution containing 177 Lu were mixed and heated to 95°C and maintained for 10 minutes. The labeling was verified by radio-HPLC (within 5 minutes, 0-100% ACN aqueous solution, C18 column).

[0235] Synthesis of compound CR19425

[0236] 1. Under N2 protection, 441.4 mg of CR19420 (structure as shown in the reaction steps above) and 8.0 mL of DMF were added to a reaction flask, stirred to dissolve, cooled in an ice bath, and then 459.2 mg of HATU and 380 μL of DIPEA were added. The mixture was stirred for half an hour. Then, 500.0 mg of CR19419 (structure as shown in the reaction steps above) and 190 μL of DIPEA were added, and the reaction was carried out at room temperature until completion. After the reaction was complete, the reaction solution was poured into acetic acid water, and a solid precipitated. The solid was filtered, and the filter cake was washed with acetic acid water, washed with water, and dried under vacuum to obtain 835.7 mg of CR19421 (structure as shown in the reaction steps above), a brown powder. The HPLC purity was 90.0%, and the yield was 90.6%.

[0237] 2. Under N2 protection, 835.7 mg of CR19421 and 16 mL of 10% guanidine DMF solution were added to the reaction flask, and the reaction was carried out at room temperature for half an hour. After the reaction was completed, the reaction solution was poured into TFA / MTBE, a solid precipitated, filtered, the filter cake was washed with MTBE, and dried under vacuum to obtain 599.7 mg of CR19422 (structure as shown in the reaction steps above), a grayish-brown powder, HPLC purity: 84.7%, yield: 83.0%.

[0238] 3. Under N2 protection, 599.7 mg CR19422, 586.7 mg CR19424 (structures as shown in the reaction steps above), and 15 mL DMF were added to the reaction flask. The mixture was stirred to dissolve, cooled in an ice bath, and then 495.7 mg HATU and 410 μL DIPEA were added. The reaction was carried out at room temperature. After the reaction was completed, the reaction solution was purified. Acetonitrile was removed from the purified solution under reduced pressure, and the solution was extracted with a mixed solvent of dichloromethane and methanol. The extract was concentrated and dried to obtain 674.9 mg CR19423 (structure as shown in the reaction steps above), a yellow powder. HPLC purity: 88.4%, yield: 63.1%.

[0239] 4. Under N2 protection, add 14.8 mg CR19423, 3.0 mL of PBS buffer (pH 6.6), and 3.0 mL of acetonitrile to the reaction flask. Stir to dissolve, add 32.9 mg CBSM09, and adjust the pH to 6.6-6.8 with Na2HPO4. React for half an hour. After the reaction is complete, purify the reaction solution. Freeze-dry the pure product to obtain 21.8 mg CR19425, a yellow powder. HPLC purity: 95.6%, yield: 48.8%.

[0240] Synthesis of compound CR19426

[0241] 1. Under N2 protection, add 25.2 mg CR19423, 3.0 mL of PBS buffer (pH 6.6), and 3.0 mL of acetonitrile to a reaction flask. Stir to dissolve, add 55.5 mg 18G-SM09, and adjust the pH to 6.6-6.8 with Na2HPO4. React for half an hour. After the reaction is complete, purify the reaction solution. Freeze-dry the pure product to obtain 44.6 mg CR19426 as a yellow powder. HPLC purity: 95.4%, yield: 55.2%.

[0242] Synthesis of compound CR19428

[0243] 1. Under N2 protection, add 486 mg CR19423, 3.0 mL of PBS buffer (pH 6.6), and 3.0 mL of acetonitrile to a reaction flask. Stir to dissolve, add 712 mg 20BK-SM09, and adjust the pH to 6.6-6.8 with Na2HPO4. React for half an hour. After the reaction is complete, purify the reaction solution. Freeze-dry the pure product to obtain 508 mg CR19428 as a yellow powder. HPLC purity: 96.7%, yield: 42.3%. [Example] [2] [Affinity determination of complex compounds to target proteins]

[0244] Affinity assay of CB-20BK binding to protein FOLR1

[0245] Experimental instruments, materials and reagents BIAcore T200 (GE) CM5 chip (GE, part number: 29104988) Buffer solution: HBS-EP+ buffer 10X (GE, catalog number: BR100669), dilute 10 times with deionized water before use. Amino Complex Reagent Kit (GE, Catalog No.: BR100050) Regeneration reagent: 10mM Glycine 2.0 (GE, catalog number: BR100355) 10mM Glycine 3.0 (GE, Part No.: BR100357)

[0246] Experimental steps

[0247] Experiments were conducted according to the BIAcore T200 (GE) user manual to determine the affinity of analytes Biotin-CB-20BK, folic acid (FA), and FA-MMAE for FOLR1. The CM5 crystal complex ligand FOLR1 (R&D System, catalog number 5646-FR) was used. The experimental results are shown in Table 4. Table 4. Affinity of CB-20BK and related compounds to FOLR1 [ka] [kd] [KD] [FA] (Folic acid) 3.335×10⁶ M⁻¹ s⁻¹ 2.258×10⁻⁴ s⁻¹ 6.770×10⁻¹¹ M [FA-MMAE] 1.789×10⁶ M⁻¹ s⁻¹ 1.150×10⁻⁴ s⁻¹ 6.429×10⁻¹¹ M [CB-20BK] 1.033×10⁶ M⁻¹ s⁻¹ 1.312×10⁻⁴ s⁻¹ 1.269×10⁻¹⁰ M

[0248] Table 4 shows that CB-20BK binds to FOLR1 and exhibits good affinity.

[0249] Affinity assay of CB-20BK binding to protein FOLH1

[0250] It binds to FOLH1 and shows good affinity. [Example] [3] [Studies on the binding and endocytosis of ligand complexes to target cells]

[0251] Cell binding and endocytosis assays of the complex compound CB-20BK

[0252] Synthesis of labeled samples Cy5-pep-20BK, Cy5-FA(CONH2) and Cy5-pep-20AK

[0253] 1. Weigh 2 g of Rink Resin with a degree of substitution of 0.45 mmol / g and load it into a solid-phase reaction column. Add DCM and bubble nitrogen to the solvent, allowing the resin to swell for 30 minutes. Remove the solvent, remove the Fmoc protecting group with DBLK, and wash 5 times with DMF. Weigh 0.96 g (1.8 mmol) of Fmoc-Lys(Dde)-OH and 0.3 g (2.2 mmol) of HOBt, dissolve them in DMF, add 0.33 ml (2.2 mmol) of DIC under an ice-water bath at 0°C, mix and activate for 5 minutes, add to the reaction column, react for 3 hours, dry under vacuum, and wash 3 times. Then remove the Fmoc protecting group with DBLK.

[0254] 2. Repeat the above steps, sequentially compounding Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and intermediate 108 according to their structures:

[0255] 3. Remove the Dde protecting group twice with 2% hydrazine hydrate / DMF, 10 minutes each time, followed by washing 5 times with DMF. Then, sequentially compound Fmoc-Lys(Dde)-OH, Fmoc-Glu-OtBu, and pteroic acid.

[0256] 4. Then, the Dde protecting group was removed twice with 2% hydrazine hydrate / DMF for 10 minutes each time, followed by washing with DMF 5 times. After compounding with Cy5-COOH, it was washed twice with DMF, and finally condensed twice with methanol and dried to obtain 3.6g of protected peptide resin.

[0257] 5. Add 3.6g of the peptide resin obtained in the previous step to a 50ml single-necked flask. Prepare 29ml of lysis buffer with a TFA:H2O:TIS ratio of 95:3:2 (volume ratio) and weigh 0.4g of DTT into the lysis buffer. Add the lysis buffer to the flask and react at room temperature for 2.5 hours. Filter off the resin, wash the resin with 8ml of TFA, combine the filtrates, and add them to 173ml of anhydrous diethyl ether to precipitate a yellow solid. Centrifuge, wash the solid with anhydrous diethyl ether, and vacuum dry to obtain 1.9g of blue solid. The crude product yield is 89.6%. The HPLC purity is 76.3%. HPLC preparative separation (preparation conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile, elution gradient (20-28)%B, time 50 minutes, collect the fraction), and freeze-dry the fraction containing the qualified product to obtain 736mg of Cy5-pep-20BK with a purity of 93.4%.

[0258] Similarly, compounds Cy5-FA(CONH2) and Cy5-pep-20AK can be obtained through steps similar to those described above. (Cy5-pep-20BK) (Cy5-pep-20AK) (Cy5-FA(CONH2))

[0259] Cells: NCI-H460 human lung cancer cells, DU145 human prostate cancer cells, LNcap human prostate cancer cells, KB human oral epidermoid cancer cells

[0260] Experimental design / procedure:

[0261] The fluorescently labeled compound was incubated with cells at 37°C for 15 and 30 minutes. After three washes, the fluorescence intensity of the cells was detected by flow cytometry.

[0262] Results and Analysis Table 5. Expression levels of FOLR1 and FOLH1 in different cell lines (Data referenced from Depmap, source: https: / / depmap.org / portal / ) cell lines LNCap DU145 NCI-H460 KB Hela SKOV-3 NCI-H460 A549 FOLR1 5+ - - + 8+ 6+ + / - + / - FOLH1 10+ - - - N / A N / A N / A N / A

[0263] According to Depmap data, rounding indicates the performance status. Data of 0 or negative number is represented by "-", data of 0.001-0.499 is represented by "+ / -", data of 0.500-1.499 is represented by "+", data of 1.500-2.499 is represented by "2+", and so on. "N / A" indicates that no data was found.

[0264] The fluorescence intensity of the cells was measured, and the results are plotted in Figures 2A and 2B. It can be seen that sample Cy5-pep-20BK bound to and internalized different cells at 15 and 30 minutes. In LNCaP and KB cell lines with relatively high FORL1 expression, the fluorescence intensity of bound and internalized Cy5-pep-20BK was significantly higher than that in the DU145 and NCI-H460 cell lines with relatively low FORL1 and FOLH1 expression. Cy5-pep-20AK only has a FOLH1 ligand and lacks the FORL1 ligand FA; therefore, Cy5-pep-20AK only showed high levels of binding and internalization in LNCaP cells, a cell line with high FOLH1 expression. The degree of ligand complex binding and internalization with cells was positively correlated with the expression level of cell-associated receptors.

[0265] Binding and endocytosis of the single-ligand complex Cy5-FA with cells

[0266] Labeled sample: Cy5-FA(CONH2)

[0267] Cell lines: HeLa cervical cancer cells, SKOV3 human ovarian cancer cells, and A549 human lung cancer cells.

[0268] Experimental design / procedure:

[0269] As shown in Figure 3, the fluorescence intensity of sample Cy5-FA after binding to and endocytosis with cells at 15 and 30 minutes was significantly higher in the FOLR1-high expression cell lines HeLa and SKOV-3 than in the relatively low FOLR1 expression cell line A549. The degree of ligand complex binding and internalization with cells was positively correlated with the expression level of cell-related receptors. [Example] [4] [Cellular Expansion Inhibition Experiment of Complex Compound]

[0270] [1. CB-20BK] [Tumor Cell Proliferation Inhibition Experiment]

[0271] Sample Information: CB-20BK

[0272] Cell lines: KB human oral epidermal carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human liver cancer cells, and LNCaP human prostate cancer cells.

[0273] Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0274] Experimental procedure:

[0275] 1) Cell plating

[0276] Prepare the cells in advance, digest them with Trypsin, collect and count them. Dilute KB cells to 2.5 × 10⁴ cells / ml, T-47D cells to 1.3 × 10⁴ cells / ml, NCI-H460 cells to 3.5 × 10⁴ cells / ml, CALU-3 cells to 4.0 × 10⁴ cells / ml, HuH-7 cells to 3.0 × 10⁴ cells / ml, and LNCaP cells to 8.0 × 10⁴ cells / ml using complete cell culture medium. Plate the cells into 96-well plates, adding 100 μl of diluted cell solution to each well. Include negative and blank control wells in each plate. Incubate the cell-filled 96-well plates overnight at 37°C in a 5% CO₂ incubator.

[0277] 2) Dilution and sample loading

[0278] Dilute the sample to the required concentration using culture medium (see Table 6). Add 50 μl per well to a 96-well plate after overnight incubation, with 3 replicates. Include negative and blank controls. Incubate at 37°C in a 5% CO2 incubator for 72 hours. Table 6: Concentration of sample (CB-20BK) after dilution Pipe number Concentration after dilution (μmol / L) 1 201 2 50 3 13 4 3.1 5 0.8 6 0.2 7 0.05 8 0.01 9 0.003 10 0.0008

[0279] 3) Color reading disc

[0280] Take CCK-8 chromogenic solution and add 15 μl (10% of the liquid volume in each well) to each well. Incubate at 37°C for an appropriate time (try to keep the OD value in the range of 1.0-2.0). Remove the culture plate cover from the 96-well plate and place it in a microplate reader (Molecular Devices Spectra MAX Plus) and read the value at 450 nm.

[0281] 4) Data processing

[0282] Use SoftMax Pro to edit the data and plot the four-parameter fitting curve.

[0283] 5) Experimental Results and Analysis

[0284] According to the four-parameter fitting curve (Figure 4A, where the C value corresponds to IC50), CB-20BK inhibited the growth and expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7, and LNCaP tumor cell lines. The inhibitory effect varied due to differences in the expression levels of the corresponding receptors for the complex compound in the cells. The IC50 values ​​for cell lines with relatively high receptor expression (T-47D, KB, LNCaP, HuH-7, and CALU-3) were significantly lower than those for cell line NCI-H460, which had relatively low receptor expression. CB-20BK demonstrated a correlation between its tumor-suppressive effect and the expression levels of cell-related receptors.

[0285] [2.] [Complex Compound] [CB-20BK] [and related compounds on tumor cell lines] [LNCaP] [(human prostate cancer cells) and] [22RV1] [Inhibition experiment on the proliferation of human prostate cancer cells]

[0286] Because different cell lines exhibit varying sensitivities to the cell proliferation-inhibiting toxicity of the payload in the ligand complex, it can occasionally interfere with quantitative analysis. We selected a series of cell lines with known receptor expression levels for different ligands and simultaneously examined their responses to the cell proliferation-inhibiting effects of the ligand complex and the payload alone. This interference was removed by taking the ratio of the IC50 of the payload alone to that of the ligand complex in a single cell line.

[0287] Relevant samples: MMAE, CB-20BK, CB-20AK, and FA-MMAE

[0288] Experimental Procedure: LNCaP and 22RV1 cells were pre-prepared and seeded into 96-well cell culture dishes at densities of 4 × 10⁴ cells / ml and 2.0 × 10⁴ cells / ml, respectively, 100 μl / well. Each dish included corresponding negative and blank control wells. After overnight adhesion, 50 μl of diluted test sample was added to each well. The dishes were incubated at 37℃ in a 5% CO₂ incubator for 68-72 hours. CCK8 chromogenic buffer was added at 15 μl per well (10% of the total liquid volume), and incubated at 37℃ for 45-70 minutes. Values ​​were read at 450 nm using a microplate reader. Data were edited using SoftMax Pro, and a 4-P curve was plotted. The calculated data are shown in Table 7. Table 7. Inhibitory effects of FA-MMAE, CB-20BK, and CB-20AK on cell line proliferation and expression levels of receptor genes in cell lines (refer to Depmap data). cell lines FOLR1 gene expression level FOLH1 gene expression level IC50 (MMAE) IC50 (FA-MMAE) IC50 (CB-20AK) IC50 (CB-20BK) IC50 ratio (MMAE / FA-MMAE) IC50 ratio (MMAE / CB-20AK) IC50 ratio (MMAE / CB-20BK) LNCaP + / - 10+ 0.0026 0.264 0.102 0.0958 0.0098 0.025 0.0266 22RV1 + / - 6+ 0.0065 1.01 1.05 0.502 0.0064 0.00617 0.0129

[0289] As shown in the table above, the FOLH1 expression levels of LNCaP and 22RV1 are not significantly different, but the FOLH1 expression level of LNCaP is significantly higher than that of 22RV1. CB-20BK (containing both FOLH1 and FOLH1 ligands) and CB-20AK (containing only FOLH1 ligands) both inhibited the expansion of LNCaP and 22RV1 tumor cell lines. CB-20BK and CB-20AK showed different degrees of inhibition based on the receptor FOLH1 expression level. In the LNCaP cell line with relatively high receptor FOLH1 expression, the IC50 ratio (MMAE / CB-20AK or MMAE / CB-20BK) was 2-4 times higher than that of the 22RV1 cell line with relatively low receptor expression. FA-MMAE also inhibited the expansion of both LNCaP and 22RV1 tumor cell lines. Because the expression levels of FOLR1 in LNCaP and 22RV1 are very low, the expression level in LNCaP cells (FOLR1 gene expression level 0.3219, according to Depmap data) is slightly higher than that in 22RV1 cells (FOLR1 gene expression level 0.0704, according to Depmap data). The difference in the IC50 ratio (MMAE / FA-MMAE) between LNCaP and 22RV1 is only 1.5-fold. These data indicate that the cell proliferation inhibition effect is positively correlated with the expression levels of the cell receptors FOLH1 and FOLR1.

[0290] [3.] [Complex Compound] [CB-20BK] [and related compounds on tumor cell lines] [PANC-1] [(human pancreatic cancer cells) and] [CFPAC-1] [Inhibition experiment on the proliferation of human pancreatic cancer cells]

[0291] Related samples: MMAE and CB-20BK

[0292] Experimental Procedure: Prepare cell counting in advance. Seed PANC-1 and CFPAC-1 at a cell density of 4 × 10⁴ cells / ml in 96-well plates, 100 μl / well. Include corresponding negative and blank control wells in each culture plate. After overnight adhesion, add 50 μl / well of diluted test sample and incubate at 37℃ in a 5% CO₂ incubator for 68-72 hours. Add 15 μl of CCK8 chromogenic solution (10% of the liquid volume in each well) to each well and incubate at 37℃ for 45-70 minutes. Read the values ​​at 450nm using a microplate reader. Edit the data using SoftMax Pro and plot the 4-P curve. The calculated data are shown in the table below: Table 8. Inhibitory effect of CB-20BK on cell line proliferation and expression levels of receptor genes in cell lines (refer to Depmap data) cell lines FOLR1 gene expression level FOLH1 gene expression level IC50 (MMAE) IC50 (CB-20BK) IC50 (MMAE) / IC50 (CB-20BK) CFPAC-1 4+ + / - 0.015 0.557 0.027 PANC-1 + / - + / - 0.002 0.126 0.020

[0293] Table 8 shows that CFPAC-1 cell line exhibits very low FOLH1 expression, while its FOLR1 expression is significantly higher than that of the PANC1 cell line. CB-20BK inhibits the proliferation of both PANC-1 and CFPAC-1 tumor cell lines. The degree of inhibition varies among cells with different FOLR1 expression levels. In the CFPAC-1 cell line with relatively high FOLR1 expression, the IC50 ratio is significantly higher than that of the PANC-1 cell line with relatively low FOLR1 expression, indicating a positive correlation between cell proliferation inhibition and the expression level of the cell-associated receptor FOLR1.

[0294] Experiments 2 and 3 demonstrate that the two ligands in CB-20BK and their receptors (FOLR1 and FOLH1) expressed on the cell surface play an important role in the process by which this compound inhibits tumor cell proliferation.

[0295] [4. CB-20B] [Tumor Cell Proliferation Inhibition Experiment]

[0296] Sample Information: CB-20B

[0297] Cell lines: A549 human lung cancer cells, HuH-7 human liver cancer cells, KB human oral epidermoid carcinoma cells, LNCaP human prostate cancer cells, DU145 human prostate cancer cells, and T-47D human breast cancer cells.

[0298] Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0299] Experimental procedure:

[0300] 1) Cell plating

[0301] Prepare the cells in advance, digest them with Trypsin, collect and count them, and use complete cell culture medium. Seed A549 cells, HuH-7 cells, KB cells, and DU145 cells at a density of 2 × 10⁴ cells / ml; seed T-47D cells at a density of 1 × 10⁵ cells / ml; and LNCaP cells at a density of 6 × 10⁴ cells / ml in 96-well plates. Add 100 μl of diluted cell solution to each well. Include negative and blank control wells in each plate. Incubate the cell-filled 96-well plates overnight at 37°C in a 5% CO₂ incubator.

[0302] 2) Dilution and sample loading

[0303] Dilute the sample to the required concentration using culture medium (see Table 9). Add 50 μl per well to a 96-well plate after overnight incubation, with 3 replicates. Include negative and blank controls. Incubate at 37°C in a 5% CO2 incubator for 72 hours. Table 9: Concentration of sample (CB-20B) after dilution Pipe number Concentration after dilution (μmol / L) 1 200 2 66.7 3 22.2 4 7.4 5 2.5 6 0.8 7 0.3 8 0.09 9 0.03 10 0.01

[0304] 3) Color reading disc

[0305] Take CCK-8 chromogenic solution and add 15 μl (10% of the liquid volume in each well) to each well. Incubate at 37°C for an appropriate time (try to keep the OD value in the range of 1.0-2.0). Remove the culture plate cover from the 96-well plate and place it in a microplate reader (Molecular Devices Spectra MAX Plus) and read the value at 450 nm.

[0306] 4) Data processing

[0307] Use SoftMax Pro to edit the data and plot the four-parameter fitting curve.

[0308] 5) Experimental Results and Analysis

[0309] According to the four-parameter fitting curve (Figure 4B, where the C value corresponds to IC50), CB-20B inhibited the growth and expansion of A549, HuH-7, KB, LNcap, DU145, and T-47D tumor cell lines. The inhibitory effect varied due to differences in the expression levels of the corresponding receptors for the complex compound in the cells. The IC50 values ​​for T-47D, LNcap, KB, HuH-7, and DU145, which showed relatively high receptor expression, were significantly lower than those for A549, which showed relatively low receptor expression. CB-20B demonstrated a correlation between its antitumor effect and the expression levels of cell-related receptors.

[0310] [5. CB-10S] [Tumor Cell Proliferation Inhibition Experiment]

[0311] Sample Information: CB-10S

[0312] Cell lines: KB human oral epidermal carcinoma cells, NCI-H460 human lung cancer cells, RT4 human bladder cancer cells, T-47D human breast cancer cells, and LNcap human prostate cancer cells.

[0313] Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0314] Experimental procedure:

[0315] 1) Cell plating

[0316] Prepare the cells in advance, digest them with Trypsin, collect and count them. Dilute KB cells to 3.5 × 10⁴ cells / ml, LNCaP cells to 8.0 × 10⁴ cells / ml, T-47D cells to 1.2 × 10⁴ cells / ml, NCI-H460 cells to 2.5 × 10⁴ cells / ml, and RT4 cells to 1.2 × 10⁴ cells / ml using complete cell culture medium. Take a 96-well plate and add 100 μl of diluted cell solution to each well. Set up negative and blank control wells on each plate. Incubate the 96-well plates with cells overnight at 37°C in a 5% CO₂ incubator.

[0317] 2) Dilution and sample loading

[0318] Dilute the sample to the required concentration using culture medium (see Table 10). Add 50 μl per well to a 96-well plate after overnight incubation, with 3 replicates. Set up negative and blank controls. Incubate at 37°C in a 5% CO2 incubator for 72 hours. Table 10: Concentration of sample (CB-10S) after dilution Pipe number Concentration after dilution (μmol / L) 1 302 2 43 3 6 4 0.9 5 0.1 6 0.02 7 0.003 8 0.0004 9 0.00005 10 0.000007

[0319] 3) Color reading disc

[0320] Take CCK-8 chromogenic solution and add 15 μl (10% of the liquid volume in each well) to each well. Incubate at 37°C for an appropriate time (try to keep the OD value in the range of 1.0-2.0). Remove the culture plate cover from the 96-well plate and place it in a microplate reader (Molecular Devices Spectra MAX Plus) and read the value at 450 nm.

[0321] 4) Data processing

[0322] Use SoftMax Pro to edit the data and plot the four-parameter fitting curve.

[0323] 5) Experimental Results and Analysis

[0324] According to the four-parameter fitting curve (Figure 4C, where the C value corresponds to IC50), CB-10S inhibited the growth and expansion of KB, LNCaP, T-47D, RT4, and NCI-H460 tumor cell lines. The inhibitory effect varied due to differences in the expression levels of the corresponding receptors for the complex compounds within the cells. The IC50 values ​​for cell lines with relatively high receptor expression (KB, LNCaP, T-47D, and RT4) were significantly lower than those for cell line with relatively low receptor expression (NCI-H460). CB-10S demonstrated a correlation between its tumor-suppressive effect and the expression levels of cell-related receptors.

[0325] [6. CB-60S] [Tumor Cell Proliferation Inhibition Experiment]

[0326] Sample Information: CB-60S

[0327] Cell lines: KB human oral epidermal carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human liver cancer cells, and LNCaP human prostate cancer cells.

[0328] Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0329] Experimental procedure:

[0330] 1) Cell plating

[0331] Prepare the cells in advance, digest them with Trypsin, collect and count them. Dilute KB cells to 2.0 × 10⁴ cells / ml, T-47D cells to 1.5 × 10⁴ cells / ml, NCI-H460 cells to 2.0 × 10⁴ cells / ml, CALU-3 cells to 3.0 × 10⁴ cells / ml, HuH-7 cells to 4.0 × 10⁴ cells / ml, and LNCaP cells to 8.0 × 10⁴ cells / ml using complete cell culture medium. Take a 96-well plate, add 100 μl of diluted cell solution to each well, and set up negative and blank control wells for each plate. Incubate the 96-well plates with cells overnight in a 37°C, 5% CO₂ incubator.

[0332] 2) Dilution and sample loading

[0333] Dilute the sample to the required concentration using culture medium (see Table 11). Add 50 μl per well to a 96-well plate after overnight incubation, with 3 replicates. Include negative and blank controls. Incubate at 37°C in a 5% CO2 incubator for 72 hours. Table 11: Concentration of sample (CB-60S) after dilution Pipe number Concentration after dilution (μmol / L) 1 320 2 80 3 20 4 5 5 1.3 6 0.3 7 0.08 8 0.02 9 0.005 10 0.001

[0334] 3) Color reading disc

[0335] Take CCK-8 chromogenic solution and add 15 μl (10% of the liquid volume in each well) to each well. Incubate at 37°C for an appropriate time (try to keep the OD value in the range of 1.0-2.0). Remove the culture plate cover from the 96-well plate and place it in a microplate reader (Molecular Devices Spectra MAX Plus) and read the value at 450 nm.

[0336] 4) Data processing

[0337] Use SoftMax Pro to edit the data and plot the four-parameter fitting curve.

[0338] 5) Experimental Results and Analysis

[0339] The four-parameter fitting curve (Figure 4D, where C corresponds to IC50) shows that CB-60S inhibits the growth and expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7, and LNCaP tumor cell lines. The inhibitory effect varies depending on the level of receptor expression of the complex compound in the cells. The IC50 values ​​for LNCaP and HuH-7, which have relatively high receptor expression, are significantly lower than those for NCI-H460, KB, T-47D, and CALU-3, which have relatively low receptor expression. CB-60S demonstrates a correlation between its antitumor effect and the level of expression of cell-related receptors.

[0340] [7. CB-60SK] [Tumor Cell Proliferation Inhibition Experiment]

[0341] Sample Information: CB-60SK

[0342] Cell lines: KB human oral epidermal carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human liver cancer cells, and LNCaP human prostate cancer cells.

[0343] Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0344] Experimental procedure:

[0345] 1) Cell plating

[0346] Prepare the cells in advance, digest them with Trypsin, collect and count them. Dilute KB cells to 2.5 × 10⁴ cells / ml, T-47D cells to 1.3 × 10⁴ cells / ml, NCI-H460 cells to 3.5 × 10⁴ cells / ml, CALU-3 cells to 4.0 × 10⁴ cells / ml, HuH-7 cells to 3.0 × 10⁴ cells / ml, and LNCaP cells to 8.0 × 10⁴ cells / ml using complete cell culture medium. Add 100 μl of diluted cell solution to each well. Include negative and blank control wells in each dish. Incubate the 96-well dishes with cells at 37°C in a 5% CO₂ incubator overnight.

[0347] 2) Dilution and sample loading

[0348] Dilute the sample to the required concentration using culture medium (see Table 12). Add 50 μl per well to a 96-well plate after overnight incubation, with 3 replicates. Set up negative and blank controls, and incubate at 37°C in a 5% CO2 incubator for 72 hours. Table 12: Concentration of sample (CB-60SK) after dilution Pipe number Concentration after dilution (μmol / L) 1 289 2 72 3 18 4 4.5 5 1.1 6 0.3 7 0.07 8 0.02 9 0.004 10 0.001

[0349] 3) Color reading disc

[0350] Take CCK-8 chromogenic solution and add 15 μl (10% of the liquid volume in each well) to each well. Incubate at 37°C for an appropriate time (try to keep the OD value in the range of 1.0-2.0). Remove the culture plate cover from the 96-well plate and place it in a microplate reader (Molecular Devices Spectra MAX Plus) and read the value at 450 nm.

[0351] 4) Data processing

[0352] Use SoftMax Pro to edit the data and plot the four-parameter fitting curve.

[0353] 5) Experimental Results and Analysis

[0354] According to the four-parameter fitting curve (Figure 4E, where the C value corresponds to IC50), CB-60SK inhibited the growth and expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7, and LNCaP tumor cell lines. The inhibitory effect varied due to differences in the expression levels of the corresponding receptors for the complex compounds within the cells. The IC50 values ​​for LNCaP and HuH-7, cell lines with relatively high receptor expression, were significantly lower than those for T-47D, NCI-H460, CALU-3, and KB, cell lines with relatively low receptor expression. CB-60SK demonstrated a correlation between its antitumor effect and the expression levels of cell-related receptors.

[0355] [8. CB-18G] [Tumor Cell Proliferation Inhibition Experiment]

[0356] Sample Information: CB-18G

[0357] Cell lines: A549 human lung cancer cells, HeLa human cervical cancer cells, SCLC-21H small cell lung cancer cells, U-2 OS human osteosarcoma cells, T-47D human breast cancer cells, and NCI-H460 human lung cancer cells.

[0358] Main reagents: IMDM medium, fetal bovine serum, p-penicillin-streptomycin solution, L-glutamine, CCK8

[0359] Experimental procedure:

[0360] 1) Cell plating

[0361] Prepare the cells in advance, digest them with Trypsin, collect and count them, and use complete cell culture medium. Seed A549 cells, HeLa cells, SCLC-21H cells, and U-2 OS cells at a density of 2 × 10⁴ cells / ml; seed T-47D cells and NCI-H460 cells at a density of 3 × 10⁴ cells / ml in 96-well plates. Add 100 μl of diluted cell solution to each well of each 96-well plate. Include negative and blank control wells in each plate. Incubate the cell-filled 96-well plates overnight at 37°C in a 5% CO₂ incubator.

[0362] 2) Dilution and sample loading

[0363] Dilute the sample to the required concentration using culture medium (see Table 13). Add 50 μl per well to a 96-well plate after overnight incubation, with 3 replicates. Set up negative and blank controls. Incubate at 37°C in a 5% CO2 incubator for 72 hours. Table 13: Concentration of sample (CB-18G) after dilution Pipe number Concentration after dilution (μmol / L) 1 100 2 25 3 6.25 4 1.56 5 0.39 6 0.10 7 0.02 8 0.006 9 0.002 10 0.0004

[0364] 3) Color reading disc

[0365] Take CCK-8 chromogenic solution and add 15 μl (10% of the liquid volume in each well) to each well. Incubate at 37°C for an appropriate time (try to keep the OD value in the range of 1.0-2.0). Remove the culture plate cover from the 96-well plate and place it in a microplate reader (Molecular Devices Spectra MAX Plus) and read the value at 450 nm.

[0366] 4) Data processing

[0367] Use SoftMax Pro to edit the data and plot the four-parameter fitting curve.

[0368] 5) Experimental Results and Analysis

[0369] According to the four-parameter fitting curve (Figure 4F, where C corresponds to IC50), CB-18G inhibited the growth and expansion of A549, HeLa, SCLC-21H, U-2 OS, T-47D, and NCI-H460 tumor cell lines. The inhibitory effect varied due to differences in the expression levels of the corresponding receptors for the complex compound in the cells. The IC50 for the HeLa cell line, with relatively high receptor expression, was lower than that for the NCI-H460 cell line, with relatively low receptor expression. CB-18G demonstrated a correlation between its antitumor effect and the expression levels of cell-related receptors.

[0370] [9.] [CB-50S] [Tumor Cell Proliferation Inhibition Experiment]

[0371] Sample Information: CB-50S

[0372] Cell lines: KB human oral epidermal carcinoma cells, NCI-H460 human lung cancer cells, RT4 human bladder cancer cells, T-47D human breast cancer cells, and LNCaP human prostate cancer cells.

[0373] Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0374] Experimental procedure:

[0375] 1) Cell plating

[0376] Prepare the cells in advance, digest them with Trypsin, collect and count them. Dilute KB cells to 3.5 × 10⁴ cells / ml, LNCaP cells to 8.0 × 10⁴ cells / ml, T-47D cells to 1.2 × 10⁴ cells / ml, NCI-H460 cells to 2.5 × 10⁴ cells / ml, and RT4 cells to 1.2 × 10⁵ cells / ml using complete cell culture medium. Take a 96-well plate and add 100 μl of diluted cell solution to each well. Set up negative and blank control wells on each plate. Incubate the 96-well plates with cells overnight at 37°C in a 5% CO₂ incubator.

[0377] 2) Dilution and sample loading

[0378] Dilute the sample to the required concentration using culture medium (see Table 14). Add 50 μl per well to a 96-well plate after overnight incubation, with 3 replicates. Include negative and blank controls. Incubate at 37°C in a 5% CO2 incubator for 72 hours. Table 14: Concentration of Sample (CB-50S) after Dilution Pipe number Concentration after dilution (μmol / L) 1 314 2 45 3 6 4 0.9 5 0.1 6 0.02 7 0.003 8 0.0004 9 0.00005 10 0.000008

[0379] 3) Color reading disc

[0380] Take CCK-8 chromogenic solution and add 15 μl (10% of the liquid volume in each well) to each well. Incubate at 37°C for an appropriate time (try to keep the OD value in the range of 1.0-2.0). Remove the culture plate cover from the 96-well plate and place it in a microplate reader (Molecular Devices Spectra MAX Plus) and read the value at 450 nm.

[0381] 4) Data processing

[0382] Use SoftMax Pro to edit the data and plot the four-parameter fitting curve.

[0383] 5) Experimental Results and Analysis

[0384] According to the four-parameter fitting curve (Figure 4G, where the C value corresponds to IC50), CB-50S inhibited the growth and expansion of KB, LNCaP, T-47D, RT4, and NCI-H460 tumor cell lines. The inhibitory effect varied due to differences in the expression levels of the corresponding receptors for the complex compounds in the cells. The IC50 values ​​for cell lines with relatively high receptor expression (KB, LNCaP, T-47D, and RT4) were significantly lower than those for cell line NCI-H460 with relatively low receptor expression. CB-50S demonstrated a correlation between its tumor-suppressive effect and the expression levels of cell-related receptors.

[0385] [10.] [Complex Compound] [CB-1020] [Tumor Cell Proliferation Inhibition Experiment]

[0386] Experimental objective: To test the inhibitory effects of CB-1020 and related compounds on the proliferation of tumor cell lines LNCaP (human prostate cancer cells), SK-BR-3 (human breast cancer cells), NCI-H226 (human lung cancer cells), CFPAC-1 (pancreatic cancer cells), and PANC-1 (pancreatic cancer cells).

[0387] Related samples: MMAE and CB-1020

[0388] Experimental Procedure: LNCaP, SK-BR-3, NCI-H226, CFPAC-1, and PANC-1 cells were counted in advance. LNCaP, SK-BR-3, and CFPAC-1 cells were diluted to 4 × 10⁴ cells / ml, NCI-H226 to 2.0 × 10⁴ cells / ml, and PANC-1 to 3.0 × 10⁴ cells / ml using complete culture medium (IMDM + 10% FBS + 1X L-Glutamine + 1X P / S). 100 μl / well was seeded into 96-well plates, with negative and blank control wells in each plate. After overnight adhesion, 50 μl of the diluted test sample was added to each well. The plates were then incubated at 37℃ with 5% CO₂. Incubate for 68-72 hours in an incubator, add CCK8 chromogenic solution (15 μl per well, 10% of the liquid volume), and incubate at 37°C for 45-70 minutes. Read the values ​​at 450 nm using a microplate reader. Edit the data using SoftMax Pro and plot the 4-P curve. The data are shown in the table below: Table 15. Inhibitory effect of CB-1020 on cell line proliferation and expression levels of cell line receptor genes. (Referencing Depmap data) cell lines TRPV6 gene expression level FOLH1 gene expression level IC50 (MMAE) IC50 (CB-1020) IC50 (MMAE) / IC50 (CB-1020) LNCap 5+ 10+ 0.003 0.168 0.0179 SK-BR-3 4+ 2+ 0.002 0.287 0.0070 CFPAC-1 + + / - 0.015 3.000 0.005 PANC-1 + / - + / - 0.002 0.458 0.0044 NCI-H226 + / - - 0.006 1.570 0.00384

[0389] As shown in the table above, both LNCaP and SK-BR-3 expressed TRPV6 and FORRH1, but LNCaP showed relatively higher expression levels of both receptors. NCI-H226, CFPAC-1, and PANC-1 showed low or weak expression of both receptors. CB-1020 inhibited the growth and expansion of tumor cell lines including LNCaP, SK-BR-3, NCI-H226, CFPAC-1, and PANC-1. The IC50 ratio of CB-1020 in the relatively high receptor expression cell line LNCaP was higher than that in the moderately expressed receptor cell line SK-BR-3; the IC50 ratio of SK-BR-3 was higher than that of CFPAC-1 (relatively low receptor expression) and both NCI-H226 and PANC-1 (weak receptor expression). The cell proliferation inhibition effect was positively correlated with the expression levels of both receptors in the cells.

[0390] [11.] [Complex Compound] [CB-1320] [Tumor Cell Proliferation Inhibition Experiment]

[0391] Experimental objective: To test the inhibitory effects of CB-1320 and related compounds on the proliferation of tumor cell lines LNCaP (human prostate cancer cells), SK-BR-3 (human breast cancer cells), MDA-MB-468 (human breast cancer cells), and CFPAC-1 (pancreatic cancer cells).

[0392] Relevant samples: MMAE and CB-1320

[0393] Experimental Procedure: LNCaP, SK-BR-3, MDA-MB-468, and CFPAC-1 cells were counted using pre-prepared culture medium (IMDM + 10% FBS + 1X L-Glutamine + 1X P / S). The cells were diluted to 4 × 10⁴ cells / ml using complete culture medium (IMDM + 10% FBS + 1X L-Glutamine + 1X P / S). 100 μl of the diluted medium was seeded into 96-well plates, with negative and blank control wells in each plate. After overnight adhesion, 50 μl of the diluted test sample was added to each well. The plates were incubated at 37°C in a 5% CO₂ incubator for 68-72 hours. CCK8 chromogenic solution was added to each well (15 μl of the total liquid volume), and the plates were incubated at 37°C for 45-70 minutes. Values ​​were read at 450 nm using a microplate reader. Data were edited using SoftMax Pro, and a 4-P curve was plotted. Specific data are shown in the table below. Table 16. Inhibitory effect of CB-1320 on cell line proliferation and expression levels of cell line receptor genes. (Referencing Depmap data) cell lines GNRH1 gene expression level FOLH1 gene expression level IC50 (MMAE) IC50 (CB-1320) IC50(MMAE) / IC50(CB-1320) LNCaP + 10+ 0.003 0.060 0.043 SK-BR-3 + 2+ 0.002 0.093 0.016 CFPAC-1 2+ + / - 0.015 1.090 0.014 MDA-MB-468 + + / - 0.002 0.212 0.011

[0394] As shown in the table above, the GNRH1 expression levels of LNCaP and SK-BR-3 cells are roughly equivalent, while the FOLH1 expression level of LNCaP is significantly higher than that of the SK-BR3 cell line. CB-1320 inhibits the proliferation of both LNCaP and SK-BR-3 tumor cell lines. CB-1320 exhibits varying degrees of cell inhibition based on the level of receptor FOLH1 expression. In the LNCaP cell line with relatively high FOLH1 expression, the IC50 ratio is significantly higher than that of the SK-BR-3 cell line with relatively low FOLH1 expression. The cell proliferation inhibition effect is positively correlated with the expression level of the cell-related receptor FOLH1. The expression levels of GNRH1 and FOLH1 in CFPAC-1 and MDA-MB-468 were roughly equivalent. CB-1320 inhibited the growth and expansion of both MDA-MB-468 and CFPAC-1 tumor cell lines. The IC50 ratios of the two cell lines were roughly equivalent. The cell proliferation inhibition effect was still correlated with the expression levels of the two cell-related receptors.

[0395]

[12] [Complex Compounds] [CB-1820] [Tumor Cell Proliferation Inhibition Experiment]

[0396] Experimental objective: To test the inhibitory effects of CB-1820 and related compounds on the proliferation of tumor cell lines LNCaP (human prostate cancer cells), MDA-MB-468 (human breast cancer cells), CFPAC-1 (pancreatic cancer cells), and PANC-1 (pancreatic cancer cells).

[0397] Relevant samples: MMAE and CB-1820

[0398] Experimental Procedure: LNCaP, MDA-MB-468, CFPAC-1, and PANC-1 cells were counted in advance. LNCaP, MDA-MB-468, and CFPAC-1 cells were diluted to 4 × 10⁴ cells / ml using complete culture medium (IMDM + 10% FBS + 1X L-Glutamine + 1X P / S); PANC-1 was diluted to 3.0 × 10⁴ cells / ml. 100 μl of each medium was seeded into a 96-well plate, with negative and blank control wells in each plate. After overnight adhesion, 50 μl of the diluted test sample was added to each well. The plates were incubated at 37°C in a 5% CO₂ incubator for 68-72 hours. CCK8 chromogenic buffer was added to each well (10% of the total liquid volume), and the plates were incubated at 37°C for 45-70 minutes. Values ​​were read at 450 nm using a microplate reader. Data were edited using SoftMax Pro, and a 4-P curve was plotted. The specific values ​​are shown in the table below:

[0399] Table 17. Inhibitory effect of CB-1820 on cell line proliferation and expression levels of cell line receptor genes. (Referencing Depmap data) cell lines SSTR2 gene expression FOLH1 gene expression level IC50 (MMAE) IC50 (CB-1820) IC50 (MMAE) / IC50 (CB-1820) LNCap + / - 10+ 0.003 0.021 0.1429 MDA-MB-468 + / - + / - 0.002 0.062 0.0323 CFPAC-1 + / - + / - 0.015 0.356 0.0421 PANC-1 + / - + / - 0.002 0.055 0.0364

[0400] As shown in the table above, the SSTR2 expression levels of LNCaP, MDA-MB-468, CFPAC-1, and PANC-1 were roughly equivalent, while the FOLH1 expression level of LNCaP was significantly higher than that of other cell lines. CB-1820 inhibited the proliferation of all four tumor cell lines. The degree of inhibition of CB-1820 varied depending on the level of FOLH1 expression. In LNCaP, a cell line with relatively high FOLH1 expression, the IC50 ratio was significantly higher than that of other cell lines with relatively low FOLH1 expression, indicating a positive correlation between cell proliferation inhibition and the expression level of the cell-related receptor FOLH1.

[0401]

[13] [Complex Compounds] [CR19425] [、] [CR19426] [and] [CR19428] [Tumor Cell Proliferation Inhibition Experiment]

[0402] Experimental objective: To test the inhibitory effects of CR19428, CR19425, CR19426 and related compounds on the expansion of tumor cell lines NCI-H226 (human lung cancer cells), CFPAC-1 (human pancreatic cancer cells), and MDA-MB-468 (human breast cancer cells).

[0403] Relevant samples: SN-38, Dxd0017, CR19428, CR19425 and CR19426

[0404] Experimental Procedure: Cell counts were performed on pre-prepared NCI-H226, CFPAC-1, and MDA-MB-468 cells. The cell density for MDA-MB-468 and CFPAC-1 was 2 × 10⁴ cells / ml, and the cell density for NCI-H226 was 1 × 10⁴ cells / ml. 100 μl / well was seeded into 96-well plates, with negative and blank control wells on each plate. After overnight adhesion, 50 μl of diluted test sample was added to each well. The plates were incubated at 37°C in a 5% CO₂ incubator for 68-72 hours. CCK8 chromogenic buffer was added to each well (15 μl, 10% of the total liquid volume), and the plates were incubated at 37°C for 45-70 minutes. Values ​​were read at 450 nm using a microplate reader. Data were edited using SoftMax Pro, and a 4-P curve was plotted. Specific data are shown in the table below.

[0405] Table 18. Inhibitory effects of CR19428, CR19425 and CR19426 on cell line proliferation cell lines IC50 (SN-38) IC50 (Dxd0017) IC50 (CR19428) IC50 (CR19425) IC50 (CR19426) CFPAC-1 0.0055 0.0064 0.9060 0.6210 1.6600 MDA-MB-468 0.0136 0.0061 1.0700 0.9510 2.3000 NCI-H226 0.0173 0.0080 1.6200 1.0100 4.3900 [Example] [5] [Compound compounds in] [CDX] [Pharmacodynamic studies in the model]

[0406] [1. CB-20BK] [exist] [CDX] [Pharmacodynamic studies in the model] [1]

[0407] 1) Sample preparation:

[0408] Weigh out CB-20BK lyophilized powder, dissolve it in PBS to prepare a sample stock solution, and dilute the stock solution with physiological saline for injection to the working concentration sample solution for later use.

[0409] 2) CDX Model Construction

[0410] Major cell lines: KB human oral epidermoid carcinoma cells, MIA paca-2 human pancreatic carcinoma cells, HCC1954 human breast cancer cells, CALU-3 human lung adenocarcinoma cells, and DU145 human prostate cancer cells.

[0411] Model construction: Resuscitate and culture cells, collect and count them, and inject them subcutaneously into the right limb of BALB / c-nude mice. When the tumor grows and expands to 80-160 mm3, administer the drug in groups or take rapidly expanded tumor blocks to transfer and expand the mouse tumor model.

[0412] 3) Grouped drug administration observation

[0413] Grouping: Once the tumors reached an average size of approximately 80-160 mm3, they were divided into a model control group and groups receiving different doses of medication.

[0414] Administration: Inject via tail vein.

[0415] Experimental observation and measurement: Routine monitoring after tumor inoculation included tumor growth and the impact of treatment on the animals' normal behavior. Specific content included the experimental animals' activity level, feeding and water intake, weight gain or loss (weight measured twice a week), and any abnormalities in the eyes, coat, or other areas.

[0416] Mouse body weight and the major and minor diameters (a and b) of the tumor were measured twice a week. Tumor volume (TV) was calculated using the formula: TV = 1 / 2 × a × b².

[0417] 4) Experimental Results and Analysis

[0418] Based on the changes in mouse tumor volume (Figures 5A-5E), it can be seen that CB-20BK has a good inhibitory effect on mouse KB, MIA aca-2, HCC1954, CALU-3 and DU145 cell line CDX tumor models.

[0419] [2. CB-20BK] [exist] [CDX] [Pharmacodynamic studies in the model] [2]

[0420] Experimental Objective: To investigate the efficacy of the complex compound CB-20BK in subcutaneous allogeneic transplantation (CDX) models of human LNCaP, DU145, and NCI-H460 cell lines.

[0421] Main CDX models: LNCaP, DU145, and NCI-H460

[0422] Experimental protocol:

[0423] Prepared cells or tumor tissue blocks were subcutaneously inoculated into the anterior right limb of BALB / c-nude mice. When the tumor volume increased to 80-160 mm³, the mice were randomly divided into a model control group and a treatment group. Treatment began on day 1 of grouping, with the dosage adjusted according to the mice's most recent body weight. The medication was administered via tail vein injection at a volume of 10 μl / g. After treatment, monitoring included tumor growth and the effect of treatment on normal animal behavior, specifically the animals' activity level, food and water intake, weight gain or loss, and any abnormalities in the eyes, coat, or other areas. Mouse body weight was measured twice weekly after treatment, and the rate of weight change was calculated. Simultaneously, the tumor's long and short diameters were measured using calipers, and tumor volume, relative tumor proliferation rate, and tumor volume inhibition rate were calculated. The tumor volume formula was TV = 0.5 a × b², where a is the long diameter of the tumor and b is the short diameter. Table 19. Growth inhibitory effect of CB-20BK on CDX model and expression levels of receptor genes in each model. (Based on data from Crown Bioscience, China) Model FOLR1 FOLH1 Tumor inhibition rate (TGI) (%) Dosage / Time LNCaP 5+ 10+ 95.68 3mg / kg D1,8,15 DU145 - - 52 5mg / kg D1,4,7 NCI-H460 - - 32 10mg / kg D1,8,15

[0424] As shown in the table above, the CB-20BK sample exhibited varying degrees of tumor growth inhibition under tail vein administration. For the LNCaP, DU145, and NCI-H460 human cell line CDX models, all showed some anti-tumor growth effects compared to the negative control group. The FOLR1 and FOLH1 dual-expression model LNCaP, administered at a dose of 3 mg / kg on days 1, 8, and 15 (D1, D8, and D15), showed a TGI value of 95.68%, demonstrating excellent anti-tumor growth effects, significantly superior to the DU145 and NCI-H460 models with relatively low FOLR1 and FOLH1 expression. The tumor-inhibiting effect showed a certain correlation with the expression levels of cell-related receptors.

[0425] [3.] [Complex Compound] [CB-20BK] [right] [HuPrime, ® , ] [Allogeneic transplantation] [PDX] [Tumor Suppression Experiment of the Model] [1]

[0426] Experimental Objective: To study the pharmacodynamics of the complex compound CB-20BK in a PDX model.

[0427] Main models: LU1206, LU1380 and LU0367

[0428] Experimental Protocol: Prepared tumor tissue blocks were subcutaneously inoculated into the anterior right limb of BALB / c-nude mice. When the tumor volume increased to 80-160 mm³, the mice were randomly divided into a model control group and a treatment group. Treatment began on day 1 of grouping, with the dosage adjusted according to the mice's most recent body weight. The medication was administered via tail vein injection at a volume of 10 μl / g and a dose of 3 mg / kg. After treatment, monitoring included tumor growth and the effects of treatment on normal animal behavior, specifically the animals' activity level, food and water intake, weight gain or loss, and any abnormalities in the eyes, coat, or other areas. Mouse body weight was measured twice weekly after treatment, and the rate of weight change was calculated. Simultaneously, the tumor's long and short diameters were measured using calipers, and tumor volume, relative tumor proliferation rate, and tumor volume inhibition rate were calculated. The tumor volume formula was TV = 0.5 a × b², where a is the long diameter of the tumor and b is the short diameter. Table 20. Inhibitory effect of CB-20BK on the growth of lung cancer PDX models and expression levels of receptor genes in various models. (Based on data from Crown Bioscience, China) Model FOLR1 gene expression level FOLH1 gene expression level TGI (%) dose LU1206 4+ 2+ 90.89 3mg / kg LU1380 - 4+ 30.02 3mg / kg LU0367 - 5+ 71.34 3mg / kg

[0429] As shown in Table 20, the tested sample CB-20BK (3 mg / kg) exhibited certain anti-tumor effects in the LU1206, LU1380, and LU0367 human lung cancer PDX models. The TGI value of the LU1206 model (dual expression of FOLR1 and FOLH1) was 90.89%, while the TGI values ​​of the LU1380 and LU0367 single expression models were 30.02% and 71.34%, respectively. In the tumor suppression experiment, the tumor-suppressing effect showed a certain correlation with the expression levels of cell-related receptors.

[0430] [4.] [Complex Compound] [CB-20BK] [right] [HuPrime, ® , ] [Allogeneic transplantation] [PDX] [Tumor Suppression Experiment of the Model] [2]

[0431] Experimental Objective: To study the pharmacodynamics of the complex compound CB-20BK in a PDX model.

[0432] Main models: BR1283 and BR0438

[0433] Experimental Protocol: Prepared tumor tissue blocks were subcutaneously inoculated into the anterior right limb of BALB / c-nude mice. When the tumor volume increased to 80-160 mm³, the mice were randomly divided into a model control group and a treatment group. Treatment began on day 1 of grouping, with the dosage adjusted according to the mice's most recent body weight. The medication was administered via tail vein injection at a volume of 10 μl / g and a dose of 3 mg / kg. After treatment, monitoring included tumor growth and the effects of treatment on normal animal behavior, specifically the animals' activity level, food and water intake, weight gain or loss, and any abnormalities in the eyes, coat, or other areas. Mouse body weight was measured twice weekly after treatment, and the rate of weight change was calculated. Simultaneously, the tumor's long and short diameters were measured using calipers, and tumor volume, relative tumor proliferation rate, and tumor volume inhibition rate were calculated. The tumor volume formula was TV = 0.5 a × b², where a is the long diameter of the tumor and b is the short diameter. Table 21. Inhibitory effect of CB-20BK on the growth of breast cancer PDX models and expression levels of receptor genes in various models. (Based on data from Crown Bioscience, China) Model FOLR1 gene expression level FOLH1 gene expression level TGI (%) dose BR1283 7+ 6+ 96.49 3mg / kg BR0438 5+ 4+ 70.74 3mg / kg

[0434] As shown in the table above, the TGI values ​​of the tested sample CB-20BK at a dose of 3 mg / kg in the FOLR1 and FOLH1 dual-expression models BR1283 and BR0438 were 96.49% and 70.74%, respectively, demonstrating excellent anti-tumor growth effects. Moreover, CB-20BK showed even better TGI in BR1283, which exhibited higher FOLR1 and FOLH1 expression.

[0435] [5. CB-20B] [exist] [CDX] [Pharmacodynamic studies in the model]

[0436] 1) Sample preparation:

[0437] Weigh out CB-20B lyophilized powder, dissolve it in PBS to prepare a sample stock solution, and dilute the stock solution with physiological saline for injection to the working concentration sample solution for later use.

[0438] 2) CDX Model Construction

[0439] Main cell lines: KB human oral epidermal carcinoma cells, PC-9 human lung cancer cells, and DU145 human prostate cancer cells.

[0440] Model construction: Resuscitate and culture cells, collect and count them, and inject them subcutaneously into the right limb of BALB / c-nude mice. When the tumor grows and expands to 80-160 mm3, administer the drug in groups or take rapidly expanded tumor blocks to transfer and expand the mouse tumor model.

[0441] 3) Grouped drug administration observation

[0442] Grouping: Once the tumors reached an average size of approximately 80-160 mm3, they were divided into a model control group and groups receiving different doses of medication.

[0443] Administration: Inject via tail vein.

[0444] Experimental observation and measurement: Routine monitoring after tumor inoculation included tumor growth and the impact of treatment on the animals' normal behavior. Specific content included the experimental animals' activity level, feeding and water intake, weight gain or loss (weight measured twice a week), and any abnormalities in the eyes, coat, or other areas.

[0445] Mouse body weight and the major and minor diameters (a and b) of the tumor were measured twice a week. Tumor volume (TV) was calculated using the formula: TV = 1 / 2 × a × b².

[0446] 4) Experimental Results and Analysis

[0447] Based on the changes in tumor volume in mice (Figures 6A-6C), it can be seen that CB-20B has a good inhibitory effect on mouse KB, PC-9 and DU145 cell line CDX tumor models.

[0448] [6. CB-18G] [exist] [CDX] [Pharmacodynamic studies in the model]

[0449] 1) Sample preparation:

[0450] Weigh out CB-18G lyophilized powder, dissolve it in PBS to prepare a sample stock solution, and dilute the stock solution with physiological saline for injection to the working concentration sample solution for later use.

[0451] 2) CDX Model Construction

[0452] Main cell lines: KB human oral epidermoid carcinoma cells, PC-9 human lung cancer cells, SPC-A1 human lung adenocarcinoma cells, CALU-3 human lung adenocarcinoma cells, and DU145 human prostate cancer cells.

[0453] Model construction: Resuscitate and culture cells, collect and count cells, inject cell fluid subcutaneously into the right limb of BALB / c-nude mice, and administer drugs in groups or take rapidly expanded tumor blocks to establish mouse tumor models when the tumor grows and expands to 80-160 mm3.

[0454] 3) Grouped drug administration observation

[0455] Grouping: Once the tumors reached an average size of approximately 80-160 mm3, they were divided into a model control group and groups receiving different doses of medication.

[0456] Administration: Inject via tail vein.

[0457] Experimental observation and measurement: Routine monitoring after tumor inoculation included tumor growth and the impact of treatment on the animals' normal behavior. Specific content included the experimental animals' activity level, feeding and water intake, weight gain or loss (weight measured twice a week), and any abnormalities in the eyes, coat, or other areas.

[0458] Mouse body weight and the major and minor diameters (a and b) of the tumor were measured twice a week. The tumor volume (TV) was calculated using the formula: TV = 1 / 2 × a × b².

[0459] 4) Experimental Results and Analysis

[0460] Based on the changes in mouse tumor volume (Figures 7A-7E), it can be seen that CB-18G has a good inhibitory effect on mouse KB, PC-9, SPC-A1, CALU-3 and DU145 cell line CDX tumor models.

[0461] [7.] [Complex Compound] [CB-1020] [、] [CB-1320] [and] [CB-1820] [Regarding human sources] [HPAF-II] [、] [NCI-H226] [and] [SCLC-21H] [Cell line subcutaneous allogeneic transplantation model] [CDX] [) tumor suppression experiment]

[0462] Experimental Objective: To study the pharmacodynamics of ligand complexes CB-1020, CB-1320, and CB-1820 in a CDX model.

[0463] Main CDX models: HPAF-II (human pancreatic cancer cells), NCI-H226 (human lung cancer cells), and SCLC-21H (small cell lung cancer cells).

[0464] Experimental protocol:

[0465] Prepared cells or tumor tissue blocks were subcutaneously inoculated into the anterior right limb of BALB / c-nude mice. When the tumor volume increased to 80-160 mm³, the mice were randomly divided into a model control group and a treatment group. Treatment began on day 1 of grouping, with the dosage adjusted according to the mice's most recent body weight. The administration was via tail vein injection at a volume of 10 μl / g. After treatment, monitoring included tumor growth and the effects of treatment on normal animal behavior, specifically the animals' activity level, food and water intake, weight gain or loss, and any abnormalities in the eyes, coat, or other areas. Mouse body weight was measured twice weekly after treatment, and the rate of weight change was calculated. Simultaneously, the tumor's long and short diameters were measured using calipers, and tumor volume, relative tumor proliferation rate, and tumor volume inhibition rate were calculated. The tumor volume formula was TV = 0.5 a × b², where a is the long diameter of the tumor and b is the short diameter. Table 22. Inhibitory effects of CB-1020, CB-1320, and CB-1820 on the CDX model and expression levels of receptor genes in each model (referencing data from Crown Bioscience). Model TRPV6 gene expression level GNRH1 gene expression level SSTR2 gene expression FOLH1 gene expression level TGI (%) (CB-1020) TGI (%) (CB-1320) TGI (%) (CB-1820) p-value dose HPAF-II + + + / - + / - 0.283 0.461 3mg / kg 0.445 0.153 5mg / kg 0.899 0.023 10mg / kg NCI­H226 + / - + + / - - 0.823 0.011 3mg / kg 0.962 0.006 3mg / kg 0.992 0.005 10mg / kg SCLC-21H 2+ 3+ 3+ - 0.995 0.131 3mg / kg 0.361 0.610 3mg / kg 0.985 0.134 10mg / kg

[0466] CB-1020 showed significant anti-tumor growth effects on human HPAF-II, CB-1320 on NCI-H226 and SCLC-21H, and CB-1820 on SCLC-21H cell line subcutaneous allogeneic transplantation models.

[0467] [8.] [Complex Compound] [CR19428] [Regarding human sources] [CALU-3] [、] [SCLC-21H] [and] [SPC-A1] [Cell line subcutaneous allogeneic transplantation model] [CDX] [) tumor suppression experiment]

[0468] Experimental Protocol: Prepared cells were subcutaneously inoculated into the anterior right limb of BALB / c-nude mice. When the tumor volume increased to 80-160 mm³, the mice were randomly divided into a model control group and a treatment group. Treatment began on day 1 of grouping, with the dosage adjusted according to the mice's most recent body weight. The treatment was administered via tail vein injection at a volume of 10 μl / g, twice weekly for 3 weeks. Post-treatment monitoring included tumor growth and the impact of treatment on normal animal behavior, specifically activity levels, food and water intake, weight gain or loss, and any abnormalities in the eyes, coat, or other areas. Mouse body weight was measured twice weekly after treatment, and the rate of weight change was calculated. Simultaneously, the tumor's long and short diameters were measured using calipers, and tumor volume, relative tumor proliferation rate, and tumor volume inhibition rate were calculated. The tumor volume formula was TV = 0.5 a × b², where a is the long diameter of the tumor and b is the short diameter. Table 23. Inhibitory effect of CR19428 on lung cancer PDX model Model TGI (%) Dosage / Time CALU-3 51 50mg / kg q2 / w*3 SCLC-21H 66 50mg / kg q2 / w*3 SPC-A1 75 50mg / kg q2 / w*3 SPC-A1 91 100mg / kg q2 / w*3

[0469] CR-19428 is a drug complex containing ligands for FOLR1 and FOLH1 and a drug effectively loaded with Dxd. As shown in the table above, it exhibits significant anti-tumor growth effects in subcutaneous allogeneic transplantation models of human CALU-3, SCLC-21H, and SPC-A1 cell lines.

[0470] [9.] [Complex Compound] [CBP-1018] Lung cancer [LU2505] [Model Validity Study()] [PDX] [Model)]

[0471] This experiment used the third-generation PDX model LU2505 (from an Asian female patient) for lung cancer, a rapidly growing tumor model. BALB / c nude mice with subcutaneous tumors, approximately 150 mm³ in volume, were divided into six groups: low, medium, and high doses of the test product; a control group consisting of small molecule MMAE and the targeting peptide 20BK-SM09; and a blank preparation control group, for a total of six groups (n=8 per group). Drug administration was performed on days 1, 8, and 15 after grouping, with a 14-day observation period following the last dose. The active ingredient in the test product CBP-1018 was CB-20BK, obtained by lyophilizing a mixture of CB-20BK and excipients. Table 24. Pharmacodynamic results of CBP-1018 for injection in the LU2505 lung cancer model (first trial) Test sample / Reference dose (mg / kg) weight (g) tumor volume (mm3) Tumor inhibition rate TGI% Tumor weight (mg) Blank preparation control group / 24.0 2219.62 / 2031.4(D22) CBP-1018 4.5 22.5 0.00 100.00 0.0(D29) 1.5 22.1 386.16 82.60 400.7(D29) 0.5 23.6 1634.50 26.36 2084.7(D26) 20BK-SM09 10 23.8 2024.75 8.78 1811.5(D22) MMAE 0.375 23.5 1132.61 48.97 1597.1(D29)

[0472] Note: 1) Due to animal welfare requirements, the average tumor volume within the group reached 2000 mm3, and the animals needed to be euthanized. Therefore, the euthanasia time varied for each group. 2) The data on body weight, tumor volume, and tumor inhibition rate are from day 22 (D22).

[0473] As shown in Table 24 and Figure 8A:

[0474] No abnormal clinical manifestations or deaths were observed in any of the groups of animals after administration; the weight of the animals in each group increased slowly.

[0475] Animals in the blank preparation control group, the 20BK-SM09 group, and the CBP-1018 low-dose group were euthanized on days 22, 22, and 26 (D26) respectively, because their average tumor volume exceeded 2000 mm3. Therefore, the body weight, tumor volume, and tumor inhibition rate data in Table 24 are all from D22.

[0476] The efficacy of CBP-1018 for injection showed a clear dose-related effect: the low-dose group was ineffective, while the medium and high-dose groups were effective. In the high-dose group, the tumors were completely cured by day 19 (D19) and no growth was observed by day 29 (D29).

[0477] No significant antitumor effect was observed in the 20BK-SM09 peptide group, suggesting that a single targeting peptide is insufficient to produce a clear antitumor effect.

[0478] The MMAE group showed a clear tumor-suppressing effect. The dose of 0.375 mg / kg was compared with that of CBP-1018 at 1.5 mg / kg containing 1 mole of MMAE. The comparison showed that CBP-1018 at 1.5 mg / kg was significantly better than the MMAE group, indicating the advantage of ligand targeting (82.60% vs 48.97%).

[0479] [10.] [Complex Compound] [CBP-1018] Lung cancer [LU1206] [Model Validity Study()] [PDX] [Model)]

[0480] This experiment used the 5th generation of the lung cancer PDX model LU1206 (from an Asian female patient), a rapidly growing tumor model. BALB / c nude mice with subcutaneous tumors, when the tumor volume was approximately 150 mm³, were divided into six groups: low, medium, and high dose groups of the test substance; a control group containing small molecule MMAE and the targeting peptide 20BK-SM09; and a blank preparation control group, for a total of 6 groups (8 mice per group). Drug administration was performed on days 1, 8, and 15 after grouping, and observation continued for 14 days after the last dose. Table 25. Pharmacodynamic results of CBP-1018 for injection in the LU1206 lung cancer model (first trial) Group dose (mg / kg) weight (g) Tumor volume (mm3) tumor volume Tumor inhibition rate (%) Tumor weight (mg) Tumor weight Inhibition rate (%) Blank preparation control group / 22.7 1216.67 / 856.14 / CBP-1018 4.5 23.4 31.44 97.42 13.55 98.42 1.5 23.7 301.64 75.21 185.10 78.38 0.5 22.7 1118.30 8.08 852.19 0.46 20BK-SM09 10 23.3 1158.80 4.76 914.36 -6.80 MMAE 0.375 23.6 778.32 36.03 437.73 54.53

[0481] As shown in Table 25 and Figure 8B:

[0482] No abnormal clinical manifestations or deaths were observed in any group of animals after administration. All animals were euthanized on day 29. The body weight of the animals in each group remained basically unchanged, and was slightly higher than on the first day of administration.

[0483] The efficacy of CBP-1018 for injection showed a clear dose-related effect: the low-dose group was ineffective (tumor inhibition rate of 8.08%), while the medium- and high-dose groups were effective, with tumor inhibition rates of 75.21% and 97.42%, respectively. The difference between the low and medium doses was relatively large.

[0484] The 20BK-SM09 peptide group did not show significant antitumor activity, suggesting that a single targeted peptide is insufficient to produce a clear antitumor effect in this model.

[0485] The MMAE group showed a clear antitumor effect. The dose of 0.375 mg / kg was CBP-1018 containing 1.5 mg / kg of MMAE; the comparison showed that CBP-1018 at 1.5 mg / kg was significantly better than the MMAE group, suggesting the advantage of ligand targeting (tumor volume inhibition rate: 75.21% vs 36.03%, tumor weight inhibition rate: 78.38% vs 54.53%).

[0486] [11.] [Tissue distribution in tumor-bearing mice] [PDX] [Model] [LU2505] [)]

[0487] Twelve female tumor-bearing mice inoculated with HuPrime® lung cancer LU2505 model tumors and twelve healthy male BALB / c nude mice were administered a single intravenous injection of 1.5 mg / 140 μCi / kg of [3H]CBP-1018 (isotope-labeled on MMAE). Three males and three females were harvested at 0.5, 2, 6, and 24 hours, anesthetized in induction boxes with an appropriate amount of carbon dioxide, and blood was collected via cardiac puncture. The mice were then immediately euthanized for blood collection. Table 26. Total radioactivity in various tissues at different time points after a single intravenous administration of [3H]CBP-1018 organize Radioactivity concentration (ng Eq. / g) 0.5 hours 2 hours 6 hours 24 hours (%Cmax) ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ tumor 763 / 643 / 566 / 413 / esophagus 915 784 628 508 521 172 286 91.7 Body fat 341 479 288 276 231 177 67.2 43.7 skeletal muscle 321 311 207 137 188 95.5 90.8 61.5 spleen 744 693 701 524 671 421 253 163 stomach wall 658 623 414 370 460 259 211 123 Whole brain 66.3 67.9 53.8 39.5 82.0 48.2 76.4 68.3 testis and epididymis 350 494 279 298 183 248 92.3 64.8 Heart 785 795 415 326 219 143 114 69.2 lungs 1493 1807 997 920 416 392 109 93.7 kidney 5682 6118 3300 4197 1227 1391 241 238 liver 1423 1306 1257 865 1138 599 712 518 Small intestine wall 726 778 641 816 490 334 210 90.9 large intestine wall 550 520 672 823 1074 490 215 128 Whole blood 2087 3194 684 691 211 132 86.5 64.6 plasma 7372 7610 1844 1760 495 306 214 140

[0488] As shown in the table above, no difference in tissue distribution was observed between males and females. After administration, the drug was mainly distributed in the kidneys, whole blood (primarily in plasma), liver, and lungs. The highest concentrations were observed in all tissues at 0.5 hours, followed by rapid elimination, with the liver and tumors showing the slowest elimination. This slow elimination within tumors may explain the efficacy advantage of CBP-1018.

[0489] [12.] [Rat Excretion Test]

[0490] Six normal SD rats, half male and half female, were administered [3H]CBP-1018 via a single tail vein injection at 0.75 mg / 70 μCi / kg. Urine, feces, cage rinsing / cleaning fluid, and carcasses were collected from whole rats before and 0–168 hours after administration, and bile, urine, feces, and cage rinsing / cleaning fluid were collected from BDC rats before and 0–72 hours after administration. All samples were frozen at -10℃ to -30℃.

[0491] After adding an appropriate amount of scintillation fluid to each sample and mixing thoroughly, the radioactivity level was determined using a liquid scintillation counter. The radioactivity levels measured in samples such as bile, urine, feces, cage rinsing and cleaning fluids, and corpses were used to calculate the percentage of the administered dose. The radioactivity level in plasma samples was used to calculate the total radioactivity per gram of sample. The main pharmacokinetic parameters of the total plasma radioactivity were calculated using WinNonLin software (version 7.0, Pharsight) according to a non-compartmental model. Table 27. Results of material balance study after administration to whole rats Gender (number of animals) urine (%) stool (%) Cage flushing / cleaning Wash solution (%) corpse (%) Total recovery rate (%) Females (n=3) 50.26±6.11 23.93±0.98 5.70±2.63 4.51±1.19 84.40±4.02 Male (n=3) 53.60±3.68 20.61±1.74 4.24±2.16 4.83±0.42 83.28±0.71 female and male (n=3 females and 3 males) 51.93±4.87 22.27±2.21 4.97±2.30 4.67±0.82 83.84±2.65

[0492] As shown in the table above, CBP-1018 is mainly excreted in urine, accounting for approximately 57% of the total dosage; less than 25% is excreted in feces. The fact that it is mainly excreted in urine through the kidneys is consistent with the tissue distribution in nude mice, where it is abundantly distributed in the kidneys.

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

Claims

1. A complex compound or a pharmaceutically acceptable salt thereof, said complex compound or a pharmaceutically acceptable salt thereof comprising an effective load and two targeting molecules, wherein said two targeting molecules are a synergistic molecule and a prostate-specific membrane antigen ligand, wherein said synergistic molecule binds to FOLR1, said synergistic molecule is folic acid or an analogue thereof, and wherein said prostate-specific membrane antigen ligand comprises the following structures: , , or.

2. The complex compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the folic acid analog is selected from the group consisting of 5-methyltetrahydrofolate, 5-methoxytetrahydrofolate, methotrexate, and 5,10-methylenetetrahydrofolate.

3. The complex compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the complex compound or a pharmaceutically acceptable salt thereof comprises one, two, three, four or more effective loads.

4. The complex compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the effective load is selected from the group consisting of small molecule compounds, nucleotides, peptides and proteins.

5. The complex compound or a pharmaceutically acceptable salt thereof as claimed in claim 4, wherein the effective load is a small molecule compound.

6. The complex compound or a pharmaceutically acceptable salt thereof as claimed in claim 5, wherein the small molecule compound is selected from the group consisting of: camptothecin and any derivative thereof, aurestatin and any derivative thereof, maytansine and any derivative thereof, radioisotope complexes, cyclooxygenase-2 inhibitors, paclitaxel and any derivative thereof, epothilone and any derivative thereof, bleomycin and any derivative thereof, dermatomycin and any derivative thereof, purcamycin and any derivative thereof, and mitomycin C.

7. The complex compound or a pharmaceutically acceptable salt thereof as claimed in claim 6, wherein the small molecule compound is camptothecin and any derivative thereof, aureatine and any derivative thereof, a radioisotope complex, or a cyclooxygenase-2 inhibitor.

8. The complex compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the payload is linked to at least one of the target molecules via a linker.

9. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker is a peptide linker, a disulfide linker, a pH-dependent linker, or a combination of the above linkers.

10. The complex compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein the peptide linker is cleaved by protease or reductive cleavage under specific physiological conditions.

11. The complex compound or a pharmaceutically acceptable salt thereof according to claim 9 or 10, wherein the peptide linker is selected from the group consisting of: cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-lysine, cysteine-glutamic acid, aspartic acid-aspartic acid, and aspartic acid-aspartic acid-lysine, optionally, the carboxylic acid in the above amino acids is acetylated.

12. The complex compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein the disulfide linker is selected from the group consisting of DMDS, MDS, DSDM and NDMDS.

13. The complex compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein the pH-dependent linker is maleic aconitine.

14. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a structure or a combination of a structure and a peptide linker:

15. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a structure or a combination of a structure and a peptide linker:

16. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a structure or a combination of a structure and a peptide linker:

17. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a structure or a combination of a structure and a peptide linker:

18. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a structure or a combination of a structure and a peptide linker:

19. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a structure or a combination of a structure and a peptide linker:

20. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a structure or a combination of a structure and a peptide linker:

21. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a structure or a combination of a structure and a peptide linker:

22. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a structure or a combination of a structure and a peptide linker:

23. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a structure or a combination of a structure and a peptide linker:

24. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a structure or a combination of a structure and a peptide linker:

25. The complex compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the linker comprises a structure or a combination of a structure and a peptide linker:

26. The complex compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the two target molecules are connected by a spacer region.

27. The complex compound of claim 26 or a pharmaceutically acceptable salt thereof, wherein the spacer region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1-14, Arg-Arg, Ala-Ser-Asn, Ala-Ala-Ala, Ser-Ser-Arg, Pro-Arg, and Pro-Leu-Gly.

28. A complex compound or a pharmaceutically acceptable salt thereof, wherein the complex compound is selected from the group consisting of: (CB-20B), (CB-20BK), (CB-60S), (CB-60SK), (CB-20C), (CB-1020), (CB-1320), (CB-1820), (CR19428), and (20R-SM09) (CB-20R), wherein M is a radioactive isotope.

29. A pharmaceutical composition comprising a complex compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-28, and a pharmaceutically acceptable carrier.

30. The pharmaceutical composition according to claim 29, wherein, The composition is intended for intravenous, subcutaneous, oral, intramuscular, or intraventricular administration.

31. Use of a complex compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-28, or a pharmaceutical composition according to claim 29 or 30, in the preparation of a medicament for delivery of a payload to a subject in need; wherein the subject in need has a disease selected from the group consisting of cancer, immune diseases, cardiovascular diseases, metabolic diseases, and neurological diseases.

32. Use of a complex compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-28, or a pharmaceutical composition according to claim 29 or 30, in the preparation of a medicament for treating a disease in a subject; wherein the disease is selected from the group consisting of cancer, immune diseases, cardiovascular diseases, metabolic diseases, and neurological diseases.

33. The use according to claim 31 or 32, wherein said cancer is selected from the group consisting of: prostate cancer, breast cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, testicular cancer, skin cancer, lymphoma, and multiple myeloma.

34. The use as described in claim 31 or 32, wherein the immune disease is an autoimmune disease.

35. The use as described in claim 34, wherein the autoimmune disease is selected from the group consisting of connective tissue diseases, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

36. The use according to claim 31 or 32, wherein the cardiovascular disease is selected from the group consisting of: angina pectoris, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia and congenital heart disease.

37. The use according to claim 31 or 32, wherein the metabolic disease is selected from the group consisting of diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.

38. The use as described in claim 31 or 32, wherein the neurological disease is selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma, and epilepsy.

39. The use according to claim 31 or 32, wherein said use further comprises administering one or more therapeutic agents in combination with the complex compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

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