Camptothecin derivatives that bind to the DDX5 protein and their prodrugs

Abstract

The present invention relates to: (a) an active camptothecin derivative represented by Chemical Formula 1, which is designed to bind to DDX5 protein and an E3 ligase; (b) a prodrug thereof, preferably an antibody-drug conjugate (ADC) thereof, which is designed to release the active camptothecin derivative (a) from an in vivo target site; or (c) a ligand-containing complex targeting DDX5 protein, in which the type I topoisomerase inhibitory ability in the active camptothecin derivative (a) or the FL118 compound of Chemical Formula 2 is inactivated via a linker linkage. The active camptothecin derivative designed according to the present invention can bind to DDX5 protein intracellularly and induce cell death through DDX5 protein degradation.

Description

Technical Field

[0001] The present invention relates to (a) an active camptothecin derivative represented by the following Chemical Formula 1, which is designed to bind to a DDX5 protein and an E3 ligase; (b) a prodrug thereof, preferably a carrier-drug complex (CDC), particularly an antibody-drug complex (ADC), which is designed to release the active camptothecin derivative (a) from an in-vivo target site; or (c) a ligand-containing complex that targets a DDX5 protein, in which the type I topoisomerase inhibitory ability in the active camptothecin derivative (a) or the FL118 compound of Chemical Formula 2 is inactivated via a linker linkage.

[0002] The active camptothecin derivative designed according to the present invention can bind to the DDX5 protein intracellularly and induce cell death through DDX5 protein degradation.

Background Art

[0003] Cancer Genes originally exist to regulate normal cell functions, but when mutations occur or they are abnormally regulated, they refer to genes that can cause cancer. One of the normal cell functions is the cell death function, and related to this, there are cell death promoting factors and cell death inhibiting factors.

[0004] For example, Cancer Genes include (i) oncogenes that actively promote cell division when mutations occur, and (ii) tumor suppressor genes that do not suppress cell division when mutations occur. Examples of oncogenes include Src, EGFR, HER2, RAS, APC, BCL-2, etc., and examples of tumor suppressor genes include p53, BRCA, Rb, PTEN, BAX, etc.

[0005] Cancer is a group of diseases in which the cell cycle of growth and division is not regulated. As shown in Figure 29, cancer may occur when the programmed cell death (e.g., apoptosis) mechanism is damaged.

[0006] Cancer occurs due to the occurrence of mutations in genes whose protein products are involved in the regulation of the cell cycle. Many cancers are associated with the overexpression of specific genes or the abnormal activities of mutant protein products (Oncogenes).

[0007] A single mutation generally does not induce cancer. Generally, various genes that regulate cell growth are mutated before the cancerous state occurs.

[0008] HER2-positive breast cancer, in which HER2 is abnormally highly expressed in breast epithelial cells, accounts for about one-fourth of all breast cancer patients. Compared with female hormone-related breast cancer, it has a higher recurrence rate and a worse prognosis during chemotherapy with chemotherapeutic agents. Herceptin (Herceptin, ingredient name: trastuzumab), a HER2-targeted therapeutic agent, treats breast cancer by reducing the number of HER2 on the cell surface and assisting immune cells to kill HER2-positive breast cancer cells.

[0009] HER2-positive breast cancer is breast cancer that harbors amplification or overexpression of the HER2 (ErbB2) cancer gene, which is a member of the ErbB receptor. Trastuzumab (Herceptin) has increased the therapeutic effect of HER2-positive breast cancer, but HER2-positive breast cancer shows a more aggressive aspect compared to other breast cancers, and more than half of the patients are refractory to existing targeted therapies or develop resistance during treatment. ion).

[0010] The ligands of ErbB receptors attached to the cell membrane include NRG1 and HB-EGF. The activated ErbB ligands bind to these receptors (EGFR, HER3), catalyze phosphorylation (P) by receptor-receptor binding (EGFR-HER2, HER2-HER3), and induce intracellular growth and proliferation signals. Such excessive activation of ErbB receptors confers resistance to anti-HER2 therapeutic agents.

[0011] Note that dysregulated transcription factors can reduce the sensitivity of cancer cells to chemotherapy and radiotherapy. Drug resistance in cancer treatment can be intrinsic or acquired. Essential drug resistance in cancer treatment may be caused by abnormally expressed transcription factors that are critical regulators of cell growth and cell death. Abnormal overexpression of NF-κB, STAT3, HIF-1, AR, or ER and inactivation of p53 and FOXO3a in tumor tissues protect cancer cells from cell death by anticancer drugs and exhibit essential drug resistance.

[0012] However, chemotherapy and radiotherapy also induce acquired drug resistance through the activation of dysregulated transcription factors. It has been revealed that ionizing radiation can stimulate the activation and induction of various other transcription factors including STAT3, NF-κB, and HIF-1. Upregulation of NF-κB and STAT3 induces the transcription of anti-apoptotic genes in cancer cells, promotes cell cycle entry and proliferation of cancer cells, and generates radiation resistance. Also, upregulation of the transcription factors NF-κB and HIF-1 by radiation can induce genes responsible for the maintenance of EMT cells and cancer stem cells that are resistant to cancer treatment. In addition, activation of the transcription factor STAT3 also promoted drug resistance to cancer treatment. Feedback activation of STAT3 by chemotherapy and radiotherapy has been revealed to be a molecular mechanism of cancer treatment resistance. Therefore, co-targeting dysregulated transcription factors and their major targets can be a promising approach to overcome drug resistance in cancer treatment.

[0013] The STAT (abbreviation for signal transducer and activator of transcription) transcription factor family includes STAT1, STAT2, STAT3, STAT4, STAT5, and STAT6. STAT proteins can be activated by JAK kinases when stimulated by growth factors, cytokines, interferons, or oncogenes and can regulate interferon signaling. The DNA-binding domain of STAT is an immunoglobulin-like structure that mediates binding to specific DNA target sequences. Phosphorylation of STAT3 by JAK kinases during activation activates dimerization. Next, the dimer moves to the nuclear compartment, binds to the sequences of the target promoter, and induces the expression of the target gene. STAT3 target genes include Survivin, bcl-xl, mcl-1, Cyclin D1, MMP2, MMP9, VEGF, Myc, Sox2, etc. STAT proteins regulate many biological processes including cell growth, cell death, differentiation, and immunity. Within the STAT system, STAT3 and STAT5 are involved in cancer progression.

[0014] Dysregulated STAT3 and activated STAT5 are considered oncogenes, increasing angiogenesis and improving the survival of cancer cells. In a study using many samples from the cBioPortal database, STAT3 was shown to be more frequently overexpressed in lung cancer, ovarian cancer, gastric cancer, hematopoietic cancer, and brain cancer compared to normal tissues, and the over expression of STAT3 was associated with a low overall survival rate. Phosphorylation of STAT3 by JAK kinases is the first step in STAT3 activation. In another study involving 90 glioblastoma patients, it was found that high p-STAT3 values were significantly associated with a decrease in progression-free survival and overall survival. Multivariate survival analysis suggested that high p-STAT3 levels could play a role as an important prognostic indicator for poor progression-free survival and overall survival.

[0015] Inhibitors of type I topoisomerase (such as irinotecan and topotecan) have an anti-cancer mechanism that has been verified for efficacy and safety in clinical practice, and excellent anti-cancer efficacy has been verified in clinical practice for various refractory solid cancers such as colorectal cancer, lung cancer, breast cancer, and ovarian cancer.

[0016] In this connection, camptothecin derivatives, which are low-molecular-weight compounds that inhibit type I topoisomerase (topoisomerase-1) and exhibit anti-tumor effects, are known.

[0017] Camptothecin (CPT) has low solubility in water. To prepare for clinical trials, the National Cancer Institute (NCI) produced a water-soluble sodium salt (NSC100880). Phase I and II clinical trials were not completed due to the high toxicity (hemorrhagic cystitis, gastrointestinal toxicity, such as nausea, vomiting, diarrhea, and myelosuppression, especially leukopenia and thrombocytopenia) exhibited by the said compound.

[0018] Subsequently, a number of CPT analogs were synthesized to obtain compounds with lower toxicity and higher water solubility. There are two drugs, irinotecan (CPT-11) and topotecan.

[0019] Irinotecan (irinotecan, CPT-11), jointly developed by Dallchi and Yakult in Japan, was the world's first camptothecin-based anti-cancer drug in 1994. Its effectiveness was proven for lung cancer (small cell, non-small cell lung cancer), and it was launched in Europe and Japan. In 1995, its effectiveness for colorectal cancer and breast cancer was further proven. Also, topotecan, developed by Glaxo Smith Kline, had its effectiveness for metastatic ovarian cancer approved by the US FDA in April 1995 and was launched. Recently, CKD-602 (belotecan), a new camptothecin derivative developed in South Korea, successfully overcame the toxicity due to the existing poor solubility as a water-soluble substance in addition to the effect of a strong type I topoisomerase inhibitor.

[0020] All camptothecin derivatives confirmed to date contain a parent structure with five rings essential for cytotoxicity (Figure 1). Structurally, the E-ring and the A- and B-ring regions were identified as important sites. Camptothecin contains a pentacyclic lactone structure in the E-ring essential for cytotoxicity. Among these, the lactone group and the alpha-hydroxyl group located at the 20th carbon of the E-ring are important for the stability of the type I topoisomerase-DNA byproduct, and it has been proven that the modification of the A- and B-rings can increase water solubility and activity. Modifications on the first ring, for example, in the case of the drugs mentioned above, have been proven to increase solubility in water and allow for greater tolerance.

[0021] CKD-602 also attempted substitution at the B-ring position of carbon 7 to increase water solubility and anti-cancer effects. Lee et al. stated that CKD-602 has superior anti-cancer effects compared to camptothecin and topotecan in a wide range of cancer cell lines. Also, in the L1210 leukemia nude mouse model, it was confirmed that the maximum tolerated dose (MTD) is 25 mg / kg and it is a relatively safe drug. The side effects of commonly known camptothecin-based drugs are significant and can be classified into hematological side effects and non-hematological side effects. As hematological side effects Then, there are neutropenia with fever, sepsis, bleeding, etc. As non-hematological side effects, there are skin-related side effects such as nausea, vomiting, hair loss, and toxicity to the gastrointestinal tract, kidneys, and nervous system. Kim et al. found no abnormal drug reactions except for increased gastric juice secretion even when administering a dose more than 10 times higher than the clinically applicable dose among the side effects of such camptothecin drugs in the animal study of CKD-602. Also, recently, in Korean clinical studies, relatively stable evidence has been shown, such as only reversible and adjustable levels of side effects of neutropenia and leukopenia being reported rather than severe systemic toxicity. However, until now, it has been used restrictively mainly for the treatment of patients who have failed standard chemotherapy or for whom standard chemotherapy cannot be performed, that is, refractory or recurrent ovarian cancer and colorectal cancer that have recurred or deteriorated after standard treatment and for whom it is judged difficult to see the effect of further anticancer chemotherapy or surgery, the treatment of refractory or recurrent limited disease small cell lung cancer that has failed primary chemotherapy, and the treatment of extensive disease small cell lung cancer.

[0022] SN-38 is a potent topoisomerase I inhibitor with an IC 50 value in the nanomolar range in various cell lines. It is the active form of irinotecan, a prodrug used in the treatment of colorectal cancer, and also shows activity in lung cancer, breast cancer, and brain cancer. In the case of Trop-2-SN-38 ADC, although it has been successfully developed in many cancer tumors such as TNBC, bladder cancer, and gastric cancer, there are still remaining resistance problems due to overexpression of drug efflux transporters, epigenetic silencing of Top1, and increased anti-apoptosis proteins.

[0023] Incidentally, Daiichi Sankyo used DXD (exatecan), a cytotoxic drug that is about 10 times more active in cancer cells than SN-38, in the development of Enhertu (registered trademark). DXD has good solubility, is relatively safe, and has a high peripheral cell killing effect, so it has advantages in the treatment of heterogeneous tumors. However, its half-life, which can reduce the off-target effect, is short. DXD was covalently conjugated to the cysteine residue of the anti-HER2 antibody with a maleimide linker, and the homogeneous DAR value reached 8. Despite the high DAR value, DXD was highly stable, with only 2.1% released from plasma over 21 days (Ogitani et al., 2016). Enhertu was approved by the US FDA in 2019, and the target patients are adult patients with unresectable metastatic Her2-positive breast cancer who have a history of receiving HER2-targeted treatment two or more times in the past.

[0024] Camptothecin derivatives such as SN-38 and Dxd (derivatives of the clinical-stage topoisomerase I inhibitor exatecan) have recently been successfully developed as new payloads for antibody-drug conjugates (ADCs). As a result, two ADCs using camptothecin-based payloads (Trodelvy (sacituzumab govitecan) containing the SN-38 payload and Enhertu (trastuzumab deruxtecan) containing the xd payload) have recently received FDA approval. Such camptothecin-based payloads have moderate cytotoxicity compared to existing ultra-toxic payload ADCs (DAR = 2 or 4) such as MMAE and MMAF, but due to their excellent safety profiles, higher drug-antibody ratios (DARs) such as 8 can be used. In addition, a linker system with a faster drug release profile such as the CL-2A or GGFG linker system can be used via such camptothecin-based payloads. New ADCs with a wider therapeutic range have been developed with such combinations of components.

[0025] ​Antibody-drug conjugates (ADCs) that use camptothecin derivatives such as trastuzumab deruxtecan and enhertu as payloads have succeeded in delivering camptothecin compounds that exhibit potent anti-cancer efficacy selectively only in cancer cells, resulting in excellent anti-cancer efficacy without severe systemic side effects. However, they still show various deficiencies. Most typically, resistance occurs due to overexpression of the ABCG2 drug efflux pump. Many camptothecin derivatives are rapidly excreted extracellularly by ABCG2, which shows that the therapeutic efficacy is severely reduced in cancer cells overexpressing ABCG2 compared to cancer cells that do not express ABCG2 or express it at low levels at normal levels. Such overexpression of ABCG2 can be a major cause of reduced therapeutic efficacy not only in small molecule camptothecin compounds such as irinotecan and topotecan, but also in ADCs such as trastuzumab deruxtecan and enhertu that use camptothecin compounds as payloads.

Summary of the Invention

Problems to be Solved by the Invention

[0026] The present invention aims to provide a camptothecin derivative designed to bind to DDX5 protein and E3 ligase as a drug candidate for killing target cells.

[0027] The inventors synthesized the FL118 compound (Figure 2), a camptothecin-based drug having a dual mechanism of action (dual MoA) that degrades the tumor protein (oncoprotein) DDX5 (p68) along with excellent type I topoisomerase inhibitory ability, and continued various studies on its physicochemical properties. However, the FL118 compound had limitations in dosage form development due to limitations in its physicochemical properties.

[0028] Based on this, the inventors attempted to design and synthesize a new structure of camptothecin derivative that exhibits a dual mechanism of action (dual MoA) from the viewpoints of type I topoisomerase inhibition and DDX5 degradation, which are the advantages of FL118, based on the structure of FL118 that is differentiated from the SN38 drug.

[0029] Therefore, the present invention aims to newly design and provide an active camptothecin derivative having a dual mechanism of action (dual MoA) that degrades the oncoprotein DDX5 together with type I topoisomerase inhibitory ability and / or a carrier drug conjugate (CDC), particularly an antibody-drug conjugate (ADC), which is a prodrug designed to release the active camptothecin derivative in vivo.

[0030] Furthermore, the present invention aims to provide an anticancer mechanism as a new modality that kills cancer cells and / or surrounding cells before intrinsic or acquired drug resistance by the active camptothecin derivative binding to DDX5 protein intracellularly and inducing cell death through DDX5 protein degradation together with the bystander effect.

Means for Solving the Problems

[0031] The first aspect of the present invention provides (a) an active camptothecin derivative represented by the following Chemical Formula 1, designed to bind to DDX5 protein and an E3 ligase; or (b) a prodrug thereof, preferably a carrier drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC), designed to release the active camptothecin derivative (a) from a target site in vivo; or (c) a ligand-containing conjugate targeting DDX5 protein, in which the type I topoisomerase inhibitory ability in the active camptothecin derivative (a) or the FL118 compound of Chemical Formula 2 is inactivated via a linker linkage.

[0032]

Chem.

[0033] X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and X1 and X3 may be the same or different. X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X1, (X2)n and X3 can form a 6- or 7-membered ring (value of n = 1 - 2), Y1, Y2 and Y3 can each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0034] In a ligand-containing complex that targets the DDX5 protein, the ligand that targets the DDX5 protein can be a ligand that binds to the DDX5 protein.

[0035] The active camptothecin derivative can be designed to adjust its hydrophobicity or cell membrane permeability as desired through X1, (X2)n, X3, Y1, Y2 and / or Y3 modifications in Chemical Formula 1, so that the bystander effect is exerted or the degree of this effect is controlled in cancer tissue.

[0036] Preferably, the active camptothecin derivative (a) designed to bind to the DDX5 protein and the E3 ligase can / may act as a binder of the ligand that binds to the E3 ligase by a molecular glue degrader; can / may kill target cells expressing the DDX5 protein through the molecular glue degrader mechanism of action (MoA); and can have a mechanism of action (MoA) that degrades the tumor protein DDX5 together with the type I topoisomerase inhibitory ability.

[0037] The second aspect of the present invention provides a pharmaceutical composition or a cancer diagnostic composition for preventing or treating cancer, containing the active camptothecin derivative (a) of the first aspect, its prodrug (b), preferably a carrier-drug complex (CDC), more preferably an antibody-drug complex (ADC) or a ligand-containing complex (c) that targets the DDX5 protein.

[0038] For example, it may be administered for treating HER2-positive cancer, breast cancer, lung cancer, and colorectal cancer.

[0039] Preferably, the pharmaceutical composition for cancer prevention or treatment can be used as a primary therapeutic agent after cancer diagnosis or administered to an individual who (over)expresses DDX5 in cancer tissue.

[0040] A third aspect of the present invention is a method for producing a ligand-containing complex that targets DDX5 protein, wherein (a) an active camptothecin derivative designed to bind to DDX5 protein and an E3 ligase, (b) a prodrug thereof designed to release the active camptothecin derivative (a) from an in vivo target site, preferably a carrier-drug complex (CDC), more preferably an antibody-drug complex (ADC), or (c) the active camptothecin derivative (a) or the FL118 compound of Chemical Formula 2, in which the type I topoisomerase inhibitory ability is inactivated via a linker linkage, the method comprising the step of designing, selecting, or synthesizing a compound such that the active camptothecin derivative (a) contains the camptothecin-based skeleton represented by Chemical Formula 1 as a nucleophile.

[0041] For example, the step of designing or selecting a compound such that the active camptothecin derivative (a) contains the camptothecin-based skeleton represented by Chemical Formula 1 as a nucleophile comprises: (1) Selecting / selecting an active camptothecin derivative designed to have a mechanism of action (MoA) that inhibits type I topoisomerase and / or a mechanism of action (MoA) that degrades the oncoprotein DDX5 from a compound library containing the camptothecin-based skeleton represented by Chemical Formula 1 as a nucleophile. (2) It may be a stage of confirming, through in vitro experiments and / or in vivo experiments, whether a compound containing a camptothecin-based skeleton represented by Chemical Formula 1 as a nucleophile inhibits type I topoisomerase and / or degrades oncoprotein DDX5.

[0042] A fourth aspect of the present invention is: (a) an active camptothecin derivative that binds to DDX5 that acts as an oncoprotein in cells and induces cell death through DDX5 proteolysis; (b) its prodrug designed to release the active camptothecin derivative (a) in vivo, preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC); or (c) a method for producing a ligand-containing conjugate that targets DDX5, in which the type I topoisomerase inhibitory ability in the active camptothecin derivative (a) is inactivated through a linker linkage, including a first step of selecting the active camptothecin derivative (a) from the group consisting of the compounds of Chemical Formula 3, the compounds of Chemical Formula 4, the compounds of Chemical Formula 5, the compounds of Chemical Formula 6, the compounds of Chemical Formula 7, the compounds of Chemical Formula 8, the compounds of Chemical Formula 9, and their isomers; and optionally, a second step of selecting its prodrug (b) designed to release the active camptothecin derivative (a) selected in the first step in vivo. A production method is provided, characterized by including these steps.

[0043]

Chem.

[0044]

Chem.

[0045]

Chem.

[0046] [Chem.]

[0047] [Chem.]

[0048] [Chem.]

[0049] [Chem.]

[0050] The production method of the third or fourth aspect may further include a step of identifying a group of patients to whom the active camptothecin derivative (a) or its prodrug (b) is to be administered by quantitatively or qualitatively analyzing the intracellular DDX5 protein using a ligand-containing complex (c) that targets the DDX5 protein in which the type I topoisomerase inhibitory ability in the FL118 compound of Chemical Formula 2 or the active camptothecin derivative (a) is inactivated via a non-cleavable linker in tissue biopsy or liquid biopsy.

[0051] The fifth aspect of the present invention provides a compound represented by the following Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8, or Chemical Formula 9, or a pharmaceutically acceptable salt thereof.

[0052] The sixth aspect of the present invention is a compound represented by the following Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8, or Chemical Formula 9, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof , preferably a carrier-drug complex (CDC), more preferably a pharmaceutical composition for preventing or treating cancer containing an antibody-drug complex (ADC).

[0053] [Chemistry]

[0054] [Chemistry]

[0055] [Chemistry]

[0056] [Chemistry]

[0057] [Chemistry]

[0058] [Chemistry]

[0059] [Chemistry]

[0060] [Chemistry]

[0061] [Chemistry]

[0062] [Chemistry]

[0063] The seventh aspect of the present invention is: (a) an active camptothecin derivative represented by the following Chemical Formula 1, which is designed to bind to the DDX5 protein and an E3 ligase; or (b) its prodrug, preferably a carrier-drug complex (CDC), more preferably an antibody-drug complex (ADC), which is designed to release the active camptothecin derivative (a) from the in-vivo target site There is provided a pharmaceutical composition for cell killing, which contains , preferably a carrier-drug complex (CDC), more preferably an antibody-drug complex (ADC).

[0064] The eighth aspect of the present invention is: (a) an active camptothecin derivative represented by the following Chemical Formula 1, which is designed to bind to the DDX5 protein and an E3 ligase; or (b) its prodrug, preferably a carrier-drug complex (CDC), more preferably an antibody-drug complex (ADC), which is designed to release the active camptothecin derivative (a) from the in-vivo target site, and is administered in a dosage that can stimulate antigen-presenting cells (APCs) through cancer cell death and induce an anti-tumor immune response, thereby providing an anti-cancer dosage form.

[0065] The ninth aspect of the present invention is a method for producing an active camptothecin derivative (a)-based anticancer agent, which controls the bystander effect and / or ADME profile of the active camptothecin derivative (a) so that T cell exhaustion does not occur due to chronic antigen exposure or is removed quickly or slowly through lymphatic drainage by controlling the hydrophobicity provision and / or aggregation to the desired degree. The active camptothecin derivative (a)-based anticancer agent is: (a) an active camptothecin derivative designed to bind to DDX5 protein and an E3 ligase with a molecular glue degrader; or (b) a prodrug thereof designed to release the active camptothecin derivative (a) from an in vivo target site, preferably a carrier-drug complex (CDC), more preferably an antibody-drug complex (ADC). The anticancer agent form contains the active camptothecin derivative (a) or its prodrug (b). The method for producing an anticancer agent is characterized by including the step of designing the active camptothecin derivative (a) or its prodrug (b) to contain a camptothecin-based skeleton represented by the following Chemical Formula 1 as a nucleophile, or selecting a compound designed in this way, or synthesizing a compound designed in this way.

[0066] Hereinafter, the present invention will be described. As used herein, a drug is any substance used for the diagnosis, cure, mitigation, treatment, or prevention of a disease (excluding food or devices) and used to affect the structure or function of the body. For example, all chemical or biological substances that affect the body and body metabolism. Preferably, as long as it binds to the DDX5 protein, it belongs to the category of drugs.

[0067] For example, the drug of the present invention can be an active camptothecin derivative or its prodrug, a payload of a prodrug such as targeted drug delivery (e.g., a drug carrier complex) that is an active camptothecin derivative, or a camptothecin derivative or its prodrug as a diagnostic agent.

[0068] In order for a drug to act effectively in vivo, the concentration of the drug at the target site in the body must be maintained within the therapeutic range for a certain period or more (Example 8, Table 6, Figure 23). If there is an excessive amount of the drug in the body, it will exhibit toxicity, and if there is too little at the target site, the therapeutic effect will not appear.

[0069] Drug efficacy means remaining in the body without being decomposed within the expected time showing an effect on the target indication (Example 8, Table 6, Figure 23). If the metabolic rate is low, the blood concentration maintenance time will be long, so the efficacy duration will be long.

[0070] In addition, the in vivo efficacy and in vivo side effects of an anticancer agent are closely related to the absorption, distribution, metabolism, and excretion (ADME) characteristics of the anticancer agent. The ADME of a drug determines the pharmacokinetics that describes the way the drug moves through the body and interacts with tissues and organs 。

[0071]

[0071] The absorption of a drug can affect efficacy and side effects. A drug that is not well absorbed cannot reach the therapeutic concentration in the target tissue, and its efficacy can be reduced. On the other hand, a drug that is well absorbed can increase systemic exposure and cause non-target side effects.

[0072] The distribution of a drug can also affect efficacy and side effects. A drug that does not distribute well to the target tissue may have no effect, and a drug that distributes widely to non-target tissues can induce toxicity in that tissue. Also, a drug with a high binding affinity to plasma proteins will have a reduced distribution to the target tissue and its efficacy can be reduced.

[0073] The distribution of drugs in the body can be affected by physicochemical properties including size, charge, and lipophilicity. The ability of a drug to penetrate tumor tissue can also be affected by factors such as the microenvironment of the tumor, including blood flow and cell density. Some anticancer drugs can accumulate in specific tissues and induce toxic effects.

[0074] Drug metabolism can also affect both efficacy and side effects. Drugs that are metabolized rapidly can have reduced efficacy due to a short half-life, and drugs that are metabolized slowly can have increased toxicity due to accumulation. Also, some metabolites can be more toxic than the parent drug and can cause side effects.

[0075] The rate and route of excretion can also affect both the efficacy and side effects of a drug. Drugs that are removed rapidly have a short half-life and can have reduced efficacy, and drugs that are removed slowly can accumulate and induce toxicity. Also, some drugs are excreted unchanged or as active metabolites, and long-term exposure or increased exposure can increase the risk of side effects.

[0076] Therefore, it is essential to analyze the ADME properties of anticancer drugs to optimize efficacy and minimize side effects in vivo. That is, when evaluating and optimizing the ADME profile, the therapeutic index of such drugs can be improved, leading to better outcomes for cancer patients.

[0077] An anti-cancer formulation or anti-cancer composition refers to a drug or therapy designed to treat cancer or prevent the growth and spread of cancer cells in the body. An anti-cancer formulation may include a combination of drugs, chemical compounds, and / or biological agents that target cancer cells by interfering with their ability to grow and divide.

[0078] The dosage form of an anti-cancer drug can reflect a complex process involving various stages, including drug discovery, pre-clinical development, clinical trials, and regulatory approval. The goal is to develop a safe and effective treatment that can be administered to patients with minimal side effects.

[0079] The development of anti-cancer dosage forms involves designing drugs or drug combinations that can identify target cancer cells and selectively target such cells while preserving healthy cells. This can be achieved by targeting specific proteins or enzymes overexpressed in cancer cells or interfering with the cell cycle, DNA replication, or other cellular processes essential for cancer cell growth and survival. Thus, it can be achieved.

[0080] Once a promising drug candidate is identified, it undergoes pre-clinical development, where the drug is tested in laboratories and animal models to evaluate its safety and efficacy. If the drug shows promise in pre-clinical studies, it can proceed to clinical trials, where it is tested in humans to determine its safety, dosage, and effectiveness.

[0081] During clinical trials, the drug dosage form is optimized to ensure safe administration to patients and achieve the desired therapeutic effect. This may include adjusting the drug dosage, administration route, or dosage form to improve efficacy and minimize side effects.

[0082] The present invention provides, as a drug candidate for killing target cells, (a) an active form of camptothecin derivative represented by the following Chemical Formula 1, designed to bind to the DDX5 protein and an E3 ligase; or (b) its prodrug, preferably a carrier-drug complex (CDC), more preferably an antibody-drug complex (ADC), designed to release the active form of camptothecin derivative (a) from an in vivo target site.

[0083] [Chemical Formula]

[0084] X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and X1 and X3 may be the same or different. X2 is carbon, oxygen, nitrogen, sulfur, a single bond, or a double bond. X1, (X2)n, and X3 can form a 6-membered or 7-membered ring (where n = 1 to 2). Y1, Y2, and Y3 are each independently hydrogen or a functional group containing oxygen, nitrogen, phosphorus, or sulfur.

[0085] Here, non-limiting examples of functional groups containing oxygen, nitrogen, phosphorus, or sulfur are -CHO, -COOH, -NH2, -SH, -CONH2, -PO3H, -PO4H2, -OPO4H, -PO2(OR 1 )(OR 2 )(R 1 、R 2 =C s H t N u O w S x P y X z 、X = -F, -Cl, -Br, or -I, 0 ≤ s ≤ 20, 0 ≤ t ≤ 2(s + u) + 1, 0 ≤ u ≤ 2s, 0 ≤ w ≤ 2s, 0 ≤ x ≤ 2s, 0 ≤ y ≤ 2s, 0 ≤ z ≤ 2s), -SO3H, -OSO3H, -NO2, -N3, -NR3OH (R = C n H 2n+1 、0 ≤ n ≤ 16), -NR3 + X - (R = C n H m 、0 ≤ n ≤ 16, 0 ≤ m ≤ 34, X = OH, Cl, or Br), NR4 + X - (R = C n H m 、0 ≤ n ≤ 16, 0 ≤ m ≤ 34, X = OH, Cl, or Br), -COSH, -COOCO-, -CORCO- (R = C l H m, 0 ≤ l ≤ 3, 0 ≤ m ≤ 2l + 1), -COOR, -CN, -N3, -N2, -NROH (R = C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, 0 ≤ s ≤ 20, 0 ≤ t ≤ 2(s + u) + 1, 0 ≤ u ≤ 2s, 0 ≤ w ≤ 2s, 0 ≤ x ≤ 2s, 0 ≤ y ≤ 2s, 0 ≤ z ≤ 2s), -NR 1 NR 2 R 3 (R 1 , R 2 , R 3 =C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, 0 ≤ s ≤ 20, 0 ≤ t ≤ 2(s + u) + 1, 0 ≤ u ≤ 2s, 0 ≤ w ≤ 2s, 0 ≤ x ≤ 2s, 0 ≤ y ≤ 2s, 0 ≤ z ≤ 2s), -CONHNR 1 R 2 (R 1 , R 2 =C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, 0 ≤ s ≤ 20, 0 ≤ t ≤ 2(s + u) + 1, 0 ≤ u ≤ 2s, 0 ≤ w ≤ 2s, 0 ≤ x ≤ 2s, 0 ≤ y ≤ 2s, 0 ≤ z ≤ 2s), -NR 1 R 2 R 3 X ′ (R 1 , R 2 , R 3 =C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, X ′ =F - , Cl - , Br -or I - where 0≦s≦20, 0≦t≦2(s + u)+1, 0≦u≦2s, 0≦w≦2s, 0≦x≦2s, 0≦y≦2s, 0≦z≦2s), -OH, -O-, >C=O, -SS-, -SO-, -NO2, -COX (X = F, Cl, Br or I), -COOCO-, -CONH-, -CN, -SCOCH3, -SCN, -NCS, -NCO, -OCN, -CN, -F, -Cl, -I, -Br, epoxy group, -hydrazone, -ONO2, -PO(OH)2, -C=NNH2, -HC=CH-, -C=C-, -C≡C- and functional groups selected from the group consisting of hydrocarbons having 2 or more carbon atoms:

[0086] The active camptothecin derivative (a) designed to bind to the DDX5 protein and E3 ligase according to the present invention can kill target cells expressing the DDX5 protein through the molecular glue degrader mechanism (MoA).

[0087] The target cells can be cancer cells or senescent cells. Senescent cells include cells that do not perform the characteristic functions of the organ.

[0088] Preferably, the active camptothecin derivative (a) designed to bind to the DDX5 protein and E3 ligase according to the present invention can have a multi - action mechanism (MoA) that degrades the oncoprotein DDX5 along with the type I topoisomerase inhibitory ability.

[0089] For example, the present invention can expose tumor antigens through cancer cell death in tumor tissues and induce an anti - tumor immune response through the stimulation of antigen - presenting cells (APCs) such as dendritic cells and macrophages.

[0090] In addition, the present invention provides various active camptothecin derivatives represented by Chemical Formula 1 designed to bind to (a) DDX5 protein and E3 ligase as drug candidates for killing cancer cells; or (b) prodrugs thereof designed to release the active camptothecin derivative (a) from in vivo target sites, so as not to cause T cell exhaustion due to chronic antigen exposure or to control lymphatic drainage so as to be removed quickly or slowly through drainage by providing a desired degree of hydrophobicity and / or controlling aggregation, and to control the bystander effect and / or ADME profile of the active camptothecin derivative (a). Thus, various active camptothecin derivative-based anticancer dosage forms, their doses, and dosages can be designed.

[0091] Applying a camptothecin derivative that exerts the effect of a type I topoisomerase inhibitor, in relation to the R1 and R2 modifications (Group A) on the A ring, which have been proven to allow greater tolerance than when designing camptothecin-based drugs as in General Formula 1, the active camptothecin derivative of the present invention is designed to be identical to the FL118 compound of Chemical Formula 2 (Example 1), and is characterized by exerting an anticancer mechanism of decomposing the oncoprotein DDX5 (Example 2).

[0092]

Chemical formula

[0093]

Chemical formula

[0094] This is based on the discovery that active camptothecin derivatives of formula 1, in which R1 and R2 in general formula 1 are designed to be the same as those of the FL118 compound, for example, compounds of formula 3, 4, or 5 below, degrade DDX5, a transcriptional cofactor and oncoprotein, thereby not only strongly inhibiting the expression of anti-apoptotic proteins (survivin, cIAP2, XIAP, etc.) in a concentration-dependent manner (Figures 11 to 14), but also killing the cells into which the compounds are introduced through cell survival experiments (Figures 15 and 16).

[0095] [ka]

[0096] [ka]

[0097] [ka]

[0098] In general formula 1 or chemical formula 2, the group C site may bind to type I topoisomerase and the group A site may bind to DNA and stabilize the covalent bond of the topoisomerase-DNA complex to prevent / prevent further ligation of the cleaved DNA fragments (Example 1), or in general formula 1 or chemical formula 2, the group A site may bind to DDX5 and the group C site may bind to E3 ligase to induce DDX5 degradation (Figures 11 to 14).

[0099] Topoisomerase I inhibitors have shown clinical efficacy This is an anti-cancer mechanism whose safety has been verified, and camptothecin-based drugs such as exatecan, exatecan derivative Dxd, and SN-38 are being developed as payloads for ADCs.

[0100] The Exatecan drug is a substance that has been confirmed to have cytotoxicity 5 to 10 times stronger than the SN-38 drug. Exatecan is different from irinotecan and does not require enzymatic activation. Also, it has stronger type I topoisomerase inhibitory activity than SN-38, which is the active ingredient of irinotecan, and topotecan used in the same clinical setting, and has stronger cytotoxic activity against various cancer cells in vitro. In particular, it was effective against cancer cells that show resistance to SN-38, etc. due to the expression of P-glycoprotein. It also showed a strong antitumor effect in a human tumor subcutaneous transplantation model in mice, and clinical trials were conducted.

[0101] The FL118 drug of Chemical Formula 2 is a hydrophobic low molecular weight substance that can permeate cell membranes. The FL118 drug is a potent type I topoisomerase inhibitor and has the same camptothecin nucleophilic structure as existing commercialized Top 1 inhibitors such as SN-38, topotecan, and exatecan (Figure 1 and Figure 2). However, when administered alone, it has a differentiated anticancer efficacy and excellent safety compared to SN-38 in various cancer cells and animal models, and is an anticancer agent that ensures a wide therapeutic window. window).

[0102] The inventors synthesized the FL118 drug, a camptothecin-based compound, and continued various studies on its physicochemical properties. However, FL118 had limitations in dosage form development due to the limitations of its physicochemical properties.

[0103] The inventors confirmed that in the case of the FL118 drug, when used as the payload of an antibody-drug conjugate (ADC), it has stronger cytotoxicity than an ADC using SN-38 as the payload and has cytotoxicity at the same level as the exatecan drug.

[0104] In the case of the FL118 drug, it is a camptothecin-based compound with excellent type I topoisomerase inhibitory ability and a dual mechanism of action (dual MoA) that degrades the oncoprotein DDX5 (p68).

[0105] Surprisingly, like the FL118 drug, the exatecan drug not only strongly inhibits the expression of anti-apoptotic proteins (such as survivin, cIAP2, XIAP, etc.), which are the main causes of resistance to other anticancer drugs, at low concentrations, but also discovers that it can block the drug resistance mechanism by doing so (Figure 10).

[0106] Therefore, based on this, as shown in Figure 1, the present invention provides (a) a novel active camptothecin derivative having a dual mechanism of action (dual MoA) that degrades the oncoprotein DDX5 (p68) together with excellent type I topoisomerase inhibitory ability from the FL118 compound, (b) its prodrug designed to release the active camptothecin derivative (a) in vivo, preferably a carrier-drug complex (CDC), more preferably an antibody-drug complex (ADC), and (c) a ligand-containing complex that targets the DDX5 protein, in which the type I topoisomerase inhibitory ability in the active camptothecin derivative (a) is inactivated via a linker linkage.

[0107] Specifically, the synthetic design concept of the active camptothecin derivative (a) having a dual mechanism of action (dual MoA) that degrades the oncoprotein DDX5 together with type I topoisomerase inhibitory ability As shown in Fig. 1, (pt) maintains the structure of Group C, which is an I-type topoisomerase inhibitory region, based on SAR (Structure Activity Relationship), and also maintains the same structure as FL118 for Group A, which is the binding site for DDX5 degradation. By adjusting the structure of Group B (R3 and R4) in General Formula 1 as shown in Chemical Formula 1, not only can the physicochemical properties of the drug be improved, but also the cytotoxicity of the drug and / or the cell membrane permeability of the drug can be regulated, and it can be designed into a structure that can regulate the bystander effect (Example 9, Figs. 25 and 26; Example 10, Fig. 27b). Also, in some cases, it can be designed into a structure that can provide the desired degree of hydrophobicity and / or control aggregation so that the active camptothecin derivative (a) released from the dead cancer cells is removed quickly or slowly through lymphatic drainage.

[0108] When simply comparing cell survival, PBX-7016 (Chemical Formula 5) newly designed and synthesized according to the present invention shows an IC level 1.5 to 2 times that of the Dxd drug. 50 However, surprisingly, when used as an ADC payload, the ADC containing PBX-7016 exhibited a cell survival-related cytotoxic effect approximately 5 times superior to that of the ADC (Enhertu) containing Dxd (Example 7, Fig. 22). The compound represented by Chemical Formula 5 (PBX-7016) and the compound represented by Chemical Formula 5-1, which is its isomer, are in a relationship of partial stereoisomers to each other.

[0109] Therefore, the present invention preferably designs an active camptothecin derivative so that the cell membrane permeability can be adjusted as desired through R3 and / or R4 modification (for example, PBX-7014, PBX-7016, PBX-7018, PBX-7020, PBX-7022, PBX-7024) in General Formula 1, and an appropriate bystander effect can be exerted in cancer tissues.

[0110] As shown in Fig. 1, the present invention modified R3 and R4 of group B in General Formula 1, and designed and synthesized a compound with a new structure such as PBX-7011 which is similar to the structure of Exatecan (Production Example 1). As shown in Fig. 2, it was confirmed that PBX-7011 of Chemical Formula 3 newly designed by the present invention has relatively high hydrophilicity compared to FL118 of Chemical Formula 2 through the calculated LogP (or cLogP) and tPSA values.

[0111] Furthermore, by adjusting group B of General Formula 1 in the newly designed PBX-7011 structure, compounds PBX-7014 and PBX-7016 with new structures were designed and synthesized (Production Example 2 and Production Example 3), and it was calculated that PBX-7014 and PBX-7016 show values similar to the hydrophilicity of Dxd derived from Exatecan (Figs. 1 and 2, Fig. 28).

[0112] Fig. 28 compares the solubility and tPSA (topological polar surface area) of FL118, Exatecan, SN-38, Dxd, Exatecan-lactate, PBX-7011 / PBX-7012, PBX-7014 / PBX-7015, and PBX-7016 / PBX-7017.

[0113] The relative hydrophilic / hydrophobic properties of drugs have an important influence on the solubility, absorption, distribution, metabolism, excretion (ADME) of drugs, especially lymphatic drainage. It is important in how easily a drug passes through the cell membrane and in the interaction with drug targets such as receptors.

[0114] The hydrophobicity of a drug is indicated by the partition coefficient (p). That's right.

[0115] Hydrophobicity has a large p-value, while hydrophilicity (polarity) has a small p-value. In fact, the log P value is used as a measure to evaluate hydrophobicity. clogP means that the log P value of a given compound has been determined through appropriate software. The smaller the cLogP value, the higher the polarity.

[0116] The molecular polar surface area (PSA) or topological polar surface area (TPSA) is defined as the surface sum for all polar atoms or molecules that mainly consist of oxygen and nitrogen and include attached hydrogen atoms. tPSA is a pharmaceutical chemistry measurement method commonly used to optimize the cell permeability of drugs. 140 Å 2 Molecules with a larger polar surface area tend not to permeate cell membranes. For a molecule to pass through the blood-brain barrier and act on receptors in the central nervous system, generally a tPSA of less than 90 Å 2 is required.

[0117] The PBX-7016 / PBX-7017 (Chemical Formula 5 and Chemical Formula 5-1) of the present invention is obtained by introducing a methyl group, which is a metabolically unstable functional group to reduce the drug lifespan, into PBX-7014 / PBX-7015 (Chemical Formula 4 and Chemical Formula 4-1). A drug that is extremely stable in metabolism and metabolized very slowly may accumulate and pose a risk of toxicity and serious side effects, so it is necessary to ensure an appropriate residence time.

[0118] The newly designed active camptothecin derivative (a) according to the present invention is a hydrophobic low-molecular compound designed by modifying R3 and R4 of General Formula 1 so as to be able to permeate cell membranes. Therefore, when it is delivered to cancer tissue, it can accumulate at a high concentration while penetrating deep into the cancer tissue. After the ADC of the active camptothecin derivative (a) or its prodrug (b) penetrates the cell membrane, the active camptothecin derivative (a) exerts cytotoxicity inside the cell, kills the cells, is released into the extracellular fluid, and continuously penetrates the cell membrane into the surrounding cells and moves into the cells, and can exert a cell death mechanism (Example 9, FIGS. 25 and 26; Example 10, FIG. 27b). Therefore, the active camptothecin derivative of the present invention can convert / convert a solid cancer with a high cell density and aggregation into a scattered cancer with a low cell density, or convert a cold tumor with low immunological activity into a hot tumor with high immunological activity. In addition, since it has a high bystander cell killing effect, it is advantageous for the treatment of heterogeneous tumors.

[0119] In short, as shown in FIGS. 1, 2, 28, and 29, the present invention can not only diversely design various camptothecin derivatives and their prodrugs (FIG. 3) with a new structure that exhibits a dual mechanism of action (dual MoA) from the viewpoints of type I topoisomerase inhibition and DDX5 proteolysis, but also regulate cell membrane permeability, lymphatic drainage, and / or bystander effect by diversely controlling hydrophilicity during the design of camptothecin derivatives.

[0120] [FL118 drug] FL118 of Chemical Formula 2 can serve as a molecular glue degrader that directly adheres to DDX5 and ubiquitination regulators and degrades DDX5.

[0121] FL118, which acts as a molecular glue, directly binds to the tumor protein DDX5, a multifunctional master regulator, via the proteasomal degradation pathway without reducing DDX5 mRNA, and has the function of dephosphorylating and degrading it. The silencing of DDX5 indicates that it is a master regulator that regulates the expression of various oncogenic proteins including survivin, Mcl-1, XIAP, cIAP2,

[0122] FL118 indirectly controls DDX5 downstream targets and potently suppresses cancer initiation, development, metastasis, recurrence and treatment resistance, as demonstrated in studies using human colorectal / pancreatic ductal adenocarcinoma cells and tumor models.

[0123] Genetic manipulation of DDX5 in PDAC cells affects tumor growth. DDX5 KO PDAC cells are resistant to FL118 treatment. FL118 has been shown to be highly effective in removing human PDAC and CRC tumors with high DDX5 expression in human tumor animal model studies, while FL118 has little effect on PDAC and CRC tumors with low DDX5 expression.

[0124] The DDX5 protein is a direct target of the FL118 drug and can serve as a biomarker for predicting tumor sensitivity to FL118 in PDAC and CRC.

[0125] In addition, the FL118 drug has Top1 inhibitory efficacy at levels equal to or higher than SN-38 in cancer cells and is 5 - 20 times more potent than SN-38, i.e., has a lower level of IC 50It shows cytotoxicity at a value, and even in the evaluation results for 140 cell lines derived from various cancer tumors, it shows a very strong anti-cancer efficacy with an IC 50 showing <100 nM against the majority of cancer cells. The FL118 drug has ensured excellent safety through GLP toxicity tests in rats and beagle dogs, and has shown excellent efficacy compared to SN-38 in various cancer cell line xenograft models. In the case of camptothecin-based anti-cancer drugs, when used in patients, they initially show excellent anti-cancer responses, but strong resistance to these drugs appears through epigenetic silencing of the Top1 gene and Top2-dependence of cancer cells. On the other hand, the FL118 drug has shown strong efficacy even in xenograft models of cancer cell lines where Top1 is not expressed through epigenetic silencing or knockout.

[0126] Also, the FL118 drug is a triple-target anti-cancer drug that directly targets type I topoisomerase, a well-established anti-cancer target, while simultaneously suppressing the Bcl family of resistance proteins such as survivin, which are involved in the resistance mechanism, and suppressing the action of efflux pumps.

[0127] Specifically, camptothecin-based anti-cancer drugs such as SN-38 show resistance in a way that the drug is excreted extracellularly due to overexpression of the ABCG2 transporter, but the FL118 drug is not affected by the ABCG2 transporter, so it is possible to overcome this resistance. The FL118 drug can block the expression of resistance by strongly suppressing the expression of anti-apoptotic proteins (survivin, cIAP2, XIAP, etc.), which are the main causes of resistance to other anti-cancer drugs, at low concentrations (Figure 10).

[0128] Therefore, the FL118 drug is not excreted extracellularly by ABCG2 of the efflux pump and can block the resistance by various anti-apoptotic proteins. As a result, the FL118 drug can overcome various resistance mechanisms of SN-38 / irinotecan.

[0129] When administered in vivo at the same amount as SN-38, the FL118 drug showed a more potent tumor regression efficacy than SN-38 in colorectal cancer / head and neck cancer / pancreatic cancer, etc.

[0130] The FL118 drug also retained a potent anti-cancer efficacy when administered with FL118 after induction of SN-38 resistance in tumor xenografts. The possibility of overcoming various resistance mechanisms of the FL118 drug to SN-38 / irinotecan was confirmed in vivo.

[0131] Furthermore, the FL118 drug has a PK / safety profile that is optimal for the application of targeted drug delivery (e.g., drug carrier complex). When the FL118 drug is administered systemically alone, it is rapidly metabolized / excreted in the blood and shows only a low concentration, but it accumulates rapidly in cancer tissues immediately after administration and maintains a high concentration for a long time. For example, it ensures the maximum selectivity between tumor tissues and normal tissues when applied to ADC. This is the same for the active camptothecin derivative (a) represented by Chemical Formula 1 (Examples 6 and 8).

[0132] In short, the FL118 drug (i) selectively inhibits / degrades DDX5, UBE2T, and USP2a, which are other anti-cancer targets in addition to Top 1, a well-verified anti-cancer target, and exhibits a potent anti-cancer effect; (ii) shows a cellular cytotoxicity that is 5 - 10 times more potent than SN-38 and a cellular cytotoxicity that is equal to or more potent than irinotecan; (iii) Different from SN-38 and exatecan, it is not a substrate of ABCG2 and P-GP, so it is possible to avoid the resistance mechanism caused by the overexpression of these transporter proteins; (iv) It exhibits excellent efficacy in animal models of cancer where epigenetic silencing of Top 1 / overexpression of Top 2, which are the main mechanisms of Top 1 inhibitor resistance, has occurred; (v) It can basically block the overexpression of anti-apoptotic proteins, which is a common resistance mechanism in many cancers, and thus avoid resistance; (vi) Biomarkers (DDX5, K-ras, p53) that can predict the anti-cancer response in patients, companion diagnostic techniques, etc. have already been established, and it is possible to perform personalized order-made treatment through companion diagnosis by securing order-made biomarkers. In particular, it shows strong efficacy in p53 / K-ras mutant cancer cells with poor prognosis.

[0133] KRAS is one of three genes related to the production of RAS proteins involved in cell growth signal transduction. Although it varies depending on the cancer type, it is known that mutations occur in overall about 20% or more. However, despite being scientifically expected to have anti-cancer efficacy, it has been treated as an undruggable target. This is because the surface of the RAS protein is smooth and no pocket suitable for binding compounds has been discovered.

[0134] [As an anti-cancer mechanism 1, a selective inhibitor of type I topoisomerase (topoisomerase-1)] Camptothecin is a selective inhibitor of type I topoisomerase, an isomerase involved in DNA replication, recombination, etc. (Figure 2). Since it was found to exhibit potent cytotoxicity in vitro, development has been initiated through clinical trials at the US National Cancer Institute (NCI) and others. However, due to the limitation of extremely poor solubility, development was interrupted because it exhibited various side effects such as myelosuppression and hemorrhagic cystitis related to it. However, after 1990, it was confirmed that camptothecin selectively inhibits DNA type I topoisomerase, different from its unique mechanism of action, i.e., the DNA type II topoisomerase (topoisomerase-2) inhibition mechanism, and exhibits antitumor effects. It has been confirmed that it selectively inhibits DNA type I topoisomerase and exhibits antitumor effects.

[0135] DNA topoisomerase is an enzyme in the nucleus and plays a role in temporarily cleaving DNA or unwinding double helices when cells require access to genetic material for replication or transcription. They also participate in various intracellular activities such as chromosome condensation and recombination, DNA repair, etc. The genetic code of topoisomerase is quite conserved among species.

[0136] Type I topoisomerase, the drug target of camptothecin, has been observed to increase in level in various malignancies. This drug does not inhibit the free enzyme but stabilizes the covalent bond of the topo-DNA complex, preventing the further ligation of the cleaved DNA fragments. Therefore, the sensitivity of cells to such topoisomerase-targeted drugs is related to the level of the enzyme present in the nucleus. By interfering with DNA recombination, this drug makes it impossible for transcription to proceed. The greater the amount of type I topoisomerase, the more cleavable complexes are formed, which means higher drug sensitivity. This has important clinical relevance, and type I topoisomerase inhibitors are used to increase the expression of type II topoisomerase, which confers further sensitivity to type II topoisomerase inhibitors. Such results are supported by the antagonistic relationship between type I topoisomerase and type II topoisomerase. Unlike type II topoisomerase, such type I topoisomerase is not closely associated with proliferation in normal tissues and is present in larger amounts in the solid cancers in the "S" phase with active cell division, such as colorectal cancer, ovarian cancer, and esophageal cancer including lymphoma, compared to the surrounding normal tissues. It is actually known that by blocking gene replication and transcription in tumor cells during the "S" phase of the cell cycle, cell death can be induced.

[0137] [As an anti-cancer mechanism 2 of a molecular glue degrader that binds to DDX5] The ubiquitin-proteasome system (UPS) is an important pathway for the degradation of intracellular proteins that regulates a wide range of cellular processes. Ubiquitin is a small protein that covalently binds to the lysine residues of substrate proteins through a series of enzymatic reactions involving ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). This process is called ubiquitination and is an important mechanism for regulating proteolysis, signal transduction, and trafficking.

[0138] Ubiquitin ligase is responsible for substrate specificity in the ubiquitination pathway. In connection with this, the active camptothecin derivative represented by Chemical Formula 1 according to the present invention is designed to bind to the DDX5 protein and the E3 ligase.

[0139] The DDX5 protein (also referred to as p68) is a multifunctional master regulator that acts through the following mechanisms: (1) a biological process of co-activating the transcription of many tumor genes through direct interaction with various transcription factors (e.g., c-Myc) in oncogenic gene promoters; (2) a biological process of regulating miRNA and pre-RNA splicing (e.g., U1, U2, U3,... snRNP); and (3) ribosome biosynthesis (e.g., 32S rRNA, pre-ribosome).

[0140] The active camptothecin derivative represented by Chemical Formula 1 according to the present invention binds to the DDX5 protein without reducing DDX5 mRNA and is functionally degraded through the dephosphorylation and proteasome degradation pathways, which suggests that the active camptothecin derivative of Chemical Formula 1 can adhere to all DDX5 and ubiquitin-involved protein stability / degradation regulat ors and serves as a "molecular adhesive degrader".

[0141] All DDX5 downstream protein targets are known to be involved in cancer initiation, development, metastasis, recurrence, and treatment resistance. Therefore, when the DDX5 downstream targets are indirectly blocked through the degradation of the DDX5 protein by the active camptothecin derivative represented by Chemical Formula 1 according to the present invention, the active camptothecin derivative represented by Chemical Formula 1 can exhibit high antitumor efficacy.

[0142] [DDX5 as a drug target and / or biomarker] Transcription factors are a group of proteins that bind to DNA strands called promoters or enhancers via DNA-binding domains and initiate gene transcription with the cooperation of RNA polymerase and other cofactors. They play important roles in regulating transcription during embryogenesis and development. The physiological state of transcription factors in other cell types is also important for maintaining cell homeostasis. Therefore, deregulation of transcription factors leads to the development of cancer cells and tumor progression. Depending on their functions in cancer cells, transcription factors can be oncogenic or tumor-suppressive. Transcription factors have also been shown to regulate cancer stem cells, epithelial-mesenchymal transition (EMT), and drug response. Therefore, measuring deregulated transcription factors is thought to be able to predict the treatment outcomes of cancer patients, and targeting deregulated transcription factors can be an encouraging strategy for cancer treatment. Deregulated major transcription factors can induce poor clinical outcomes in cancer patients. Therefore, it is useful for predicting prognosis and drug response and targeting deregulated transcription factors to design new drugs and treatment strategies for cancer treatment.

[0143] Transcription is well known to be the first step of gene expression in cells during the processes of embryogenesis, development, and homeostasis. During the transcription process, RNA transcripts with specific coding information are generated from DNA. To ensure the specificity of gene expression in different cells and tissues, a specific group of proteins, transcription factors, select the genes to be transcribed into RNA transcripts and control transcription. Transcription factors have DNA-binding domains and can bind to specific DNA sequences called promoters or enhancers of genes, and can initiate gene transcription with the cooperation of activators, mediators, cofactors, and RNA polymerase.

[0144] Transcription factors also control the transcription rate from DNA to mRNA. Consequently, the physiological state of transcription factors in different types of cells is important for maintaining cell homeostasis. Changes in the physiological state of intracellular transcription factors cause transcriptional disorders, initiate the translation of specific genes, and cause pathological changes in cells. Therefore, dysregulated transcription factors can induce many human diseases. One of the common diseases with dysregulated transcription factors is cancer.

[0145] The homeostasis control of human cells generally prevents inappropriate proliferation and suppresses the growth of cells that abnormally proliferate outside the normal niche. Since transcriptional control is a core process of homeostasis, if the levels or activities of transcription factors are not regulated, cells can deviate from homeostasis control and induce the onset and progression of human cancer. In fact, many chemotherapies can activate tumor suppressor transcription factors.

[0146] Dysregulated transcription factors play important roles in tumor formation and tumor progression. They regulate central tumor signaling pathways such as NF-κB, Notch, Wnt / β-catenin, and JAK / STAT. Dysregulated oncogenic and tumor suppressor transcription factors preferentially induce intrinsic or acquired drug resistance and tumor progression in cancer treatment for the survival of cancer cells, cancer stem cells, and EMT cells. Cells.

[0147] DDX5 (p68) is a well-known multifunctional DEAD-box type RNA helicase and a transcription cofactor. Therefore, when cancer develops due to the deregulation of transcription factors by the physiological state of DDX5, the cancer disease can be treated by selectively degrading DDX5 (p68), a transcription cofactor. Similarly, the cancer disease can be prevented by selectively degrading DDX5 (p68), a transcription cofactor.

[0148] DDX5 has been recognized as a potential biomarker and target for the treatment of various cancer types. Through years of research, the understanding of the functional diversity of DDX5 in various cancer types has been greatly expanded, and the knowledge of its MoA (Mechanism of Action) has been extended. This provides a rationale for using DDX5 as a biomarker and therapeutic target for the development of new cancer therapeutics. However, although in many published studies DDX5 has been shown to be an oncogenic target and a cancer treatment resistance biomarker, some studies have reported that in certain scenarios DDX5 can act as a tumor suppressor. The multiple functions of DDX5 make it an excellent independent oncogenic biomarker and a target for targeted cancer therapy.

[0149] The transformation of normal cells into cancerous cells occurs due to the deregulation of different metabolic pathways, including complex networks of protein-protein interactions. Cellular enzymes and DDX5 play important roles in maintaining normal cell metabolism, but if they are deregulated, they can accelerate tumor transformation. DDX5 interacts with all together hundreds of different cellular proteins, and depending on the specific pathway involved, both proteins can act as tumor suppressor genes or oncogenes.

[0150] DEAD-box RNA helicases are involved in various metabolic pathways, from transcription and translation to cell proliferation, innate immunity, and the stress response. When considering these diverse roles, deregulation of these helicases or mutations in them have been linked to various pathological conditions, including cancer. However, in some cases, loss of function of a given DEAD-box helicase promotes tumor transformation and exhibits a tumor suppressor role, while in other situations, overexpression of the same enzyme acts as a typical oncogene, favoring cancer progression. The role of DDX5 as both an oncogene and a tumor suppressor has been documented in various cancer types. Understanding the interactions of different cellular contexts defined by the molecular interaction network of DDX5 in different tumors can help explain the seemingly opposing roles of these proteins as cancer inducers or suppressors.

[0151] Table 1 shows that DDX5 frequently acts as an oncogene but can also have tumor suppressor functions in certain cancer types.

[0152] Source: Secchi, M.; Lodola, C.; Garbelli, A.; Bione, S.; Maga, G. DEAD-Box RNA Helicases DDX3X and DDX5 as Oncogenes or Oncosuppressor s: A Network Perspective. Cancers 2022, 14, 3820. https: / / doi.org / 10.3390 / cancers14153820

[0153]

Table 1

[0154] According to the present invention, the active camptothecin derivative represented by Chemical Formula 1 can avoid drug resistance, non-responsiveness to targeted therapy and / or resistance during treatment because the DDX5 protein is the drug target. In addition, the active camptothecin derivative can block the transcriptional induction of anti-apoptotic genes. Furthermore, the active camptothecin derivative can degrade the DDX5 protein, which is a transcription cofactor, and maintain or improve the sensitivity of cancer cells to chemotherapy and radiotherapy.

[0155] [Camptothecin derivatives represented by Chemical Formula 1, Chemical Formulas 3 to 9 and isomers thereof] The active camptothecin derivative represented by Chemical Formula 1, in which R1 and R2 in General Formula 1 are designed to be the same as those of the FL118 compound according to the present invention, for example, the compound of the following Chemical Formula 3, the compound of Chemical Formula 4, the compound of Chemical Formula 5, the compound of Chemical Formula 6, the compound of Chemical Formula 7, the compound of Chemical Formula 8, the compound of Chemical Formula 9 and isomers thereof can also degrade DDX5, which is a transcription cofactor and an oncoprotein, thereby not only strongly suppressing the expression of anti-apoptotic proteins (survivin, cIAP2, XIAP, etc.) in a concentration-dependent manner (Figs. 11 to 14), but also killing the cells into which the compound has been introduced intracellularly through a cell viability experiment (Figs. 15 and 16).

[0156] According to the present invention, the camptothecin derivatives represented by Chemical Formula 1, for example, Chemical Formulas 3 to 9, have a pentacyclic structure having a lactone in the E-ring essential for cytotoxicity like camptothecin shown in FIG. 1, and the lactone group and alpha-hydroxyl group located at the 20th carbon of the E-ring, which are important for the stabilization of type I topoisomerase-DNA by-products, are designed to be maintained. While maintaining the structure of the FL118 drug that maintains the advantages of the aforementioned FL118 drug and R1 and R2 (-OCH2O-(methylenedioxo) 5-sided ring) in General Formula 1, the structural characteristics of the exatecan drug (R3 and R4 in General Formula 1) are such that, compared to SN-38 in which aggregation is induced by the π-π stacking of the aromatic rings formed by the A- and B-rings, the various orientations of the continuous carbon-carbon single bonds of the 6-membered ring extended from the A- and B-rings form a dynamic equilibrium, and the stacking of the aromatic rings formed by the A- and B-rings can be weakened or suppressed.

[0157] In addition, the compound represented by Chemical Formula 3 or Chemical Formula 3-1 is designed such that the molecular bond can rotate with respect to the carbon-carbon single bond in the 6-membered ring extended from the A- and B-rings, and the highly flexible -NH2 is exposed to water (H2O) and either bears a (+) charge or forms a hydrogen bond with water to increase water dispersibility.

[0158] In addition, the compound represented by Chemical Formula 4 or Chemical Formula 4-1 is designed such that the molecular bond can rotate with respect to the carbon-carbon single bond in the 6-membered ring extended from the A- and B-rings, and the functional group CH2(OH)CONH- of -NH2 in the form of glycolic acid, which has a high degree of freedom, is exposed to water (H2O) and forms a hydrogen bond with water while rotating like a propeller to increase water dispersibility.

[0159] In addition, the compound represented by Chemical Formula 5 or Chemical Formula 5-1 is designed such that the rotation of the molecular bond is possible with respect to the carbon-carbon single bond in the six-membered ring extended from the A- and B-rings, and the functional group CH3CH(OH)CONH- in the lactic acid form of -NH2 with a large degree of freedom is exposed to water (H2O) and forms a hydrogen bond with water while rotating like a propeller to increase the water dispersibility.

[0160] The compound represented by Chemical Formula 5 or Chemical Formula 5-1 of the present invention is obtained by introducing a methyl group, which is a metabolically unstable functional group, into the compound represented by Chemical Formula 4 or Chemical Formula 4-1 in order to reduce the drug lifetime. A drug that is extremely stable in metabolism and is metabolized very slowly may accumulate and have the risk of toxicity and serious side effects, so it is necessary to ensure an appropriate residence time.

[0161] The DXd payload used in Enhertu was originally made from an exatecan compound that is hardly affected by ABCG2. However, due to the presence of the glycolic acid (α-hydroxyacetic acid) functional group used to convert exatecan to DXd, it is strongly affected by ABCG2. However, the glycolic acid functional group plays a very important role in the excellent safety / efficacy profile of Enhertu. Removing this group will cause difficulties in ADC production and at the same time lead to problems of poor ADC performance (the emergence of safety issues or a decrease in efficacy) in animal models and clinical trials. Therefore, there is a very high demand for a new camptothecin derivative that can be easily used in ADC production and is not affected by ABCG2. The compound represented by Chemical Formula 9 (PBX-7024) is a compound derived from the compound represented by Chemical Formula 3 (PBX-7011) and is a new camptothecin compound that is not affected by ABCG2 and can be easily used in ADC production.

[0162] To confirm the anti-cancer efficacy of PBX-7024, in cancer cells that do not express ABCG2 The efficacy of the camptothecin compound containing DXd was evaluated in A549, a cancer cell overexpressing a certain FaDu and ABCG2. In A549, a cancer cell line overexpressing ABCG2, as shown in Fig. 15, the camptothecin compound containing DXd showed a high IC 50 value, while it was confirmed that PBX-7016 and PBX-7024 still maintained strong efficacy.

[0163] That is, when using PBX-7016 and PBX-7024, the new camptothecin compounds according to the present invention, unlike ADCs using existing camptothecin compounds such as SN-38 and DXd, have the effect of overcoming the resistance mechanism due to the overexpression of ABCG2.

[0164] Figs. 28 and 29 compare the LogP, cLogP and tPSA (topological polar surface area) of FL118, exatecan, SN-38, Dxd, compound A, compound B, compound C, compound D, compound E, compound F, compound G, compound H, compound I, compound J, compound G’, compound H’, compound I’ and compound J’.

[0165] The compound of Chemical Formula 3 and the compound of Chemical Formula 3-1 are in a relationship of partial stereoisomers with each other. Similarly, the compound of Chemical Formula 4 and the compound of Chemical Formula 4-1; and the compound of Chemical Formula 5 and the compound of Chemical Formula 5-1 are also in a relationship of partial stereoisomers with each other. Therefore, compounds in a relationship of partial stereoisomers with the compound of Chemical Formula 6, the compound of Chemical Formula 7, the compound of Chemical Formula 8 or the compound of Chemical Formula 9 also belong to the scope of the present invention.

[0166] As exemplified in Production Example 2, PBX-7014 of Chemical Formula 4 can be synthesized from PBX-7011 of Chemical Formula 3, and PBX-7015 of Chemical Formula 4-1 can be synthesized from PBX-7012 of Chemical Formula 3-1.

[0167] As exemplified in Production Example 3, the compound of Chemical Formula 5 (PBX-7016, Compound E) can be synthesized from the compound of Chemical Formula 3 (PBX-7011, Compound A), and in the same manner, the compound of Chemical Formula 5-1 (PBX-7017, Compound F) can be synthesized from the compound of Chemical Formula 3-1 (PBX-7012, Compound B).

[0168] In the present specification, the camptothecin-based drugs of Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9 according to the present invention include not only the compounds of Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, but also pharmaceutically acceptable salts thereof, solvates thereof, prodrugs thereof, preferably carrier-drug conjugates (CDCs), more preferably antibody-drug conjugates (ADCs).

[0169] In the present specification, the pharmaceutically acceptable salts mean salts commonly used in the pharmaceutical industry, for example, salts of inorganic ions including sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc., and salts of inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, etc., and in addition, salts of organic acids such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotic acid, acetylsalicylic acid, etc., and amino acid salts such as lysine, arginine, guanidine, etc. Further, there are salts of organic ions such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium, benzethonium, etc., which can be used in pharmaceutical reactions, purification and separation processes. However, the types of salts meant in the present invention are not limited by these listed salts.

[0170] "Solvate" means a compound represented by Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, and a pharmaceutically acceptable salt thereof, which further contains a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0171] [Advantages of the drug modality of molecular glue degrader] Intracellular proteins in the body are naturally degraded within hours to days after performing their functions. All cells in the body have a purification function called the ubiquitin proteasome system (UPS) that degrades proteins. In this process, ubiquitin serves as a marker that presents the protein to be degraded, and the proteasome acts as a grinder that recognizes the ubiquitin label and destroys the protein. That is, multiple substances called ubiquitin attach like labels to the side of the protein that has performed its role, and a substance called proteasome selectively degrades only the protein with this label like a grinder. E3 ligase is an enzyme that activates the body's protein degradation system and is responsible for substrate specificity in the ubiquitination pathway.

[0172] A molecular glue degrader or molecular glue is a compound that functions as an adhesive that attaches a target protein to a specific enzyme (E3 ligase) in the human body. One of the advantages of a molecular glue is its catalytic role, which means that after degrading a target, it can be further separated and degrade other target proteins.

[0173] When an E3 ligase enzyme attaches to a tumor protein via a molecular glue, the tumor protein is degraded, and other tumor proteins are continuously degraded until the target tumor protein disappears, thus preventing the growth of cancer cells. Therefore, the molecular glue for tumor proteins overcomes the problem of drug resistance, which is a problem of target anticancer drugs, and has a high therapeutic effect even at low doses.

[0174] Not only the FL118 drug, but also the active camptothecin derivative designed by Formula 1 according to the present invention is a molecular glue degrader that binds to the DDX5 protein and the E3 ligase, that is, a molecular glue that activates the degradation of the tumor protein DDX5 (FIGS. 11 to 14).

[0175] The molecular glue degrader can not only act as a ligand (warhead) that binds to the target protein, but also act as an E3 ligase ligand (binder).

[0176] Therefore, since the active camptothecin derivative designed by Formula 1 according to the present invention is a molecular glue that activates the degradation of the tumor protein DDX5, it can be used as a ligand that targets the DDX5 protein or a ligand that binds to the DDX5 protein.

[0177] Unlike kinase inhibitors, the molecular glue degrader is "proximity-driven" and "event-driven" depending on the formation of a transient triple complex of "target protein - molecular glue - E3 ligase" with degradation-inducing ability. After the degradation occurs, the separated molecular glue can form a further triple complex with the target protein and proceed through several degradation processes until the target protein disappears.

[0178] Most protein drug targets develop resistance that evolves in response to drugs. However, molecular glue, a small molecule compound that functions as an adhesive to attach tumor proteins and E3 ligases to each other, is resistant to resistance and is thus a modality suitable for cancer therapeutics.

[0179] Each time a gene mutation occurs, the shape of the drug target protein changes little by little. It would be good if one drug could prevent the activity of all mutants, but this is not possible due to selectivity issues. Therefore, in order for one drug to prevent the activity of all mutations, the drug target protein is decomposed and removed from the beginning.

[0180] Therefore, the active camptothecin derivative represented by Chemical Formula 1 according to the present invention is a drug that can accurately bind to the DDX5 tumor protein, and can avoid the resistance problem of the target therapeutic agent through a molecular glue access method that immediately and selectively degrades the protein.

[0181] The active camptothecin derivative represented by Chemical Formula 1 according to the present invention is preferably an irreversible drug whose binding strength to the target protein, the DDX5 tumor protein, is determined by its physicochemical properties and binds very strongly and cannot return to its original state.

[0182] Therefore, the active camptothecin derivative represented by Chemical Formula 1 according to the present invention can control the binding strength to the DDX5 tumor protein, which is the target protein, through the X1, (X2) n , X3, Y1, Y2 and / or Y3 modifications in Chemical Formula 1.

[0183] The active camptothecin derivative designed according to Chemical Formula 1 by the present invention, different from existing SN38 drugs, has a high affinity for the DDX5 protein and is not easily detached. Through the function of a molecular glue, it degrades the DDX5 protein, not only irreversibly suppressing the signal transduction of cancer cells related to DDX5, but also adjusting the cell membrane permeability of the drug as desired according to its physicochemical properties, and by exerting a bystander effect or controlling the degree of this effect, it has a high bystander killing effect, is advantageous for the treatment of heterogeneous tumors, can suppress cancer progression for a long time, reduce the risk of resistance expression, and increase the treatment response rate.

[0184] [Cell Death and Drug Resistance of Anticancer Agents] The active camptothecin derivative represented by Chemical Formula 1 according to the present invention can bind to DDX5, which acts as an oncoprotein in cells, and induce cell death through DDX5 protein degradation (Figure 30).

[0185] There are two elements to determine tumor growth. The first is cell proliferation, and the second is cell death. When the cell cycle of tumor cells is stopped by cytotoxic substances, the tumor cells die by apoptosis.

[0186] Cell death plays a very important role as a mechanism for maintaining tissue homeostasis and morphology, regulating cell numbers, removing damaged or abnormal cells, and defending against infections. The living body maintains life normally by precisely regulating the processes of cell growth, differentiation, and death.

[0187] Apoptosis has been described as a complex process that induces cell death involving the expression of various genes and the activation of proteins that are their expression products. Such apoptosis is an intracellular physiological and active suicide mechanism and plays an essential role in maintaining the development, differentiation, and homeostasis of multicellular organisms. Apoptosis is diverse due to many factors It occurs in certain types of cells and, unlike cell necrosis, there is no release of lysosome enzymes, apoptotic bodies are found, and cell shrinkage, nuclear condensation, and characteristic ladder DNA segments, etc. are found.

[0188] Apoptosis is part of the normal development program of animals and is important for cancer prevention. The protease system caspases are involved in cell death and cleave many target proteins. When cell death is impaired, cells that should be killed can survive, proliferate, and form clones that can potentially become cancer.

[0189] Essential drug resistance in cancer treatment can be caused by abnormally expressed transcription factors that are critical regulators of cell proliferation and cell death. Thus, the active camptothecin derivatives of the present invention can induce DDX5 proteolysis and thereby cell death through an action mechanism as a molecular glue degrader that binds to DDX5, which is a transcription factor cofactor and an oncoprotein. At this time, DDX5 proteolysis can downregulate the transcription of anti-apoptotic genes. Therefore, unlike other targeted therapeutic agents, it can rarely or not cause drug resistance caused by abnormally expressed, cell proliferation and / or cell death-related transcription factors, or can suppress acquired drug resistance induced through the activation of transcription factors not regulated by chemotherapy and / or radiotherapy.

[0190] That is, the anti-cancer mechanism of the active camptothecin derivatives according to the present invention can bypass the molecular mechanism of cancer treatment resistance, so it can be free from the problem of drug resistance. When resistance occurs, the therapeutic effect decreases compared to before, so the active camptothecin derivatives of the present invention are preferred as standard therapeutic agents or primary therapeutic agents after cancer diagnosis.

[0191] In short, the active camptothecin derivative represented by Chemical Formula 1 according to the present invention can be a targeted anti-cancer agent that acts on the DDX5 protein, which plays an important role in cancer cell growth, survival, proliferation, metastasis, and / or metabolism.

[0192] [Clinical Results and Side Effects of Camptothecin Payload-based ADCs (ADCs with Camptothecin Payloads)] Trastuzumab deruxtecan (Enhertu) is a humanized anti-HER2 antibody conjugated to deruxtecan, a camptothecin derivative, via a stable linker. In the first phase 1 dose escalation study, 22 patients with HER-2 positive advanced or metastatic breast cancer, gastric cancer, or other HER-2 expressing solid tumors were treated with trastuzumab deruxtecan at 0.8 mg / kg to 8.0 mg / kg once every 3 weeks. Dose-limiting toxicity was not observed, and the MTD was not reached. The target drug exposure was achieved at a dose of 6.4 mg / kg selected as the recommended phase 2 dose.

[0193] The DESTINY-Breast01 clinical trial, a pivotal single-arm phase 2 clinical trial, consisted of two parts. In the first part, patients with advanced / metastatic breast cancer who had previously been treated with two or more anti-HER2 therapies were randomly assigned and treated with trastuzumab deruxtecan at doses of 5.4 mg / kg (n = 50), 6.4 mg / kg (n = 48), or 7.4 mg / kg (n = 21) once every 3 weeks. In the second part, a dose of 5.4 mg / kg was administered to 134 patients based on the efficacy and toxicity data obtained in the first part 1. Among the 184 patients treated with trastuzumab deruxtecan at 5.4 mg / kg the most frequently occurring adverse reactions in all grades (≥20%) were nausea (77.5%), fatigue (49.8%), alopecia (49.8%), vomiting (44.3%), neutropenia (40.3%), constipation (37.5%), anemia (33.6%), decreased appetite ( (33.2%), diarrhea (29.2%), leukopenia (26.9%), thrombocytopenia (24.9%). Grade 3 or higher adverse reactions occurred in 57.1% of patients, with neutropenia (20.7%), anemia (8.7%), nausea (7.6%), leukopenia (6.5%), lymphopenia (6.5%), and fatigue (6.0%) being the most frequent. Discontinuation of medication, dose reduction, and treatment interruption due to adverse reactions occurred in 35.3%, 23.4%, and 15.2% of patients, respectively, with pneumonia (11 patients) and interstitial lung disease (5 patients) being the most frequent reasons. The black box warning for patients treated with trastuzumab deruxtecan includes interstitial lung disease (ILD) and pneumonia. Treatment-related interstitial lung disease and fatal outcomes occurred in 9% and 2.6% of patients treated with trastuzumab deruxtecan, respectively. Similar to other HER-2 targeted ADCs, patients treated with trastuzumab deruxtecan also had an increased risk of embryofetal toxicity and left ventricular dysfunction. In addition, sacituzumab govitecan (Trodelvy) is a humanized anti-TOP-2 IgG conjugated to the active metabolite of irinotecan (SN-38) via a pH-sensitive linker. In the first human dose-escalation 1 / 2 phase clinical phase 1, 25 patients with various metastatic solid tumors were treated with sacituzumab govitecan at 8 mg / kg to 18 mg / kg on days 1 and 8 of a 21-day cycle. The MTD of the first cycle was determined to be 12 mg / kg, with neutropenia appearing as the dose-limiting toxicity. However, this dose level was extremely toxic in subsequent cycles, and doses of 8 mg / kg and 10 mg / kg were selected in the phase 2 clinical trial. In this phase 2 clinical trial, sacituzumab govitecan was administered at 8 mg / kg (n = 81) or 10 mg to various metastatic epithelial cancer patients who had previously received various treatments.

[0194] Note that sacituzumab govitecan (Trodelvy) is a humanized anti-TOP-2 IgG conjugated to the active metabolite of irinotecan (SN-38) via a pH-sensitive linker. In the first human dose-escalation 1 / 2 phase clinical phase 1, 25 patients with various metastatic solid tumors were treated with sacituzumab govitecan at 8 mg / kg to 18 mg / kg on days 1 and 8 of a 21-day cycle. The MTD of the first cycle was determined to be 12 mg / kg, with neutropenia appearing as the dose-limiting toxicity. However, this dose level was extremely toxic in subsequent cycles, and doses of 8 mg / kg and 10 mg / kg were selected in the phase 2 clinical trial. In this phase 2 clinical trial, sacituzumab govitecan was administered at 8 mg / kg (n = 81) or 10 mg / kg to various metastatic epithelial cancer patients who had previously received various treatments. Doses of g / kg (n = 97) were administered. The most frequent adverse reactions of all grades (≥ 25%) reported in the 8 mg / kg and 10 mg / kg cohorts were nausea (59% vs. 63%), diarrhea (53% vs. 62%), neutropenia (42% vs. 58%), fatigue (61% vs. 52%), vomiting (36% vs. 43%), anemia (38% vs. 42%), alopecia (46% vs. 37%), and constipation (33% vs. 37%). The most frequent adverse reactions of grade 3 or higher (≥ 10%) reported in the 8 mg / kg and 10 mg / kg treatment groups were neutropenia (30% vs. 36%), anemia (13% vs. 12%), diarrhea (4% vs. 10%), and leukopenia (6% vs. 12%). Dose reduction occurred in 19% and 28% of patients in the 8 mg / kg and 10 mg / kg cohorts, respectively. Neutropenia was the most frequent adverse reaction leading to dose delay or reduction. There were significantly more patients experiencing grade 3 or higher neutropenia after the first dose in the 10 mg / kg cohort than in the 8 mg / kg cohort (47% vs. 21%).

[0195] Black box warnings for severe or life-threatening neutropenia and severe diarrhea were added to the label of sacituzumab govitecan. Such side effects are likely mediated by the released ( "free") SN-38 and are associated with the same toxicities as irinotecan, the SN-38 prodrug. This is because it is highly likely to be mediated by the released ( "free") SN-38, and the same toxicity as irinotecan, the SN-38 prodrug, is associated.

[0196] All grades and grade 3 or higher neutropenia occurred in 61% and 47%, respectively, of all patients treated with sacituzumab govitecan. Febrile neutropenia occurred in 7% of patients. All grades and grade 3 or higher diarrhea occurred in 65% and 12%, respectively, of all patients treated with sacituzumab govitecan. Neutropenic colitis occurred in 0.5% of patients. In addition, patients treated with trastuzumab deruxtecan (Enhertu) had IL

[0197] ​​If experiencing D / pneumonia, apply corticosteroid treatment (https: / / www.enhertuhcp.com / en / gastric / manage-potential-risks / ild-and-pneumonitis).

[0198] Corticosteroids play an important role in immunosuppression by modulating diverse aspects of the immune response and reducing inflammation. The mechanism of action of corticosteroids contributes to suppressing excessive immune activity and alleviating organ damage under conditions where immunoregulatory disorders occur.

[0199] Immunosuppressants, particularly corticosteroids, are widely used to suppress excessive immune responses and alleviate organ damage in a variety of clinical scenarios such as autoimmune diseases, transplant rejection, and immune-related adverse events (irAE) associated with checkpoint blockade therapy.

[0200] One of the main mechanisms of corticosteroid action is the ability to suppress the transcription of pro-inflammatory genes. Within immune cells, corticosteroids bind to specific receptors in the cytoplasm, forming a corticosteroid-receptor complex. This complex moves to the cell nucleus and binds to a specific DNA sequence known as the glucocorticoid response element (GRE) located in the promoter of target genes. Thus, corticosteroids interfere with the recruitment of transcription factors and co-factors, ultimately blocking the transcriptional activation of pro-inflammatory genes. This reduces the synthesis of inflammatory mediators such as cytokines (e.g., interleukins and tumor necrosis factor alpha) and prostaglandins, which contribute to tissue damage.

[0201] In addition, corticosteroids can also directly regulate immune cell function. Corticosteroids reduce the expression of adhesion molecules on endothelial cells, downregulate chemokine receptors on immune cells, and inhibit the migration of immune cells to the site of inflammation. Corticosteroids also inhibit the activation and proliferation of T cells, which are important mediators of the immune response. They can interfere with the antigen presentation process and the generation of costimulatory molecules, weaken T cell activation, and reduce effector function. In addition, corticosteroids suppress B cell function and reduce antibody production.

[0202] Therefore, camptothecin-based payload ADCs (ADCs with Camptothecin Payloads) can also fall under the category of drugs that induce an immune response (in some cases, pneumonia / ILD side effects).

[0203] [ADME Profile of Antibody-Drug Conjugates (ADCs)] The in vivo efficacy and in vivo side effects of anticancer drugs can be affected by the absorption, distribution, metabolism, and excretion (Absorption, Distribution, Metabolism, and Excretion, ADME) characteristics of the ADC and the payload released therefrom. The ADME profile of a drug can affect its ability to reach and act on cancer cells. That is, the ADME characteristics of the ADC and the payload released therefrom can have a significant impact on in vivo efficacy and in vivo side effects.

[0204] The ability of the ADC and the payload released therefrom to penetrate tumor tissue can also be affected by factors such as the tumor microenvironment, including blood flow and cell density.

[0205] Therefore, understanding the ADME properties of the ADC and the payload to be released therefrom and optimizing drug / drug modality selection and dosage / usage can improve the therapeutic index of such ADCs and the payloads to be released therefrom, leading to better outcomes for cancer patients.

[0206] Antibodies (Abs) and antibody-drug conjugates (ADCs) have different lifetimes and ADME profiles due to their different structures and mechanisms of action.

[0207] Antibodies are large proteins that react to foreign substances (antigens) and are naturally produced by the immune system. Due to their size and complex structure, they have a long circulation half-life (from weeks to months) and can be protected from degradation and removal. Antibodies are distributed throughout the body, including in tissues, and can interact with target antigens with high specificity and affinity. Antibodies are removed mainly by the reticuloendothelial system (RES), including the liver and spleen, and by catabolism in the kidneys and other organs.

[0208] An ADC is composed of an antibody (generally a monoclonal antibody) conjugated to a cytotoxic drug molecule. The antibody component provides specificity and targeting to tumor or diseased tissue, while the drug component (the payload released from the ADC) provides cytotoxic activity to kill target cells. An ADC has a shorter half-life than an antibody, generally reaching from a few days to one week. This is because the ADC is internalized by target cells, leading to lysosomal degradation of the ADC and release of the drug payload. An ADC is mainly removed by the RES, but the drug payload released from the ADC can also undergo metabolism and excretion in the liver and kidneys via lymphatic drainage and subsequently through the circulatory system (Example 4).

[0209] As predicted by the pharmacokinetics and biodistribution of monoclonal antibodies, in ADCs as well, high-affinity mAb binding to cell membrane proteins can localize a significant portion of the mAb to the target cell population, and the chemical conjugation of the payload to the anti-cancer mAb increases the selectivity with which the payload is delivered to cancer cells and increases the therapeutic index of the payload.

[0210] Antibodies are generally administered by subcutaneous or intravenous injection and are absorbed into the bloodstream. They can distribute throughout the body, including into tissues, but are generally restricted to the extracellular space due to their size. ADCs are also administered by injection and are absorbed into the bloodstream, but following targeting to tumors or diseased tissues by the antibody component and, in some cases, receptor-mediated endocytosis, the drug payload released from the ADC can penetrate cells and tissues depending on its physicochemical properties (e.g., hydrophobicity, size, presence of aggregation) and can exert a bystander effect by non-selective uptake (Figure 31). Furthermore, the metabolism and excretion of ADCs vary depending on the specific structure and the specific drug or antibody used.

[0211] The drug-to-antibody ratio (DAR) is a very important property that determines the pharmacokinetic properties and in vivo distribution in ADC development.

[0212] The higher the DAR of an ADC, the higher the efficacy in in vitro tests. However, ADCs with inherently high DARs have unexpectedly low in vivo efficacy, presumably because the higher the number of conjugated drugs in an ADC, the higher the plasma clearance rate. As a result, for some time, the DAR of ADC formulations has been adjusted to approximately 2 - 4. Accordingly, techniques used for the drug conjugation method to the cysteine or lysine residues of the antibody are mainly used.

[0213] Since a significant number of cytotoxic drugs and linkers mainly used are hydrophobic, problems such as aggregation of ADCs, loss of affinity for target antigens, and high plasma clearance occur. Hydrophilic linkers containing sulfonate or polyethylene glycol (PEG) solve the problems caused by hydrophobic linkers. PEG linkers have the advantages of being water-soluble, having low toxicity, and low immunogenicity.

[0214] It has long been considered optimal for the DAR to be approximately 4. However, in reality, this is the case when it comes to second-generation linkers using MMAE or D This corresponds to second-generation linkers using M1 as the drug. For third-generation linkers, a higher DAR is better. Many ADCs approved in recent years have DAR values reaching nearly 8, and new ADCs currently in clinical trials have diverse DAR values ranging from 1 to 15.

[0215] Antibody-Drug Conjugates (ADCs) using camptothecin-based payloads have attracted great interest as a new approach for treating various cancers, especially solid tumors. Among such ADCs, new ADCs (e.g., Enhertu) containing newly synthesized camptothecin-based payloads (e.g., DXd) optimized for the ADC modality have recently attracted particular interest due to their successful clinical results.

[0216] The success of such ADCs is due in part to the fact that the new camptothecin has features such as (1) excellent cell growth inhibition, (2) a better safety profile than existing ADC payloads, (3) optimized bystander effect, and (4) excellent physicochemical properties suitable for generating high DAR ADCs. However, despite the remarkable success of ADCs that utilize camptothecin-based payloads such as Enhertu or DS-1062a, as described above, there are still clear requirements that have not been met, including improvement of the safety profile (minimization of interstitial lung disease (ILD) and neutropenia) and the development of new ADCs with multiple MoA payloads to address cancer heterogeneity.

[0217] To address such unmet requirements, the present invention provides an active camptothecin derivative of Chemical Formula 1 designed to bind to DDX5 protein and E3 ligase with a molecular glue degrader as a drug candidate providing various physicochemical properties. Furthermore, a new camptothecin-based payload (PBX series compound) represented by Chemical Formula 1 was synthesized (Production Examples 1 to 4) and evaluated (Examples 2 to 10).

[0218] The new PBX series compounds represented by Chemical Formula 1 have an excellent safety profile, are derivatives that nucleophilize FL118 of Chemical Formula 2, which is a potent dual Top1 / anti-cell death pathway inhibitor, show potent ex-vivo cytotoxicity comparable to Dxd, show an excellent safety profile in preliminary toxicity studies in mice, and show rapid clearance in PK studies (Example 4, Tables 4 and 5). This minimizes systemic side effects when the linker is cleaved early during use as an ADC payload.

[0219] As shown in Figure 17 (Example 5), which is the experimental result of the conversion from the lactone form to the carboxylate at pH 7.4, the PBX-7014, PBX-7016, and PBX-7024 compounds represented by Chemical Formula 1 are not only faster but also converted to the carboxylate form (inactive form) that is inactive as a TOP1 poison in the lactone form (active form) at a higher rate compared to other reference compounds (exatecan, DXd, SN-38, FL118) at pH 7.4, which is the pH of blood or extracellular fluid.

[0220] In particular, the degree (63 - 86%) and rate (0.4 - 0.8% / min) of the formation of the carboxylate form, which is inactive as a TOP1 poison at blood pH 7.4, vary greatly depending on the type of camptothecin derivative (Figure 17). PBX-7014 and PBX-7016 formed more than 86% of the carboxylate form and were relatively rapidly converted from the lactone form to the carboxylate form at a level of 0.8% per minute. The competitive drug DXd formed a carboxylate form at a level of 76.5% and was converted from the lactone form to the carboxylate form at a level of 0.6% per minute.

[0221] The formation rate of the lactone form, which is active as a TOP1 inhibitor at pH 6.0, the pH of the micro-tumor environment (TME) (measuring the carboxylate form), was confirmed to be at a similar level for all evaluated drugs (forming a carboxylate form at a level of 20% after approximately 800 minutes) (Figure 18).

[0222] Through this, compounds represented by Chemical Formula 1, such as PBX-7014, PBX-7016, and PBX-7024 compounds, form the carboxylate form, which is an inactive form rather than the lactone form that shows cytotoxicity rapidly and at a high rate in blood or extracellular fluid (normal tissues other than tumor tissues) compared to the reference compound. By having relatively low toxicity, that is, it is predicted to show high safety in normal cells. However, in the micro-tumor environment, PBX-7014, PBX-7016, and PBX-7024 compounds represented by Chemical Formula 1 all form the lactone form with activity at the same level as other TOP1 inhibitors, without factors reducing efficacy.

[0223] Additional evaluation of the ADC payloads linked to various antibodies including trastuzumab (HER2 antibody), nimotuzumab, and sacituzumab among the PBX series compounds represented by Chemical Formula 1 For this purpose, two lead compounds, PBX-7014 and PBX-7016, were selected.

[0224] Trastuzumab-7014 and trastuzumab-7016 (DAR7-8) were easily synthesized without aggregation problems by linking PBX-7014 or PBX-7016 to trastuzumab with an existing enzymatically cleavable linker in the same manner for comparative evaluation with Enhertu (dxd-trastuzumab) (Production Example 5, Figures 19 and 20).

[0225] In Her2-high cell line (MDA-MB-453) and Her2-low / mid cell line (FaDu), these two new ADCs showed superior cytotoxicity compared to DS-8201a (anti-HER2-DXd), while in Her2-negative cell line (MDA-MB-468), they showed a cytotoxicity level similar to DS-8201a (Example 7 and Figure 22).

[0226] In the NCI-N87CDX model with high Her2 expression, trastuzumab-7014 and trastuzumab-7016 not only showed similar or even better clear dose-dependent anti-cancer efficacy compared to the same dose of DS-8201 (Example 8, Figures 23 and 24), but trastuzumab-7016 also showed a bystander effect similar to that of Enhertu (Example 9, Figures 25 and 26).

[0227] Not only did trastuzumab-7014 and trastuzumab-7016 exert a tumor regression effect through the bystander effect in tumor tissues for more than 20 days after administration, but the difference in just one methyl group also resulted in different ADME for each other, showing not only dose-dependent anti-cancer efficacy differences (conc.vs.effect) in targeted tumor tissues, but also differences in anti-cancer efficacy over time (effect vs.time) (Example 8, Figures 23 and 24).

[0228] When the hydrophobicity of the linker and the drug increases, problems such as increased aggregation of ADC and the resulting decrease in the therapeutic index of the drug are caused. Surprisingly, although the hydrophobicity of trastuzumab-7016 increases due to the difference in just one methyl group compared to trastuzumab-7014, it was superior in dose-dependent anti-cancer efficacy (conc.vs.effect) and anti-cancer efficacy over time (effect vs.time) (Example 8, Figures 23 and 24).

[0229] However, in some cases, being superior in anti-cancer efficacy over time can also increase side effects (persistent T cell activation due to chronic antigen exposure and accumulation of inhibitory signals and subsequent T cell exhaustion caused thereby, side effects due to the peripheral cell killing effect on fibroblasts derived from the connective tissue around the tumor tissue, ILD, neutropenia). The side effects may also increase For example, depending on the drug modality (small molecule vs. ADC) of the active form camptothecin derivative of Chemical Formula 1, it is important to exceed a specific drug concentration (high), and in some cases, the time maintained above a specific drug concentration (low) may be important. By providing the desired degree of hydrophobicity and / or controlling aggregation, it may be important to be removed quickly or slowly through lymphatic drainage.

[0230] Therefore, considering not only various side effects but also problems such as a decrease in the therapeutic index, it is also a major feature of the present invention that the selection range of the payload of an ADC that exhibits appropriate anti-cancer efficacy can be extended to a variety of candidate groups including the active form camptothecin derivative of Chemical Formula 1, which is designed to bind to the DDX5 protein and E3 ligase with a molecular glue degrader.

[0231] Despite the surprising success of ADCs that utilize camptothecin-based payloads such as Enhertu or DS-1062a, as candidates for improving the safety profile (minimizing ILD and neutropenia) and multiple MoA payloads to address cancer heterogeneity, by diversely providing the active form camptothecin derivative represented by Chemical Formula 1, which is designed to bind to the DDX5 protein and E3 ligase according to the present invention, one or more appropriate drugs among the active form camptothecin derivatives of Chemical Formula 1 can be selected so that the desired efficacy (e.g., anti-cancer, combination therapy) and side effects (cancer-promoting inflammation, drug resistance, ILD, neutropenia) can be precisely controlled. Furthermore, an appropriate linker can be selected for this, and an appropriate dose-dosage can be applied.

[0232] [ADC Toxicity Profile, Non-Selective Uptake, and Bystander Effect] The therapeutic index of a drug is a measure that assesses the safety and efficacy of a drug in medical treatment. It is defined as the ratio between the dose of a drug that produces a therapeutic effect and the dose that causes toxicity or adverse effects. That is, it indicates the range between the therapeutic dose and the toxic dose of a drug.

[0233] A high therapeutic index indicates a wide margin of safety where the effective dose is significantly lower than the toxic dose. This means that the drug can be administered at therapeutic levels without inducing serious side effects or toxicity. Drugs with a high therapeutic index are generally regarded as safer and more preferable for clinical use.

[0234] On the other hand, a low therapeutic index means a narrow safety margin. In such cases, the effective dose and the toxic dose are relatively close, and there is a high risk of side effects and toxicity when using the drug. Drugs with a low therapeutic index require careful monitoring and accurate dosing to avoid harming the patient.

[0235] The therapeutic index is an important consideration in drug development as it helps determine the dosage range that can provide the desired therapeutic effect while minimizing the risk of side effects. When prescribing a drug, it provides useful information to evaluate the overall benefit-to-risk ratio of the drug.

[0236] Antibody-drug conjugates (ADCs) are rapidly growing anti-cancer therapeutics, and more than 100 ADCs are in clinical trials. Currently, gemtuzumab ozogamicin (Mylotarg) , Brentuximab Vedotin (Adcetris), Inotuzumab Ozogamicin (Besponsa), Trastuzumab Emtansine (Kadcyla), Polatuzumab Vedotin (Polivy), Enfortumab Vedotin (Padcev), Trastuzumab Deruxtecan (Enhertu), Sacituzumab Govitecan (Trodelvy), Twelve ADCs such as Belantamab Mafodotin (Blenrep), Loncastuximab Tesirine (Zynlonta),Tisotumab Vedotin (Tivdak), and Mirvetuximab Soravtansine (Elahere) have been approved by the US Food and Drug Administration (FDA). Furthermore, a relatively small number of payload molecules (e.g., MMAE, MMAF, DM1, DM4, Calicheamicin, SN38, Dxd, PBD) are used in many approved and developing ADCs.

[0237] Some ADCs have demonstrated sufficient efficacy and safety to receive FDA approval, but when all ADCs are used clinically, they induce significant toxicity in treated patients, and many ADCs have failed during clinical development due to unacceptable toxicity profiles. This is because off-site toxicity remains a problem and limits the ADC dose to levels below those required for substantial anti-cancer efficacy. Even in the case of FDA-approved ADCs, a significant number of treated patients require adjuvant therapy to reduce the severity of ADC-related toxicity, and many patients require dose reduction, treatment delay, or treatment interruption.

[0238] Analysis of clinical data has demonstrated that dose-limiting toxicities (DLTs) are often shared among various ADCs that deliver the same cytotoxic payload, regardless of the target antigen and / or the type of cancer being treated. DLTs are generally associated with cells and tissues that do not express the target antigen (i.e., off-target toxicity) and often limit the ADC dose to levels below those required for optimal anti-cancer effects.

[0239] An ADC has the potential to improve the efficacy of cytotoxic drugs more safely than when used as single agents. An ADC attaches a drug to an antibody so that the antibody specifically targets only the lesion site, ensuring that the drug is not delivered to normal tissues but only to the lesion site. In the case of cancer cells, a highly toxic drug is specifically delivered to cancer cells using an antibody that specifically binds to a specific antigen expressed on the surface of cancer cells, killing only the cancer cells.

[0240] For an ADC to function, it must enter the target cell. After the antibody of the ADC specifically binds to a specific antigen expressed on the surface of a target cell such as a cancer cell, it enters the target cell through the clathrin-coated pit mechanism of the cell membrane.

[0241] The ADC incorporated into the cell is separated from clathrin, fuses with other vesicles within the cell, and then follows the endosome-lysosome pathway. After reaching the endosome, the drug is separated from the antibody by specific elements of the internal environment of the specific tumor cells here. The free cytotoxic drug, which has become independent of the antibody, penetrates the lysosomal membrane and enters the cytoplasm. The activated drug exerts its pharmacological effect by binding to its molecular target in the vicinity, inducing cell death and killing cancer cells.

[0242] During such activity, some cytotoxic drugs are passively diffused, actively transported, or excreted extracellularly through dead cells. When the drug thus spread to the surroundings penetrates the cell membrane, there may be a bystander cell-killing effect in which it enters adjacent cells and kills the surrounding cells together (the so-called by-stander cell-killing phenomenon).

[0243] In contrast to Kadcyla's DM1, Enhertu's DXd has high membrane permeability, and as a result, it has also been reported that the payload released from inside the cell is transmitted to cells that do not express HER2 adjacent to the target cell. This indicates that Enhertu has a potential bystander effect due to the characteristics of the payload and can provide clinical benefits to patients who are resistant to Herceptin or Kadcyla.

[0244] A significant number of cancer-specific antigens are expressed restrictedly on the surface of cancer cells. In such cases, since it is not easy to deliver a sufficient amount of the cytotoxic drug into cancer cells with ADC, as an alternative, the intensity of the toxin is increased.

[0245] Also, since the drug must be conjugated while minimally affecting the antibody, the amount of payload that can be carried is limited. This indicates that cytotoxic drugs for application to ADC must be able to kill many tumor cells at low concentrations (nM or pM) and show a therapeutic effect while enabling the regulation of drug release.

[0246] As the cytotoxic drug conjugated to ADC, a drug stronger than general anticancer agents has been used. The potency of the cytotoxic drug (payload) conjugated to ADC is usually 100 to 1000 times greater than the potency when the toxic drug is used alone.

[0247] Most of the potent cytotoxic drugs introduced into ADC are very toxic, and the drug payload released from ADC can affect normal cells due to the bystander effect (Figure 31).

[0248] Therefore, it is necessary to develop an ADC that acts very specifically on target cancer cells without inducing serious side effects in normal cells.

[0249] Fibroblasts, which are a type of connective tissue, are the dominant cells in the stroma, especially in the cases of breast cancer, prostate cancer, and pancreatic cancer.

[0250] Stromal fibroblasts, also known as cancer-associated fibroblasts (CAFs), are a specific type of fibroblasts found within the tumor microenvironment. Since stromal fibroblasts play an important role in tumor growth, invasion, and metastasis, they have received significant attention. CAFs are responsible for the production of paracrine growth factors, proteolytic enzymes, and ECM components.

[0251] According to the present invention, the camptothecin-based drug represented by Chemical Formula 1 is a hydrophobic low-molecular-weight compound that can penetrate cell membranes. Therefore, it can penetrate deep into cancer tissues and accumulate at high concentrations. It can penetrate the cell membrane and exert cytotoxicity inside the cell. After killing the cells, it is released and can continuously penetrate the cell membrane of surrounding cells and move into the cells to act. In this case, it has a high peripheral cell killing effect, which may be advantageous for the treatment of heterogeneous tumors. However, in some cases, in order to provide an approach (modality) to alleviate or prevent the camptothecin-based payload-based ADC toxicity such as ILD, which is a type of autoimmune disease, the camptothecin-based payload released from the ADC does not exert a peripheral cell killing effect on the fibroblasts around cancer cells. For example, it can be designed / selected from a variety of candidate groups containing the active camptothecin derivative of Chemical Formula 1 that does not penetrate the cell membrane of fibroblasts, has a high IC50, or is quickly removed through lymphatic drainage and does not accumulate at high concentrations. Furthermore, its dosage and usage can be appropriately applied to precisely control the desired efficacy (e.g., anti-cancer, combination therapy) and side effects (chronic cancer antigen exposure, drug resistance, ILD).

[0252] Also, the therapeutic index of the camptothecin-based drug as the payload To increase the [index] and suppress the non-selective uptake of camptothecin-based drugs and / or ADCs released from dead cells, the present invention can select the payload of ADCs from the group of camptothecin-based drug candidates represented by Chemical Formula 1. For example, by selecting a camptothecin-based drug with a high killing effect on target cancer cells at a low concentration of the drug (payload) from the candidate drugs represented by Chemical Formula 1, through an ADC dosage that reduces the total concentration of the ADC payload, as shown in Figure 31, it is possible to suppress the by-stander cell-killing phenomenon of the free drug (payload) released from dead cells on normal cells or overcome the problem of off-target toxicity of the free camptothecin-based drug (payload) released from target / non-target apoptotic cells.

[0253] [Cell death of fibroblasts, which are members of the connective tissue surrounding cancer cells or tumor tissue, and the resulting immune response] Unlike SN-38, the camptothecin-based compound represented by Chemical Formula 1 is a small molecule drug that suppresses the Bcl family, such as survivin, which is a resistance protein involved in the resistance mechanism, or binds to and degrades the oncogenic protein DDX5 (p68), which controls c-Myc, survivin, and mutant Kras.

[0254] In addition, the camptothecin-based compound represented by Chemical Formula 1 is designed to bind to the DDX5 protein and E3 ligase, and as a camptothecin-based cytotoxic drug that degrades the DDX5 protein, it can kill cells through a mechanism of action (MoA) that degrades the oncoprotein DDX5, along with its type I topoisomerase inhibitory ability.

[0255] The signal transduction pathways that regulate cell death are a complex network of intracellular signal transduction pathways that are tightly regulated by a variety of proteins and can be activated by various internal or external stimuli such as DNA damage, oxidative stress, and growth factor deficiency. Dysregulation of such pathways can induce a variety of diseases including cancer and neurodegenerative disorders.

[0256] All of the main signal pathways that regulate cell death converge on the activation of caspases, a cysteine protease system that cleaves various cellular substrates and ultimately leads to cell death. Caspase activation is tightly regulated by a variety of proteins including Bcl-2 family proteins, which can either promote or inhibit apoptosis.

[0257] The Bcl-2 protein family includes both pro-apoptotic and anti-apoptotic members that can either promote or inhibit cell suicide, respectively. The balance between pro-apoptotic and anti-apoptotic Bcl-2 family members is important in determining the fate of the cell and its sensitivity to cell death.

[0258] Other signal pathways such as the p53 pathway and the PI3K / Akt pathway can also regulate cell death. The p53 pathway is activated in response to DNA damage and leads to the transcription of pro-apoptotic genes such as Bax and Puma. On the other hand, the PI3K / Akt pathway can phosphorylate and inactivate the same pro-apoptotic proteins as Bad and Bim, and can inhibit apoptosis.

[0259] Some forms of necroptosis can be a programmed cell response to stimuli such as infection or DNA damage.

[0260] Immunogenic cell death is accompanied by changes on the cell surface, release of cancer antigens and damage-associated molecular patterns, etc. during the process of cancer cells being damaged and dying. The released immunomodulators can stimulate dendritic cells and macrophages, which are immune cells to induce an anti-tumor immune response.

[0261] Apoptosis is a way for damaged or aged cells to die on their own, accompanied by the deformation and decomposition of various intracellular substances. In this process, cancer antigens that induce in vivo immunity against cancer cells, immune-inducing substances such as damage-associated molecular patterns (DAMPs), are damaged by proteolytic enzymes, oxidation, etc., and the immunotherapeutic effect against residual cancer and metastatic cancer after treatment can be limited.

[0262] Cell death caused by reactive oxygen species generated from chemotherapeutic agents is mainly apoptosis involving the protease caspase.

[0263] Necroptosis, a type of necrosis that causes cell death by disrupting the cell membrane, is different from apoptosis involving catabolic enzymes such as caspase, and proteolysis, oxidation, etc. do not occur, so damage to cancer markers and immune-inducing substances can be minimized.

[0264] Damage-associated molecular patterns (DAMPs) are molecules that can induce an intracellular immune response released during the process of cell death and stimulate immune cells. Typically, calreticulin, heat shock protein 70 / 90, HMG box protein 1, ATP, etc. are examples.

[0265] Note that inflammation is a natural reaction of the body to infection or injury, including the release of inflammatory cytokines and the mobilization of immune cells to the affected site. Also, if inflammation persists for a long time, it can damage tissues and increase the risk of cancer development.

[0266] T cell exhaustion can also occur due to chronic antigen exposure in relation to cancer. Tumor cells express antigens that can be recognized by T cells, but continuous exposure to such antigens can cause T cells to become exhausted. As a result, the anti-tumor response weakens and tumors can grow and spread.

[0267] Generally, the relationship between T cell exhaustion, chronic antigen exposure, and disease is complex and multifaceted.

[0268] As described above in relation to the clinical results and side effects of ADCs with Camptothecin Payloads, ADCs with Camptothecin Payloads can also fall under the category of drugs that induce an immune response (in some cases, pneumonia / ILD side effects) through cell death.

[0269] One aspect of the present invention is to administer, in a dosage that can stimulate antigen-presenting cells (APCs) through cell death of cancer cells and induce an anti-tumor immune response, (a) an active camptothecin derivative represented by the above Chemical Formula 1, which is designed to bind to DDX5 protein and an E3 ligase; or (b) its prodrug, preferably a carrier-drug complex (CDC), more preferably an antibody-drug complex (ADC), which is designed to release the active camptothecin derivative (a) from an in vivo target site.

[0270] Here, the antigen-presenting cells (APCs) can be dendritic cells and macrophages.

[0271] Also, one aspect of the present invention is chronic antigen expo To prevent T cell exhaustion (T cell exhaustion) by (sure) or to control the hydrophobicity and / or aggregation to the desired degree so that it is removed quickly or slowly through lymphatic drainage, a method for producing an active camptothecin derivative (a)-based anticancer agent in which the bystander effect and / or ADME profile of the active camptothecin derivative (a) are controlled, The active camptothecin derivative (a)-based anticancer agent is an anticancer agent form containing (a) an active camptothecin derivative designed to bind to the DDX5 protein and an E3 ligase with a molecular glue degrader; or (b) a prodrug thereof designed to release the active camptothecin derivative (a) from an in vivo target site, preferably a carrier-drug complex (CDC), more preferably an antibody-drug complex (ADC); Provided is a method for producing an anticancer agent, characterized by including the step of designing the active camptothecin derivative (a) or its prodrug (b) to contain the camptothecin-based skeleton represented by the chemical formula 1 as a mother nucleus, selecting a compound designed in this way, or synthesizing a compound designed in this way.

[0272] Related to the clinical results and side effects of trastuzumab deruxtecan (Enhertu) As described above, the anticancer cell-mediated action that can be caused by a camptothecin-based payload-based ADC can provide a treatment method that targets cancer cells through the immune system. The anticancer cell-mediated action mainly means attacking cancer cells using T cells and natural killer cells (NK cells).

[0273] Anticancer cells caused by trastuzumab deruxtecan (Enhertu) Mediation is a method that is very useful for cancer treatment, but side effects may often occur. The most frequent of these side effects is the occurrence of autoimmune diseases. Autoimmune diseases mean a state in which the immune system mistakenly attacks its own normal tissues.

[0274] During anti-cancer cell-mediated therapy, there is a possibility of developing autoimmune diseases because anti-cancer cell-mediated action can attack not only cancer cells but also normal cells. Common side effects may include inflammation, fatigue, rash, fever, etc. caused by drugs that induce immune reactions. However, in the case of severe autoimmune diseases, it can affect various tissues and organs such as the skin, joints, kidneys, liver, lungs, etc. (e.g., ILD).

[0275] The camptothecin-based payload released from the camptothecin-based payload ADC has a high peripheral cell killing effect in addition to the cell death of target cells, which may be advantageous for the treatment of heterogeneous tumors. However, in some cases, autoimmune diseases such as ILD may occur due to the cell death of fibroblasts derived from connective tissues around the tumor tissue.

[0276] Considering such points, although trastuzumab-7016 has an increased hydrophobicity due to a difference of one methyl group from trastuzumab-7014 and is excellent in dose-dependent anti-cancer efficacy and anti-cancer efficacy over time (Example 8, Figures 23 and 24), from the perspective of side effects such as T cell depletion, ILD, and / or neutropenia, in order to control the peripheral cell killing effect, not only low concentrations / dosing amounts of trastuzumab-7016 but also treatment with trastuzumab-7014 may be required.

[0277] Only by knowing the properties of a drug can it be used correctly. Pharmacodynamic and pharmacokinetic parameters of a drug are helpful for understanding the properties of the drug.

[0278] Pharmacodynamics explains the size and pattern of changes (drug effects, effects) (cell survival, clinical effects) (therapeutic effects, toxic effects, side effects) that occur in cells and the body after a drug binds to a receptor in relation to drug concentration.

[0279] PK (Pharmaco-kinetics) indicates how drug concentration changes when a drug or drug modality moves through ADME and into other compartments of the body. Examples 8, Table 6, Figures 23 and 24 can predict the drug concentration of the active form of camptothecin derivative (a) in tumor tissue over time for various drugs / modalities.

[0280] From a pharmacokinetic perspective, reasons for failure in new drug development include that toxic drugs, such as the camptothecin-based payload released from a camptothecin-based payload ADC, can accumulate, and useful drugs may not be beneficial because the doses are too low to establish treatment, and drugs can be rapidly metabolized.

[0281] Therefore, to use drugs correctly, for example, to control the peripheral cell killing effect through lymphatic drainage, not only should an appropriate camptothecin-based payload of Chemical Formula 1 be selected to control the hydrophobicity and / or aggregation of the camptothecin-based payload of Chemical Formula 1, but also an appropriate dose-dosage should be applied. The dose indicates the amount of drug administered at one time. The dose of a drug is generally prescribed by a healthcare provider based on factors such as the patient's age, weight, and health status. On the other hand, the dosage indicates the frequency and duration of drug administration. It is a measurement of the total amount of drug given over a certain period and is generally expressed as the amount per day or per week. Dose and dosage are important considerations for the safe and effective use of drugs.

[0282]

[0283] ​From such a perspective, the present invention provides various active camptothecin derivatives of Chemical Formula 1 that bind to DDX5 protein and E3 ligase with a molecular glue degrader as various drug candidates, and by selecting one or more appropriate drugs and appropriately applying their dosage and usage, the desired efficacy (e.g., anti-cancer, combination therapy) and side effects (chronic cancer antigen exposure, drug resistance, ILD) can be precisely controlled.

[0284] For example, it is important for the active camptothecin derivative of Chemical Formula 1 to exceed a specific drug concentration (high) in the tumor tissue from the perspective of controlling the peripheral cell killing effect in terms of cell death of heterogeneous tumor cells and / or fibroblasts derived from the connective tissue surrounding the tumor tissue by the drug modality (small molecule, ADC). In some cases, the time maintained above a specific drug concentration (low) in the tumor tissue may be important, and it may be important to be removed quickly or slowly through lymphatic drainage. This can be achieved by selecting an appropriate camptothecin-based payload of Chemical Formula 1 and controlling the hydrophobicity provision and / or aggregation to the desired degree. It can be realized by selecting an appropriate camptothecin-based payload of Chemical Formula 1 and controlling the hydrophobicity provision and / or aggregation to the desired degree.

[0285] [Pro-drug] As used herein, prodrugs include, but are not limited to, prodrugs that are biologically activated or further activated after being placed in a predetermined physiological environment.

[0286] For example, a physiologically active subst It refers to a compound that chemically modifies a prodrug or a therapeutically active organic compound and is designed to release or liberate the parent compound in vivo under enzymatic or other conditions. A prodrug changes into the target compound in vivo after administration. Despite being useful drugs, chemical modifications are made to those with properties that do not conform in terms of side effects, stability, solubility, absorbability, duration of action, etc. to make them clinically applicable.

[0287] As an example of a prodrug, an antibody-drug conjugate (ADC) in which a cytotoxic drug is conjugated to an antibody that is expressed on the surface of cancer cells and can be internalized by the cells is expected to selectively deliver the drug to cancer cells, accumulate the drug in cancer cells, and kill the cancer cells by being able to do so.

[0288] Among the components constituting the ADC, the linker is what binds the antibody and the cytotoxic drug.

[0289] The linker must be stable in the bloodstream, prevent the drug from dissociating from the antibody, be maintained in a prodrug state until it reaches the target, and minimize the damage to normal tissues. The most ideal linker is one that is stable while the ADC is in systemic circulation but is cleaved in target cells, appropriately releases the cytotoxic drug, safely delivers the drug to the target, and enables the ADC to have both efficacy and safety.

[0290] When binding the drug to the antibody with the linker, the drug must not affect the structural stability, substrate-binding properties, and pharmacokinetics of the antibody. One of the reasons for the early failure of ADC drugs is known to be the early release of the drug.

[0291] A significant number of ADCs currently in clinical trials employ chemical linkers such as hydrazone, disulfide, peptide, or thioether linkages. The process of drug release from the linker utilizes differences in specific pH, enzyme concentration, etc. within cancer cells. Generally, hydrazone and disulfide linkers have limited stability in plasma. On the other hand, peptide-based linkers have excellent plasma stability and are easy to regulate drug release.

[0292] Some chemical linkers regulate the balance of hydrophobicity between the antibody and the drug, preventing the aggregation of ADCs in the hydrophilic bloodstream environment. Hydrophilic linkers and spacers include cyclodextrin, polyethylene glycol (PEG), or other polymers, which play a role in stability in the bloodstream and pharmacokinetic properties.

[0293] The linkers used in ADCs can be divided into non-cleavable and cleavable types based on their cleavage ability.

[0294] The non-cleavable linker (Non-cleavable linker) is represented by the thioether linker. Compared with cleavable linkers, it has relatively high plasma stability and is resistant to proteolysis. Different from cleavable linkers, non-cleavable linkers do not undergo self-degradation. Therefore, when the antibody is degraded after intracellular introduction in the ADC form, it has the property of enabling drug release.

[0295] The drug released in this way is charged, and the possibility of a bystander effect occurring in surrounding cells is low. There is no bystander effect showing toxicity to surrounding cells, but the effect only appears in target cells after target cell internalization, which means relatively high safety. It can be seen that ADCs manufactured with non-cleavable linkers are more dependent on the intracellular biological mechanism of target cells. In many studies, ADCs with non-cleavable linkers show high stability and efficacy and are utilized as linkers for ADC development. Currently, an ADC to which this technology has been applied is Kadcyla (registered trademark).

[0296] The cleavable linker is cleaved in response to specific environmental factors to release the drug into the cytoplasm. Cleavable linkers include enzyme-cleavable and non-enzyme-cleavable types.

[0297] Enzyme-cleavable linkers are cleaved by enzymes such as cathepsin B, β-glucuronidase, phosphatase, pyrophosphatase, and sulfatase.

[0298] Peptide linkers are degraded by proteolytic enzymes. Protease inhibitors are present in plasma, so peptide linkers have high plasma stability. Cathepsin B is a typical proteolytic enzyme used in ADCs. Cathepsin B is present at high levels in tumor tissues and confers tumor selectivity to ADCs. Peptide linkers have mainly been developed as dipeptides formed by binding two amino acids and have been applied to Adcetris (registered trademark).

[0299] Peptide linkers are composed of dipeptides or tetrapeptides that are recognized and cleaved by lysosomal proteolytic enzymes when the ADC is internalized. Tetrapeptides were used in the early stages of development, but relatively slow drug release and the potential for aggregation when combined with hydrophobic drugs became apparent. Such problems have been solved with the development of dipeptide linkers such as Val-Cit, Phe-Lys, Val-Lys, and Val-Ala, and have been successfully applied to some ADCs such as Adcetris (registered trademark) and Vedotin (registered trademark). have been successfully applied to several ADCs.

[0300] β-Glucuronide linkers are degraded by β-glucuronidase, a glycolytic enzyme present in lysosomes. β-Glucuronidase is overexpressed in some tumor cells and confers tumor specificity. This enzyme has high activity at low pH but its activity decreases to 10% at neutral pH. Due to such characteristics, ADCs with β-glucuronide linkers have improved stability in plasma and prevent drug release outside the target.

[0301] For non-enzymatic cleavage types, there are acid-labile linkers and oxidation-reduction reaction linkers.

[0302] Acid-labile linkers are chemically unstable linkers that were developed in the early stage of ADC development. They have low stability but are still in use. A typical linker is the hydrazone linker, which is stable at pH 7.3 - 7.5, the neutral environment of the blood, but is hydrolyzed in weakly acidic environments such as those around tumor cells (pH 6.5 - 7.2) or endosomes (pH 5.0 - 6.5) and lysosomes (pH 4.5 - 5.0) where intracellular internalization occurs, releasing the drug. However, the acidic state is not limited to the tumor microenvironment and is often found outside cells as well, so non-specific drug release may occur.

[0303] For oxidation-reduction reaction linkers, the disulfide linker is typical. The disulfide linker is also a type of chemically unstable linker and is based on oxidation-reduction reactions. After internalization, the linker is decomposed by reducing agents such as disulfide exchange or glutathione, releasing the cytotoxic drug.

[0304] Glutathione is a low-molecular-weight thiol and is known as an antioxidant that regulates cell growth and death and protects cells from inflammation and oxidative stress. Glutathione exists intracellularly at a concentration of 0.5 - 10 mM, but in tumors in a hypoxic state, it exists at a concentration up to 1000 times higher. In plasma, it exists at a low concentration (2 - 20 μM), and the disulfide linker has high plasma stability, thereby reducing non-specific drug release and is a linker that reacts specifically with tumors relatively safely.

[0305] The use of different linkers affects the efficient release of cytotoxic drugs.

[0306] The development of linkers reflects the long half-life, which is an advantage of monoclonal antibodies (mAbs). It is important that mAbs must be stable in the systemic circulation and that the binding of the linker to the cytotoxic drug does not affect the stability and pharmacokinetics of the antibody.

[0307] Generally, the catabolic effects that can occur in the systemic blood circulation related to the linker-cytotoxic drug are as follows, and there are also various catabolic effects that have not been clearly revealed: hydrazone cleavage, protease-mediated dipeptide cleavage, esterase-mediated carbamate cleavage, hydrolysis of acetate ester, disulfide cleavage, succinimde ring opening.

[0308] The catabolic effects occurring at specific positions of the linker-cytotoxic drug may, in some cases, maintain the cytotoxic effect and show activity against the target, and may cause toxicity in the systemic blood circulation. Conversely, when the cytotoxic effect is lost, even if the drug reaches the target, it may be difficult to expect a pharmacological effect. For example, in the case of tylanstatin developed as a payload as a spliceosome inhibitor, even if ester group hydrolysis occurs, the activity is maintained, while in the case of cryptophycin and tubulysin, which are tubulin inhibitors, when ester group hydrolysis occurs, the activity is lost.

[0309] The stability of the ADC during its movement to the target is very important for achieving the desired therapeutic index regardless of the type of linker used, such as cleavable or non-cleavable linkers.

[0310] An example of a prodrug of the active camptothecin derivative (a) represented by Chemical Formula 1, which is designed to bind to the DDX5 protein and the E3 ligase according to the present invention, may be one in which the active camptothecin derivative (a) represented by Chemical Formula 1 is used instead of exatecan in DE-310 described later.

[0311] As described in WO97 / 46260 (corresponding Korean Registration No. 10-2087017B1), DE-310 is a complex in which exatecan is bound to a biodegradable carboxymethyldextran polyalcohol polymer via a GGFG peptide spacer. By formulating exatecan as a polymeric prodrug, high blood retention is maintained, and by utilizing the enhanced permeability of tumor neovessels and tumor tissue retention, the passive targeting to the tumor site is enhanced. DE-310 continuously releases exatecan, which is the active entity, and exatecan to which glycine is bound to the amino group by cleavage of the peptide spacer by an enzyme. As a result, the pharmacokinetics are improved, and in various tumor evaluation models in non-clinical trials, DE-310 showed higher efficacy than when exatecan was administered alone, even though the dose of exatecan was decreased compared to when exatecan was administered alone. Regarding DE-310, there are reports that clinical trials have been conducted, effective examples have been confirmed in humans, and it has been confirmed that the active entity accumulates in tumors rather than in normal tissues. On the other hand, there are also reports that the accumulation of DE-310 and the active entity in tumors in humans is not significantly different from the accumulation in normal tissues, and passive targeting was not observed in humans.

[0312] As a related compound of DE-310, a complex in which a structure represented by -NH(CH2)4C(=O)- is inserted between the -GGFG- spacer and exatecan, and -GGFG-NH(CH2)4C(=O)- is used as the spacer structure is also known.

[0313] An example of a prodrug of the active camptothecin derivative (a) represented by Chemical Formula 1, which is designed to bind to the DDX5 protein and E3 ligase according to the present invention, may include a linker and / or spacer designed to release the active camptothecin derivative (a) from the in vivo target site.

[0314] Non-limiting examples of self-immolative spacers are illustrated in FIG. 32.

[0315] According to the present invention, the active camptothecin derivatives represented by the following Chemical Formula 1, which are designed to bind to the DDX5 protein and E3 ligase, for example, the camptothecin-based drugs of Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9 can be used as payloads for various carrier-drug conjugates. That is, various carrier-drug conjugates in which the active camptothecin derivative is linked with the payload are a kind of prodrug of the active camptothecin derivative that releases the active camptothecin derivative in vivo. Accordingly, the present invention provides a carrier-drug conjugate comprising a compound which is the active camptothecin derivative represented by Chemical Formula 1, preferably a compound of Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9.

[0316] In the carrier-drug conjugate according to the present invention, the carrier may be, but is not limited to, an antibody, a peptide, a repebody, and / or an aptamer. [Table 2]

[0317] One type of prodrug of the present invention, an antibody-drug conjugate (ADC), comprises an immunocomplex containing an antibody or a fragment thereof containing an antigen-binding site, or a pharmaceutically acceptable salt thereof, designed to release the active camptothecin derivative (a) of the present invention in vivo.

[0318] As the monoclonal antibody used in ADC, IgG1, IgG2, and IgG4 are used, among which IgG1 is most frequently used. In traditional ADCs, full-length antigens were used. As a strategy to enhance absorption and permeation, attempts have been made to use smaller Fab, scFv, and diabody instead of monoclonal antibodies. Since the immunocomplex of the present invention specifically binds to cancer cell antigens and releases drugs inside and outside cancer cells to exhibit cytotoxicity, it can be usefully used for the treatment or prevention of cancer.

[0319] For example, an antibody-drug conjugate (ADC) is a new drug platform that selectively delivers a payload with strong anti-cancer efficacy only to cancer tissue by using the high tissue selectivity of an antibody that specifically binds to a specific antigen expressed on the surface of cancer cells. ADC can selectively deliver a powerful payload that kills cancer cells even at low concentrations in the pM level only to cancer tissue, minimizing drug exposure to the whole body and ensuring both anti-cancer efficacy and safety.

[0320]

[0321] ​The intracellular introduction of most ADCs proceeds through the clathrin-coated pit function. The ADCs that have moved into the cell separate from clathrin, fuse with other vesicles in the cell, and then proceed through the endosome-lysosome pathway. Next, proteases in the acidic environment of the endosome cleave the linker, and the activated "free" drug passes through the lysosomal membrane and moves into the cytoplasm, and then binds to the molecular target of the drug, causing the cell cycle of tumor cells to arrest and cancer cells to die by apoptosis. Among these, a certain amount of the drug diffuses passively in the cell, is actively transported, or flows out of the cell through dead cells. At this time, if the leaked drug has cell membrane permeability, it may also enter the surrounding cells, causing the so-called bystander killing phenomenon.

[0322] An aptamer-drug conjugate (ApDC) is obtained by introducing an aptamer instead of an antibody in an ADC. An aptamer is a single-stranded nucleic acid with a three-dimensional structure. It is discovered through the "SELEX" (Systematic Evolution of Ligands by Exponential enrichment) process. SELEX is a technique in which a target protein molecule is introduced into a compound library to obtain a functional nucleic acid that binds to it.

[0323] An aptamer can bind very strongly and selectively to a target and is also called a "chemical antibody". An aptamer has a size of about 20 kDa and is known to have excellent cell permeability and low immunogenicity compared to antibodies.

[0324] Since aptamers can be chemically synthesized, precise design of the conjugation position and number of conjugated drugs is possible during the production of aptamer-drug conjugates. The production cost is lower than that of ADCs.

[0325] Aptamers generally consist of natural nucleic acids, are degraded by in vivo nucleases, and have reduced in vivo stability. However, the limit of the stability of modified aptamers can be overcome by using the fact that chemical modification of aptamers is easy.

[0326] A peptide-drug conjugate (PDC) is a form in which a peptide is introduced instead of an antibody in an ADC. A peptide is composed of amino acids and has a size in the range of 500 to 5000 Da (dalton). This is a very small size compared to an antibody of 150 kDa (kilodalton) or more. Therefore, peptide-based PDCs have excellent cell penetration ability compared to ADCs and have a very low possibility of generating immunogenicity. Also, peptides can be chemically synthesized. Therefore, PDCs not only have very low production costs, but also can precisely regulate the binding position and ratio of the peptide and the drug.

[0327] Generally, peptides are easily degraded by proteolytic enzymes and thus have a short biological half-life. To overcome the limitations of such peptide-based drug conjugates, strategies have been proposed that utilize modified peptides such as cyclic peptides and introduction of unnatural amino acids.

[0328] A repebody does not have an antibody backbone but is a type of artificial antibody that has a function of recognizing an antigen like an antibody. A repebody specific to a target protein can be discovered through phage display.

[0329] The phage display method is a technique for expressing a desired protein on the surface of a bacteriophage. A repebody has a size of about 30 kDa at the 20% level of antibody drugs. Therefore, it is known to have relatively low immunogenicity and improved cell permeability compared to antibodies. In addition, it is expected that the thermal and pH stability of the repebody can be adjusted and its structural stability can be enhanced. It is also evaluated that the production cost is relatively low compared to antibodies. Due to such advantages of the repebody, interest in the development of a repebody-drug conjugate (Repebody-DC) as a strategy to change antibodies to lipibodies is also increasing.

[0330] The carrier-drug conjugate according to the present invention is characterized in that the carrier is complexed with a drug, that is, a compound which is an active camptothecin derivative represented by Chemical Formula 1, preferably a compound of Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, but is not limited thereto.

[0331] In the carrier-drug conjugate according to the present invention, the compound which is an active camptothecin derivative represented by Chemical Formula 1, preferably a compound of Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9 may be linked to a linker at an appropriate site as long as its properties such as anti-cancer activity do not change. Thus, the present invention provides a drug linker in which a compound which is an active camptothecin derivative represented by Chemical Formula 1, preferably a compound of Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9 is linked to a linker.

[0332] Preferably, the drug linker according to the present invention can have a structure of Chemical Formula 3a, Chemical Formula 3a-1, Chemical Formula 4a, Chemical Formula 4a-1, Chemical Formula 5a, Chemical Formula 5a-1, Chemical Formula 6a, Chemical Formula 7a, Chemical Formula 8a or Chemical Formula 9a, but is not limited thereto.

[0333] [Chemistry]

[0334] [Chemistry]

[0335] [Chemistry]

[0336] [Chemistry]

[0337] [Chemistry]

[0338] [Chemistry]

[0339] [Chemistry]

[0340] [Chemistry]

[0341] [Chemistry]

[0342] [Chemistry]

[0343] In the above structural formula, L means a linker.

[0344] [Target Antigen and Antibody] In an ADC, the interaction between the antibody and the target antigen is important for ensuring safety and obtaining a therapeutic effect. The two variables for antigen selection are tumor specificity and expression level. Ideally, the antigen is specifically expressed only in tumors and is not expressed or minimized in normal cells. Specificity is crucial for reducing toxicity and determining the success of an ADC. Cancer-specific antigens are expressed on the surface of tumor cells by surface receptors, within the tumor vasculature, or within the tumor microenvironment. Cancer-specific antigens are expressed on the surface of tumor cells by surface receptors, within the tumor vasculature, or within the tumor microenvironment.

[0345] When cancer-specific antigens are homogeneously expressed in tumor tissue, such as in a homogeneous tumor, and all cancer cells respond to the drug, cancer treatment becomes easier. In the case of heterogeneous tumors, non-responsive cancer cells are mixed, and there may be cancer cells that survive after ADC treatment. If the ADC has a peripheral cell killing effect, such problems of heterogeneous tumors can be overcome.

[0346] Targeting antigens internalized by tumor cells is not easy in solid tumors rich in intracellular substrates. In such cases, approach the tumor microenvironment rather than cancer cells as the target. Release cytotoxic drugs extracellularly using components such as extracellular proteins, acidic substances in the extracellular matrix, and glutathione.

[0347] Among the ADCs currently in clinical trials, a target antigen that is attractively used due to its excellent marketability is known as HER2, and three HER2-targeted ADCs are in the clinical phase III (Dean et al., 2021).

[0348] In the case of HER2-positive cancer, an ADC that is an active camptothecin derivative (a) or its prodrug targeting the DDX5 protein according to the present invention can be a new treatment method for resolving anti-HER2 treatment resistance.

[0349] To specifically deliver a potent cytotoxic drug only to specific cancer cells, determining the target antigen is the first major step in ADC development. By using an antibody, high specificity for the target and a long half-life enable long-term systemic circulation, which allows the cytotoxic drug to selectively accumulate only in tumor cells, minimizing exposure of normal tissues, reducing damage and side effects, and increasing the therapeutic effect. Therefore, a target antigen that can identify tumor cells must be sought, and the following conditions are required. First, the target antigen should be uniformly overexpressed on the surface of tumor cells and preferably have relatively low or no expression in normal cells. A typical example is the Human epidermal growth factor receptor 2 (HER2) receptor, which is known to be expressed more than 100 times more in HER2-positive breast cancer than in normal cells. Thus, before making an antibody, the tumor expression of the target antigen is analyzed through various profiling to confirm the overexpression of a specific antigen, and then a monoclonal antibody that recognizes this antigen is generated. Second, it is the binding force to the antigen. Due to the characteristic of the antibody that internalization occurs through the receptor, the stronger the binding force to the epitope of the antigen, the more internalization can occur, which can increase the therapeutic effect. Furthermore, it has low immunogenicity. Initially, first-generation ADCs were produced using antibodies produced via mice. First-generation ADCs injected mouse antibodies into humans, but side effects and antibody neutralization due to the human body's immune response to the administered mouse antibodies occurred, and the anti-cancer effect was difficult to observe. With the development of genetic engineering technology, many improvements have been shown by manufacturing chimeric antibodies, humanized antibodies, and fully human antibodies to address the problems with the immune response.

[0350] The antibody that recognizes the antigen of cancer cells must undergo cell internalization together with the drug. To enhance cell internalization in cancer cells, bispecific antibodies have been developed.

[0351] MEDI4267 (Trastuzumab-META), which is being developed by Medimmune and Astrazeneca, is a biparatopic antibody that targets two non-overlapping epitopes on HER2 and induces HER2 receptor clustering, through which it has been shown to promote endocytosis, lysosomal trafficking, and degradation.

[0352] In addition, bispecific antibodies conjugated with lysosomal markers CD63 or APLP2, or prolactin receptor, together with tumor-targeting antibodies, have been shown to improve endocytosis compared to single antibodies with tumor antigen recognition. There is a bispecific ADC targeting HER2, HER2xCD63-duostatin-3 (Creative biolabs), which has shown a strong anti-cancer effect by reducing delivery to normal tissues and enabling cancer cell-specific delivery.

[0353] When designing the antibody-drug conjugate (ADC), immunoconjugate, or carrier-drug conjugate of the present invention, the targets for targeting can be extended not only to cancer cells but also to cells associated with infectious disease organisms and / or autoimmune diseases.

[0354] Therefore, the cells targeted by the antibody or its antigen-binding site-containing fragment can be cancer cells, infectious disease organisms, and / or cells associated with autoimmune diseases.

[0355] Non-limiting examples of target antigens include antigens selectively distributed on the surface of cancers such as Her2, FolR, PSMA, and cancer cell overexpressed antigens that are also distributed in small amounts in normal tissues such as Trop2.

[0356] ​Cancer cell target antigens include, for example, 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, AFP, aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, ALCAM, ALK, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOCl, AR, aromatase , ASPH, ATX, AX1, AXL, AZGP1 (zinc-a-glycoprotein), B4GALNT1, B7, B7.1, B7.2, B7-H1, B7-H3, B7-H4, B7-H6, BAD, BAFF, BAG1, BAI1, BCR, BCL2, BCL6, BCMA, BDNF, BLNK, BLR1 (MDR15), BIyS, BMP1, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMP10, BMPR1A, BMPR1B, BMPR2, BPAG1 (plectin), BRCA1, C19orflO (IL27w), C3, C4A, C5, C5R1, CA6, CA9, CANT1, CAPRIN-1, CASP1, CASP4, CAV1, CCBP2 (D6 / JAB61), CCL1 (1-309), CCLI1 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MEP-2), SLC, exodus-2, CCL22 (MDC / STC-I), CCL23 (MPIF-I), CCL24 (MPIF-2 / eotaxin-2), CCL25 (TECT), CCL26 (eotaxin-3), CCL27 (CTACK / ILC), CCL28, CCL3 (MIP-Ia), CCL4 (MIPIb), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1 (CKR1 / HM145), CCR2 (mcp-IRB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7 (CKR7 / EBI1), CCR8 or CDw198 (CMKBR8 / TERI / CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), CD13, CD164, CD19, CDH6, CDIC, CD2, CD20, CD21, CD200, CD22, CD23, CD24, CD27, CD28, CD29, CD3, CD33, CD35, CD37, CD38, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD45RB, CD47, CD52, CD56, CD69, CD70, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD97, CD99, CD117, CD125, CD137, CD147, CD179b, CD223, CD279, CD152, CD274,

[0357] CDH1 (E-cadherin), CDH1O, CDH12, CDH13, CDH18, CDH19, CDH2O, CDH3, CDH5, CDH7, CDH8, CDH9, CDH17, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKN1A (p21Wap1 / Cip1), CDKN1B (p27Kip1), CDKN1C, CDKN2A (p16INK4a), CDKN2B, CDKN2C, CDKN3, CEA, CEACAM5, CEACAM6, CEBPB, CERI, CFC1B, CHGA, CHGB, Chitinase, CHST1O, CIK, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN6, CLDN7 (Claudin-7), CLDN18, CLEC5A, CLEC6A, CLEC11A, CLEC14A, CLN3, CLU (Clusterin), CMKLR1, CMKOR1 (RDC1), CNR1, C-MET, COL18A1, COLIA1, COL4A3, COL6A1, CR2, Cripto, CRP, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTAG1B (NY-ESO-1), CTLA4, CTL8, CTNNB1 (β-catenin), CTSB (Cathepsin B), CX3CL1 (SCYD1), CX3CR1 (V28), CXCL1 (GRO1), CXCL1O (IP-IO), CXCLI1 (1-TAC / IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16, CXCL2 (GRO2), CXCL3 (GRO3), CXCL5 (ENA-78 / LIX), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9 / CKR-L2), CXCR4, CXCR6 (TYMSTR / STRL33 / Bonzo), CYB5, CYC1, CYSLTR1, DAB2IP, DES, DKFZp451J0118, DLK1, DNCL1, DPP4, E2F1, Engel, Edge, Fennel, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, Enola, ENO2, ENO3, EpCAM, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1,EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, EPHRIN-A1, EPHRIN-A2, EPHRINA3, EPHRIN-A4, EPHRIN-A5, EPHRIN-A6, EPHRIN-B1, EPHRIN-B2, EPHRIN-B3, EPHB4, EPG, ERBB2(HER-2), ERBB3, ERBB4, EREG, ERK8, estrogen receptor, Earl, ESR2, F3(TF), FADD, FAP, farnesyl transferase, FasL, FASNf, FCER1A, FCER2, FCGR3A, FGF, FGF1(aFGF), FGF10, FGF1 1, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2(bFGF), FGF20, FGF21, FGF22, FGF23, FGF3(int-2), FGF4(HST), FGF5, FGF6(HST-2), FGF7(KGF), FGF8, FGF9, FGFR1, FGFR2, FGFR3, FGFR4, FIGF(VEGFD), FIL1(EPSILON), FBL1(ZETA), FLJ12584, FLJ25530, FLRT1(fibronectin), FLT1, FLT-3, FOLR1, FOS, FOSL1(FRA-1), FR-alpha, FY(DARC), GABRP(GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GD2, GD3, GDF5, GFI1, GFRA1, GGT1, GM-CSF, GNAS1, GNRH1, GPC1, GPC3, GPNB, GPR2(CCR10), GPR31, GPR44, GPR81(FKSG80), GRCC1O(C, 1O), GRP, GSN(Gelsolin), GSTP1, GUCY2C, HAVCR1, HAVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, Hedgehog, HER3, HGF, HIF1A, HIP1,

[0358] Histamine and histamine receptors, HLA-A, HLA-DR, HLA-DRA, HLA-E, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-α, IFNA1, IFNA2, IFNA4, IFNA5, EFNA6, BFNA7, IFNB1, IFN gamma, IFNω1, IGBP1, IGF1, IGFIR, IGF2, IGFBP2, IGFBP3, IGFBP6, DL-1, IL10, IL10RA, IL10RB, IL-1, IL1R1(CD121a), IL1R2(CD121b), IL-1RA, IL-2, IL2RA(CD25), IL2RB(CD122), IL2RG(CD132), IL-4, IL-4R(CD123), IL-5, IL5RA(CD125), IL3RB(CD131), IL-6, IL6RA, (CD126), IR6RB(CD130), IL-7, IL7RA(CD127), IL-8, CXCR1(IL8RA), CXCR2, (IL8RB / CD128), IL-9, IL9R(CD129), IL-10, IL10RA(CD210), IL10RB(CDW210B), IL-11, IL11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, DL1F9, IL1HY1, IL1R1, IL1R2, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RL1, IL1RL2, IL1RN, IL2, IL20, IL20RA, IL21R, IL22, IL22R, IL22RA2, IL23, DL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA, IL4, 1L4, IL6ST (glycoprotein 130), ILK, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (α6 integrin), ITGAV, ITGB3, ITGB4 (β4 integrin),JAG1, JAK1, JAK3, JTB, JUN, K6HF, KAI1, KDR, KIT, KITLG, KLF5 (GC Box BP), KLF6, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, KRT1, KRT19 (Keratin 19), KRT2A, KRTHB6 (Hair-specific type II keratin), L1CAM, LAG3, LAMA5, LAMP1, LEP (Leptin), Lewis Y antigen ("LeY"), LILRB1, Lingo-p75, Lingo-Troy, LGALS3BP, LRRC15, LPS, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2, LTBR, LY75, LYPD3, MACMARCKS, MAG or OMgp, MAGEA3, MAGEA6, MAP2K7 (c-Jun), MCP-1, MDK, MIB1, midkine, MIF, MISRII, MJP-2, MLSN, MK, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (Metallothioneptin-UI), mTOR, MTSS1, MUC1 (mucin), MUC16, MYC, MYD88, NCK2, NCR3LG1, Neurocan, NFKBI, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgRNogo66, (Nogo), NgR-p75, NgR-Troy, NMEI (NM23A), NOTCH, NOTCH1, NOTCH3,

[0359] NOX5, NPPB, NROB1, NROB2, NRID1, NR1D2, NR1H2, NR1H3, NR1H4, NR112, NR113, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR 4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, NY-ESO1, ODZI, OPRDI, P2RX7, PAP, PART1, PATE, PAWR, P-cadherin, PCA3, PCD1, PD-L1, PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB, PECAMI, L1-CAM, peg-asparaginase, PF4 (CXCL4), PGF, PGR, phosphacan, PIAS2, PI3 kinase, PIK3CG, PLAU (uPA), PLG, PLXDCI, PKC, PKC-beta, PPBP (CXCL7), PPID, PR1, PRAME, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PSMA, PTAFR, PTEN, PTHR2, PTGS2 (COX-2), PTN, PVRIG, RAC2 (P21Rac2), RANK, RANK ligand, RARB, RGS1, RGS13, RGS3, RNFI1O (ZNF144), Ron, ROBO2, ROR1, RXR, S100A2, SCGB 1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYE1 (endothelial mononuclear cell-activating cytokine), SDF2, SERPENA1, SERPINA3, SERPINB5 (maspin), SERPINEI (PAI-I), SERPINFI, SHIP-1, SHIP-2, SHB1, SHB2, SHBG, SfcAZ, SLAMF7, SLC2A2, SLC33A1, SLC43A1, SLC44A4, SLC34A2, SLIT2, SPP1, SPRR1B (Spr1), ST6GAL1, ST8SIA1, STAB1, STATE, STEAP, STEAP2, TB4R2, TBX21, TCP1O, TDGF1, TEK, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, THIL, THBS1 (thrombospondin-1, THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor (tissuefactor), TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TNF, TNF-a, TNFAIP2 (B94), TNFAIP3, TNFRSFI1A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6 (Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSF1O (TRAIL), TNFRSF10A, TNFRSF10B, TNFRSF12A, TNFRSF17, TNFSF1 1 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFRSF14 (HVEM), TNFSF15 (VEGI), TNFSF18, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase Iia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM2,

[0360] TROP2, TRPC6, TSLP, TWEAK, Tyrosinase, uPAR, VEGF, VEGFB, VEGFC, versican, VHL C5, VLA-4, WT1, Wnt-1, XCL1 (lymphotactin), XCL2 (SCM-Ib), XCRI (GPR5 / CCXCR1), YY1, ZFPM2, CLEC4C (BDCA-2, DLEC, CD303, CDH6, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6A (dectin-2), CLEC5A (MDL-1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), CLEC7A (dectin-1), CLEC11A, PDGFRa, SLAMF7, GP6 (GPVI), LILRA1 (CD85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), LILRB1, NCR1 (CD335, LY94, NKp46), NCR3 (CD335, L Y94, NKp46), NCR3 (CD337, NKp30), OSCAR, TARM1, CD30, CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 (CD314, NKG2D), NCR2 (CD336, NKp44), PILRB, SIGLEC1 (CD169, SN), SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPA, SIRPB1 (CD172B), TREM1 (CD354), TREM2, KLRF1 (NKp80), 17-1A, SLAM7, MSLN, CTAG1B / NY-ESO-1, MAGEA3 / A6, ATP5I (Q06185), OAT (P29758), AIFM1 (Q9Z0X1), AOFA (Q64133), MTDC (P18155), CMC1 (Q8BH59), PREP (Q8K411), YMEL1 (O88967), LPPRC (Q6PB66), LONM (Q8CGK3), ACON (Q99KI0), ODO1 (Q60597), IDHP (P54071), ALDH2 (P47738), ATPB (P56480), AATM (P05202), TMM93 (Q9CQW0), ERGI3 (Q9CQE7), RTN4 (Q99P72), CL041 (Q8BQR4), ERLN2 (Q8BFZ9), TERA (Q01853), DAD1 (P61804), CALX (P35564), CALU (O35887), VAPA (Q9WV55), MOGS (Q80UM7), GANAB (Q8BHN3), ERO1A (Q8R180), UGGG1 (Q6P5E4), P4HA1 (Q60715), HYEP (Q9D379), CALR (P14211), AT2A2 (O55143), PDIA4 (P08003), PDIA1 (P09103), PDIA3 (P27773), PDIA6 (Q922R8), CLH (Q68FD5), PPIB (P24369), TCPG (P80318), MOT4 (P57787), NICA (P57716), BASI (P18572), VAPA (Q9WV55)It may be, but is not limited to, ENV2 (P11370), VAT1 (Q62465), 4F2 (P10852), ENOA (P17182), ILK (O55222), GPNMB (Q99P91), ENV1 (P10404), ERO1A (Q8R180), CLH (Q68FD5), DSG1A (Q61495), AT1A1 (Q8VDN2), HYOU1 (Q9JKR6), TRAP1 (Q9CQN1), GRP75 (P38647), ENPL (P08113), CH60 (P63038), or CH10 (Q64433).

[0361] The target antigen may be an antigen that is distributed more than 10 times as much in cancer cells as in normal cells.

[0362] In the present invention that can be used for the antibody-drug conjugate (ADC) according to the present invention, non-limiting examples of the antibody include Urelumab, Utomilumab, Bebtelovimab, Aducanumab, Bapineuzumab, Crenezumab, Donanemab, Gantenerumab, Lecanemab, Solanezumab, Nesvacumab, Evinacumab, Enoblituzumab, Omburtamab, Belimumab, Ianalumab, Tabalumab, Bertilimumab, Mogamulizumab, Leronlimab, Siplizumab, Foralumab, Muromonab-CD3, Otelixizumab, Teplizumab, Ibalizumab, Tregalizumab, Zanolimumab, Itolizumab, Efalizumab), Inebilizumab, Tafasitamab, Tositumomab, Ocrelizumab, Ofatumumab, Rituximab, Ublituximab, Veltuzumab, Epratuzumab, Basiliximab, Daclizumab, Varlilumab, Lulizumab, Iratumumab, Lintuzumab, Daratumumab, Felzartamab, Isatuximab, Mezagitamab, Bleselumab, Dacetuzumab, Iscalimab, Lucatumumab, Mitazalimab, Sotigalimab, Dapirolizumab, Apamistamab, Ligufalimab, Magrolimab, Alemtuzumab, Crizanlizumab, Inclacumab, Cusatuzumab, Oleclumab, Milatuzumab, Galiximab, Carotuximab, Adecatumumab, Eptinezumab, Erenumab, Fremanezumab, Galcanezumab, Zolbetuximab, Onartuzumab, Eculizumab, Pozelimab, Ravulizumab, Lacnotuzumab, Axatilimab,Cabiralizumab,

[0363] Emactuzumab, Ipilimumab, Quavonlimab, Tremelimumab, Zalifrelimab, Cetuximab, Depatuxizumab, Futuximab, Imgatuzumab, Matuzumab, Modotuximab, Necitumumab, Nimotuzumab, Panitumumab, Tomuzotuximab, Zalutumumab, Batoclimab, Nipocalimab, Rozanolixizumab, Burosumab, Farletuzumab, Dinutuximab, Naxitamab, Ragifilimab, Gimsilumab, Lenzilumab, Mavrilimumab, Namilumab, Otilimab, Plonmarlimab, Codrituzumab, Margetuximab, Pertuzumab, Trastuzumab, Datopotamab, Patritumab, Seribantumab, Duligotuzumab, Ficlatuzumab, Rilotumumab, Alomfilimab, Anifrolumab, Emapalumab, Ligelizumab, Omalizum ab) Cixutumumab, Dalotuzumab, Figitumumab, Ganitumumab, Teprotumumab, Bermekimab, Canakinumab, Gevokizumab, Briakinumab, Ustekinumab, Anrukinzumab, Cendakimab, Lebrikizumab, Tralokinumab, Brodalumab, Bimekizumab, Ixekizumab, Secukinumab, Brazikumab, Guselkumab, Mirikizumab, Risankizumab, Tildrakizumab, Nemolizumab, Imsidolimab, Spesolimab, Pascolizumab, Dupilumab, Depemokimab, Mepolizumab, Reslizumab, Benralizumab, Clazakizumab, Olokizumab, Siltuximab, Sirukumab, Ziltivekimab, Levilimab, Sarilumab, Satralizumab, Tocilizumab,

[0364] Abituzumab, Favezelimab, Fianlimab, Ieramilimab, Relatlimab, Simtuzumab, Abagovomab, Oregovomab, Tanezumab, Ivuxolimab, Rocatinlimab, Tavolimab, Telazorlimab, Vonlerolizumab, Alirocumab, Bococizumab, Ebronucimab, Evolocumab, Frovocimab, Ongericimab, Tafolecimab, Dostarlimab, Balstilimab, Camrelizumab, Cemiplimab, Geptanolimab, Nivolumab, Pembrolizumab, Penpulimab, Pidilizumab, Prolgolimab, Retifanlimab, Sasanlimab, Serplulimab, Sintilimab, Spartalizumab, Tislelizumab, Toripalimab, Ezabenlimab, Zimberelimab, Atezolizumab, Avelumab, Cosibelimab, Sugemalimab, Durvalumab, Envafolimab, Suvratoxumab, Denosumab, Zilovertamab,Elotuzumab, Domvanalimab, Etigilimab, Ociperlimab, Tiragolumab, Vibostolimab, , Surzebiclimab, Cobolimab, Sabatolimab, Concizumab, Marstacimab, Adalimumab, Golimumab, Infliximab, Certolizumab, Conatumumab, Tigatuzumab, Tezepelumab, Gatipotuzumab, Cabiralizumab, Bevacizumab, Brolucizumab, Ranibizumab, Olinvacimab, Icrucumab, Ramucirumab, Caplacizumab, Abrilumab, Etrolizumab, Vedolizumab, Intetumumab, Natalizumab, Obrindatamab, Elranatamab,

[0365] Linvoseltamab, Teclistamab, Epcoritamab, Glofitamab, Mosunetuzumab, Odronextamab, Flotetuzumab, Vibecotamab, Catumaxomab, Cibisatamab, Talquetamab, Ubamatamab, Emfizatamab, Blinatumomab, Amivantamab, Emicizumab, Zenocutuzumab, Zanidatamab, Tibulizumab, Naptumomab, Belantamab, Pivekimab, Praluzatamab, Coltuximab, Denintuzumab, Loncastuximab, Ibritumomab, Inotuzumab, Epratuzumab, Moxetumomab, Brentuximab, Gemtuzumab, Vadastuximab, Lorvotuzumab, Polatuzumab, Tusamitamab, Terisot Zumab (Telisotuzumab), Rovalpituzumab, Depatuxizumab, Farletuzumab, Mirvetuximab, Disitamab, Anetumab, Enfortumab, Sacituzumab, Vobarilizumab, Cadonilimab, Vudalimab, Tebotelimab, Ivonescimab, Erfonrilimab, Ozoralizumab, Faricimab, Vanucizumab, Navicixizumab, and the like.

[0366] [Pharmaceutical composition for cancer prevention or treatment] The present invention provides a pharmaceutical composition for cancer prevention or treatment, which contains, as an active ingredient, a compound represented by the aforementioned Chemical Formula 1, for example, Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8, or Chemical Formula 9, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof (pro drug), preferably a carrier-drug conjugate (CDC) thereof, more preferably an antibody-drug conjugate (ADC).

[0367] According to the present invention, the active form camptothecin derivative (a) represented by Chemical Formula 1 or its prodrug (b), preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC), can be included as an active ingredient in a pharmaceutical composition for administration to a patient group in which DDX5 acts as an oncoprotein intracellularly.

[0368] For example, in tissue biopsy or liquid biopsy, the ability of the FL118 compound of Chemical Formula 2 or the active camptothecin derivative (a) to inhibit type I topoisomerase, preferably a ligand-containing complex (c) that targets the DDX5 protein and is inactivated via a non-cleavable, linker-linked manner, is used to quantitatively or qualitatively analyze the intracellular DDX5 protein, thereby identifying a patient group to whom the active camptothecin derivative (a) or its prodrug (b) is to be administered.

[0369] The active camptothecin derivative (a) represented by Chemical Formula 1 according to the present invention, for example, the compounds represented by Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, are camptothecin derivatives with a new structure. Therefore, through this, drug candidate substances having more preferable properties in terms of the efficacy, toxicity, selectivity, action time, administration, handling, stability and / or producibility, etc. of Dxd drugs can be provided. Also, the interaction between the target and the drug candidate substance, that is, the pharmacodynamic properties can be optimized, and the access of the drug to the target, that is, the pharmacokinetic ability can be improved.

[0370] Also, one specific example of the present invention provides a method for treating or preventing cancer, which includes administering a therapeutically effective amount of a compound represented by Chemical Formula 1, for example, a compound represented by Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, its pharmaceutically acceptable salt, its solvate, or its prodrug, preferably a carrier-drug complex (CDC), more preferably an antibody-drug complex (ADC), to a subject in need thereof. The subject can be a mammal including a human.

[0371] In the present invention, the cancer includes all cancers that can be treated by inhibition of topoisomerase I and / or suppression of any one or more cancer-associated survival genes selected from the group consisting of DDX5, survivin, Mcl-1, XIAP, and cIAP2, and can be solid cancer or hematological cancer. For example, caustic mucinous tumor, intrahepatic bile duct cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, fungating polyp, acute myeloid leukemia, acute lymphoblastic leukemia, basal cell cancer, epithelial ovarian cancer, ovarian germ cell tumor, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, bile duct cancer, colorectal cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, cancer of the Vater ampulla, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, paranasal and nasal cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, heart cancer , duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoid, gastrointestinal stromal tumor, Wilms tumor, breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal tumor, rectal cancer, rectal carcinoid, vaginal cancer, spinal cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi sarcoma, Paget's disease, tonsil cancer, squamous cell cancer, lung aden ocarcinoma, lung cancer, squamous cell lung cancer, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, hematological cancer, thymic cancer, and can be one or more selected from the group consisting of, but not limited to, these. In addition, the cancer includes not only primary cancer but also metastatic cancer.

[0372] As used herein, the terms "patient", "subject", and "subject entity" refer to animals such as mammals. In certain embodiments, the patient is a human. In other embodiments, the patient is a non-human animal such as a dog, cat, livestock (e.g., horse, pig or donkey), chimpanzee or monkey.

[0373] As used in the present invention, the term "therapeutically effective amount" refers to an amount of the active form of camptothecin derivative (a) represented by Chemical Formula 1 that is effective for the treatment or prevention of cancer, for example, Chemical Formula 3, a compound represented by Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5 or Chemical Formula 5-1, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof. Specifically, "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined by factors such as the type and severity of the individual, age, gender, type of disease, activity of the drug, drug sensitivity, administration time, administration route and excretion rate, treatment period, factors including drugs used concomitantly, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with commercially available therapeutic agents. Also, single or multiple administrations can be performed. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects considering all the above factors, and the compounds represented by Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9 of the present invention and pharmaceutically acceptable salts thereof exhibit dose-dependent effects, so the administration dosage can be easily determined by those skilled in the art according to various factors such as the patient's condition, age, gender and complications. Since the active ingredient of the pharmaceutical composition of the present invention is excellent in safety, it can be used even above the determined administration dosage.

[0374] Also, according to one specific example of the present invention, the present invention provides the use of an active camptothecin derivative (a) represented by Chemical Formula 1, for example, a compound represented by Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, its pharmaceutically acceptable salt, its solvate, or its prodrug, preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC), for use in the manufacture of a medicament for the treatment or prevention of cancer. The compound represented by Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, its pharmaceutically acceptable salt, its solvate, or its prodrug for the manufacture of a medicament can be mixed with acceptable adjuvants, diluents, carriers, etc., and can be manufactured into a combined preparation together with other active preparations and can have a synergistic effect of the active ingredients.

[0375] The matters referred to in the use, composition, and treatment method of the present invention are applied identically as long as they do not contradict each other.

[0376] In the present specification, the anti-cancer effect or therapeutic effect of an anti-cancer agent can refer to an effect of reducing the severity of cancer, reducing the size of a tumor, or delaying or blunting the progression of cancer that occurs while a patient is suffering from a specific cancer.

[0377] For example, the anti-cancer effect of an anti-cancer agent is the cell viability of cancer cells after treating the cancer cells with the anti-cancer agent in vitro and / or in vivo It can be (the degree of cytotoxicity or the change in the number of cells). For example, it can be indirectly confirmed through drug response tests using cell lines or non-clinical animal models (xenografts). Also, in cancer patients, the anti-cancer effect of anti-cancer drugs can be directly confirmed, and related data can be derived and used in the database. Additionally, when designing the dosing guidelines for anti-cancer drugs, animal model PK parameters and / or toxicity profiles can be considered in parallel.

[0378] The anti-cancer effect of an anti-cancer drug can be inferred from in-vitro data such as the % maximum effect of the anti-cancer drug, for example, IC 50 、IC 60 、IC 70 、IC 80 and IC 90 and can also be confirmed in non-clinical animal models and clinical cancer patients through in-vivo data such as the maximum plasma concentration (Cmax) of the drug and / or the area under the curve (AUC) of the drug plasma concentration-time curve.

[0379] The responsiveness of an anti-cancer drug means clinical sensitivity from the perspective of the anti-cancer effect.

[0380] When referring in relation to treatment with anti-cancer drugs, "sensitive" and "sensitive to" are relative terms that refer to the degree of the effect of a compound in alleviating or reducing the progression of the tumor or disease being treated.

[0381] "Effective patient anti-cancer effect / response" can be, for example, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more suppression in patient response, as measured by any suitable means, such as gene expression, cell count, analysis results, etc.

[0382] In the present specification, the administration dose is a dose at which a medicinal effect is expected. In the present invention, the medicinal effect can be an anti-cancer effect. The reactivity (anti-cancer effect) of an anti-cancer drug is the degree of reaction, and is expressed as the % maximum effect of the anti-cancer drug, for example, IC 50 ,I C 60 ,I C 70 ,I C 80 and I.C. 90 , the value at which toxicity is exerted on normal cells (LC 50 ).

[0383] For example, oral dosage forms can be formulated using a variety of formulation techniques known in the art. For example, they may include a biodegradable (hydrolyzable) polymeric carrier that is used to adhere to the oral mucosa. They are fabricated to slowly erode over a predetermined period of time, where drug delivery is essentially provided throughout.

[0384] Drug delivery in oral dosage forms avoids the drawbacks encountered with oral drug administration, such as slow absorption, degradation of the active agent by fluids present in the digestive tract, and / or initial pain and inactivation in the liver. For biodegradable (hydrolyzable) polymeric carriers, virtually any such carrier can be used as long as the desired drug release profile is not compromised, and the carrier is compatible with any other components present in the oral dosage unit. Generally, polymeric carriers include hydrophilic (water-soluble and water-swellable) polymers that adhere to the moist surface of the oral mucosa. Examples of polymeric carriers useful herein include acrylic acid polymers (e.g., carbomer). In some embodiments, non-limiting examples of other components that may be incorporated into oral dosage forms include disintegrants, diluents, binders, lubricants, flavoring agents, coloring agents, preservatives, and the like. In some embodiments, the oral dosage form may be in the form of a tablet, lozenge, or gel formulated in a conventional manner for buccal or sublingual administration.

[0385] In some embodiments, if the patient's condition improves, administration of the compound is subsequently provided at the discretion of the physician; alternatively, the dosage of the drug administered may be temporarily reduced or discontinued. can be interrupted for any length of time (i.e., "drug holiday"). The length of the drug holiday can vary from 2 days to 1 year, and by way of example only, includes 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. In some embodiments, the dosage reduction during the drug holiday is from 10% - 100%, and by way of example only, includes 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0386] When improvement in the patient's condition occurs, maintenance dosages are administered as needed. Thereafter, the dosage or dosing frequency, or both, can be decreased as a function of symptoms to a level at which the improved disease, disorder or condition is maintained. However, the patient may require long-term intermittent treatment upon any recurrence of symptoms.

[0387] The amount of a given formulation corresponding to such amounts will vary depending on factors of the subject in need of treatment, such as, for example, the particular compound, the severity of the disease, identity (e.g., weight), but can nevertheless be routinely determined in the art in a manner known in the art depending on, for example, the dosage form administered, the route of administration, and the particular circumstances surrounding the subject being treated. Generally, however, dosages used in the treatment of adults can typically range from 0.02 - 5000 mg / day, or about 1 - 1500 mg / day.

[0388] The single dose herein can be provided as a single dose or simultaneously, for example, as divided doses administered as sub-doses of 2, 3, 4 or more than that.

[0389] In some embodiments, an oral dosage form is a unit dosage form suitable for single administration of an accurate dosage. In the unit dosage form, the dosage form is divided into unit dosages containing an appropriate amount of one or more compounds. In some embodiments, the unit dosage is in the form of a package containing separate amounts of the dosage form. Non-limiting examples are packaged tablets or capsules, and powder vials or ampoules. An aqueous suspension composition can be packaged in a single-dose non-resealable container. Optionally, a multi-dose resealable container can be used, in which case it is typical to include a preservative in the composition.

[0390] In some embodiments, a parenteral injection dosage form is provided in a unit dosage form, including but not limited to ampoules, or a multi-dose container, together with an added preservative.

[0391] Typically, it is manufactured in a unit-dose injectable form with a pharmaceutically acceptable parenteral vehicle for parenteral administration, i.e., bolus, intravenous, and intratumoral injection. It is optionally mixed in a lyophilized formulation or aqueous solution form with a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer (Remington’s Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.).

[0392] [Ligand targeting DDX5 protein] Not only FL118, but also the active camptothecin derivative represented by Chemical Formula 1 according to the present invention can be used as a ligand targeting the DDX5 protein or a ligand binding to the DDX5 protein because it is a molecular adhesive that activates tumor protein DDX5 degradation. Therefore, a ligand-containing complex targeting the DDX5 protein in which the type I topoisomerase inhibitory ability in the FL118 compound or the active camptothecin derivative (a) is inactivated via a linker linkage can be provided.

[0393] The linker may be a non-cleavable linker, and non-limiting examples of non-cleavable linkers are And there is a thioether linker.

[0394] The FL118 compound or the active form of camptothecin derivative (a) represented by Chemical Formula 1 binds to the DDX5 protein, and the DDX5 protein is the direct target of the active form of camptothecin derivative (a).

[0395] Therefore, DDX5 can be used as a biomarker for predicting drug sensitivity or tumour sensitivity.

[0396] By using a ligand-containing complex (c) that targets the DDX5 protein, in which the type I topoisomerase inhibitory ability of the FL118 compound of Chemical Formula 2 or the active form of camptothecin derivative (a) represented by Chemical Formula 1 is inactivated via a linker linkage, to quantitatively or qualitatively analyze the intracellular DDX5 protein, it is possible to determine the patient group to be administered the active form of camptothecin derivative (a) or its prodrug (b), or to predict the recurrence rate and / or prognosis during chemotherapy with chemotherapeutic agents.

[0397] [Cancer diagnostic composition] The present invention provides a cancer diagnostic composition containing a ligand-containing complex (c) that targets the DDX5 protein, in which the type I topoisomerase inhibitory ability of the FL118 compound of Chemical Formula 2 or the active form of camptothecin derivative (a) represented by Chemical Formula 1 is inactivated via a linker linkage.

[0398] When the cancer diagnostic composition of the present invention is used, it can be used to determine the patient group to be administered the active form of camptothecin derivative (a) or its prodrug (b), or to predict the drug reaction, recurrence rate and / or prognosis during chemotherapy with chemotherapeutic agents.

Effects of the Invention

[0399] The camptothecin derivatives represented by Chemical Formula 1 according to the present invention, for example, the camptothecin derivatives represented by Chemical Formulas 3 to 9 or their isomers, are compounds with novel structures. Through this, it is possible to provide drug candidate substances having more favorable properties in terms of the efficacy, toxicity, selectivity, action time, administration, handling, stability, and / or producibility, etc. of Dxd drugs. Also, the interaction between the target and the drug candidate substance, that is, the pharmacodynamic properties can be optimized, and the access to the drug target can be optimized, that is, the pharmacokinetic ability can be improved.

[0400] The present invention provides a compound of Chemical Formula 1, for example, a compound of Chemical Formulas 3 to 9 or its isomer, which maintains the structure of the FL118 drug and can maximize the efficacy of major drug target regulation through (i) simultaneously suppressing / suppressing the Bcl family such as survivin, a resistance protein, or (ii) further mechanisms of action that suppress the action of efflux pumps, thereby maintaining the advantages of the FL118 drug.

[0401] Therefore, the compound of Chemical Formula 1, for example, the compound of Chemical Formulas 3 to 9 or its isomer, deviates from the existing drug development method in which one drug targets one drug target and the therapeutic efficacy is limited, and is designed based on a well-designed polypharmacology-based access method that targets various drug targets that need to be controlled simultaneously to obtain excellent therapeutic efficacy, and can treat intractable diseases for which existing therapeutic agents have not shown a clear therapeutic effect. Also, recurrent / resistant cancers have a problem that sufficient efficacy cannot be obtained by simply regulating the activity of one drug target, and safety cannot be ensured when targeting an unspecified number of drug targets simultaneously. However, like the FL118 drug, by utilizing two or more well-selected drug targets, showing synergy from the perspective of drug efficacy, and simultaneously ensuring sufficient safety.

Brief Description of the Drawings

[0402]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20a

Figure 20b

Figure 20c

Figure 20d

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27a

Figure 27b

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Mode for Carrying Out the Invention

[0403] Hereinafter, the present invention will be described more specifically based on examples. However, the following examples are merely for clearly illustrating the technical features of the present invention and do not limit the protection scope of the present invention.

[0404] Production Example 1: Synthesis of Active Camptothecin Derivatives of Chemical Formula 3 or Chemical Formula 3-1 (PBX-7011 and PBX-7012)

Chem.

[0405] 1-1. Synthesis of Compound 2

Chem.

[0406] To a solution of 5-nitrobenzo[d][1,3]dioxole (25 g, 150 mmol) in a dichloromethane (748 mL) flask, silver triflate (57.7 g, 224 mmol) and iodine (57.0 g, 224 mmol) were added. This solution was stirred in a dark room at room temperature under a nitrogen atmosphere.

[0407] AgI was removed by filtration, and the solid was washed with dichloromethane (100 mL). The solvent It was removed under reduced pressure, and the residue was partitioned between EtOAc (250 mL) and 5% (v / v) NH4OH / H2O solution (200 mL). The organic layer was separated, washed with 1 M Na2SO 3 (5 x 200 mL) and brine (200 mL), dried over Na2SO4, treated with activated carbon, and filtered through celite. The solvent was evaporated under reduced pressure to afford the crude product as a brown solid. This was triturated from ice-cold EtOH (400 mL), filtered, and the solid was washed with ice-cold EtOH (100 mL) to afford the product as a pale brown-gray solid (14.3 g). The solvent was removed from the filtrate, and the crude product was triturated a second time from ice-cold EtOH (300 mL). The solid was collected by filtration and washed with EtOH (50 mL) to afford an additional amount of product (10.2 g) as a dark brown-gray solid. The two batches were used as such without further purification. SC_ACID: m / z 294.2 [M+H] +

[0408] 1 - 2. Synthesis of Compound 3

Chemical Structure

[0409] A suspension of 4-iodo-6-nitrobenzo[d][1,3]dioxole (8.75 g, 29.9 mmol) in a mixture of water (80 mL), methanol (40.0 mL) and tetrahydrofuran (40.0 mL) was added iron powder (6.67 g, 119 mmol) and ammonium chloride (6.39 g, 119 mmol). The suspension was heated at 75 °C and stirred for 16.5 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting black solid was suspended in ethyl acetate (100 mL) and the solution was decanted. The residue was washed with EtOAc (3 x 50 mL). The mixture was transferred to a separatory funnel, water was added and the aqueous layer was removed. The organic layer was washed with saturated aqueous NaHCO3 solution (150 mL) and brine (150 mL). The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure to give a brown solid (4.75 g, 60% yield). The iron residue was washed thoroughly with ethyl acetate to recover additional product. Next, the organic fraction was washed with saturated aqueous NaHCO3 solution (150 mL), brine (150 mL) and dried over Na2SO4. The solvent was removed under reduced pressure to give a brown solid (1.74 g, 22% yield). SC_ACID: m / z 264.0 [M+H] +

[0410] 1 - 3. Synthesis of Compound 4

Chemical Structure

[0411] To a solution of 7-iodobenzo[d][1,3]dioxol-5-amine (6.39 g, 24.29 mmol) in dichloromethane (49 mL) were added acetic anhydride (2.75 mL, 29.2 mmol) and triethylamine (4.06 mL, 29.2 mmol). The reaction mixture was stirred overnight at room temperature. After several hours, additional DCM (10 mL) was added. The suspension was filtered through a sintered funnel and washed with ice-cold DCM (10 mL). The product was further dried under reduced pressure to afford a light gray solid (4.46 g). The filtrate was concentrated under reduced pressure, the residue was dissolved in EtOAc, washed with water and brine, dried over Na2SO4, and concentrated under reduced pressure to give a brown solid (~3 g). The brown solid was purified by flash column chromatography (80 g Si, 0 - 100% ethyl acetate in heptane). All batches of the product were triturated from ice-cold EtOAc (5 - 10 mL). Two batches were combined and further dried to give an off-white solid. Total yield: 5.0 g, 66% SC_ACID: m / z 306.0 [M+H] + 1 H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H), 7.39 (d, J = 1.9 Hz, 1H), 7.17 (d, J = 1.9 Hz, 1H), 6.04 (s, 2H), 1.99 (s, 3H).

[0412] 1 - 4. Synthesis of Compound 5

Chemical Structure

[0413] In a three-necked flask equipped with a condenser, a slurry of N-(7-iodobenzo[d][1,3]dioxol-5-yl)acetamide (5.06 g, 16.59 mmol), but-3-enoic acid (1.69 mL, 19.90 mmol) and potassium carbonate (2.98 g, 21.56 mmol) in acetonitrile (40 mL) was cooled to 0 - 5 °C. Water (13.33 mL) was slowly added to generate gas. When gas generation was interrupted, the mixture was degassed with Ar for 30 minutes. Tri-o-tolylphosphine (0.505 g, 1.659 mmol) and palladium acetate (0.186 g, 0.829 mmol) were added, and the mixture was degassed for another 30 minutes and then heated under reflux under Ar. The reaction was cooled to room temperature and filtered through Celite. The filter cake was washed with H2O and EtOAc. The organic solvent was removed from the filtrate under vacuum, and the aqueous material was acidified with a concentrated solution. The pH was adjusted to 1 - 2 with HCl. The aqueous layer was extracted with EtOAc, the combined organic phases were washed with brine, dried over Na2SO4, filtered, concentrated under reduced pressure to obtain the crude product. The crude product was triturated from ice-cold EtOAc (30 mL), and the solid was collected by filtration to give the product as a brown solid. The mother liquor was concentrated under reduced pressure and purified by flash chromatography (80 g Si, 0 - 100% EtOAc in heptane). The product fraction was concentrated under reduced pressure to obtain a light brown foam. Total yield: 3.46 g, 75%. A mixture of E / Z isomers was obtained. SC_ACID: m / z 264.4 [M+H] +

[0414] 1 - 5. Synthesis of Compound 6

Chemical Structure

[0415] In tetrahydrofuran (50 mL) / water (50 mL), a suspension of 4-(6-acetamidobenzene[d][1,3]dioxol-4-yl)but-3-enoic acid (3.47 g, 13.18 mmol) was degassed with N2 for 15 minutes. Before heating at 40 °C under H2 (balloon), Pd / C (2.97 g, 1.397 mmol) was added and the suspension was degassed with H2 for 5 minutes. After 24 hours, additional Pd / C (2.97 g, 1.397 mmol) was added to the reaction mixture under nitrogen. The reaction mixture was bubbled with hydrogen for 10 minutes and stirred overnight at 45 °C under hydrogen. The reaction was filtered through Celite. The filter cake was washed with H2O (50 mL). EtOAc (50 mL) and the organic solvent were removed from the filtrate under vacuum. The aqueous solution was acidified with concentrated HCl to pH = 1 and the dark brown / green precipitate was collected by filtration through a sintered funnel. The aqueous filtrate was extracted. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and the solvent was removed under vacuum to give a brown oil. Since the product recovery was low, the filter cake was added to a flask containing EtOAc (50 mL), water (200 ml), and MeOH (400 ml), and the water / EtOAc flash was added to the solid from the MeOH flash. The aqueous layer was acidified to a high concentration. HCl was added until pH = 1 and an off-white precipitate formed, which was collected by filtration through a sintered funnel. The aqueous filtrate was extracted with EtOAc (3 x 200 mL), and LCMS indicated that all products were removed in the aqueous phase. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and the solvent was removed under vacuum to give a light brown solid. All product batches were combined and purified by flash column chromatography (40 g Si, 0 - 10% MeOH in DCM). The product fractions were concentrated to give a light brown solid. Yield: 2.52 g, 72%. SC_ACID:266.2[M+H] + 11H NMR (400 MHz, DMSO-d6) δ 12.07 (s, 1H), 9.78 (s, 1H), 7.14 (d, J = 2.0 Hz, 1H), 6.80 (d, J = 2.2 Hz, 1H), 5.95 (s, 2H), 2.50 - 2.46 (m, 2H), 2.23 (t, J = 7.4 Hz, 2H), 1.98 (s, 3H), 1.84 - 1.70 (m, 2H).

[0416] 1 - 6. Synthesis of Compound 7

Chemical Structure

[0417] A suspension of 4-(6-acetamidobenzod[d][1,3]dioxol-4-yl)butanoic acid (150 mg, 0.565 mmol) in TFA (433 μL, 5.65 mmol) was cooled on ice. TFAA (157 μL, 1.131 mmol) was added. The mixture was stirred at 0 - 5 °C for 1 h and turned into a dark solution over time. The reaction mixture was added dropwise to ice-cooled saturated aqueous NaHCO3 solution (10 mL), and the aqueous solution was extracted with ethyl acetate (3 x 25 mL). The combined organic layers were dried over Na2SO4 with sat. NaHCO3 and brine, filtered, and the solvent was removed under vacuum to obtain a pale pink solid. Yield: 140 mg, 100%. SC_ACID: m / z 248.2 [M + H] + 1 1H NMR (400 MHz, DMSO-d6) δ 12.34 (s, 1H), 8.11 (s, 1H), 6.13 (s, 2H), 2.80 (t, J = 6.2 Hz, 2H), 2.66 - 2.58 (m, 2H), 2.12 (s, 3H), 2.01 - 1.91 (m, 2H).

[0418] 1 - 7. Synthesis of Compound 8

Chemical Structure

[0419] A suspension of N-(6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide (50 mg, 0.202 mmol) in tetrahydrofuran (1.2 mL) was cooled to 0 °C, and potassium tert-butoxide (27.2 mg, 0.243 mmol) and isoamyl nitrite (35.0 μL, 0.263 mmol) were added. The dark green mixture was stirred on ice (<5 °C) for 1.5 h. Acetic acid (170 μL, 2.94 mmol), acetic anhydride (170 μL, 1.810 mmol) and zinc, dust (66.1 mg, 1.011 mmol) were added to the reaction mixture. The suspension was stirred at 0 °C for 2 h. The reaction mixture was filtered through Celite and flushed with DCM. The filtrate was concentrated under reduced pressure to give a black oily substance. The crude product was purified using flash column chromatography (4 g Si, in DCM, 0 - 4% MeOH). The product fractions were concentrated to give a gray solid (32 mg, 52%) with a purity of 80 - 90%. MPL for purification Purification by C can give a sample with even higher purity. SC_ACID: m / z 305.4 [M+H] + 1 H NMR (400 MHz, DMSO-d6) δ 12.10 (s, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.12 (s, 1H), 6.15 (d, J = 9.3 Hz, 2H), 4.66 - 4.56 (m, 1H), 2.93 (dd, J = 8.9, 4.0 Hz, 2H), 2.14 (s, 3H), 2.13 - 1.93 (m, 2H), 1.91 (s, 3H).

[0420] 1 - 8. Synthesis of Compound 9

Chemical Structure

[0421] N,N'-(6-Oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxole-5,7-diyl)diacetamide (244 mg, 0.802 mmol) was suspended in 2 M hydrochloric acid (4.69 mL, 9.38 mmol) in ethanol / water (5 / 1). The mixture was heated at 55 °C for 4 h. The black mixture was cooled to 0 - 5 °C. Triethylamine (1.4 mL, 10.04 mmol) was added dropwise with stirring. Next, the mixture was diluted with EtOH and evaporated to dryness. The residue was partitioned between water and DCM. The layers were separated and the aqueous layer was extracted once with DCM. The combined organic layers were dried over Na2SO4 and concentrated to give the product as a brown solid (178 mg, 73%) with 86% purity. SC_ACID: m / z 263.0 [M+H] + 1 1H NMR (400 MHz, DMSO) δ 8.04 (d, J = 8.0 Hz, 1H), 6.20 (s, 1H), 5.94 (d, J = 6.0 Hz, 2H), 4.48 - 4.41 (m, 1H), 3.08 (s, 2H), 2.88 - 2.69 (m, 2H), 2.15 - 2.03 (m, 1H), 1.88 (s, 3H), 1.86 - 1.75 (m, 1H).

[0422] 1 - 9. Synthesis of Compound 10

Chemical Structure

[0423] (4S)-4-Ethyl-7,8-dihydro-4-hydroxy-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (146 mg, 0.555 mmol) and N-(5-amino-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-7-yl)acetamide (112 mg, 0.427 mmol) were added to dry toluene (4.5 ml), PPTS (21 mg, 0.085 mmol) was added, and the reaction mixture was stirred at 115 °C for 40 h. The reaction mixture was cooled to room temperature. The suspension was diluted with 2 mL of DCM and filtered. A black residue (210 mg) was obtained. The crude product was purified by column chromatography (12 g Si, in DCM, 0 - 7% methanol) to afford the product (45 mg, 21%) as a brown solid. LCMS analysis showed two diastereoisomers. SC_ACID: m / z 490.2 [M+H] + 1 H NMR (400 MHz, DMSO-d6) δ 8.47 (t, J = 9.3 Hz, 1H), 7.42 (s, 1H), 7.24 (s, 1H), 6.49 (s, 1H), 6.29 (d, J = 5.2 Hz, 2H), 5.57 - 5.49 (m, 1H), 5.41 (s, 2H), 5.23 - 5.07 (m, 2H), 3.09 - 3.00 (m, 2H), 2.11 - 2.01 (m, 2H), 1.91 (s, 3H), 1.89 - 1.79 (m, 2H), 0.87 (t, J = 7.1 Hz, 3H).

[0424] 1 - 10. Synthesis of PBX - 7011 and PBX - 7012

Chemical Structure

[0425] N - ((10S)-10 - ethyl - 10 - hydroxy - 11,14 - dioxo - 2,3,10,11,14,16 - hexahydro - 1H,13H - benzo[de][1,3]dioxolo[4,5 - g]pyrano[3’,4’:6,7]indolizino[1,2 - b]quinolin - 1 - yl)acetamide (73.5 mg, 0.150 mmol) was dissolved in 1.0 mL of 6N HCl and stirred at 90 °C for 8 h followed by overnight stirring at room temperature. The reaction mixture was concentrated under reduced pressure and purified by performing MPLC for acidic fraction twice (Luna 2 - 30). The fractions containing the separated diastereoisomers were acidified with 5 drops of 3N HCl and lyophilized to afford the product as a yellow solid. The first eluted isomer: PBX - 7011, 23 mg, 34% yield

[0426] U_AN_ACID: m / z 448.4 [M+H] U_AN_ACID: m / z 448.4 [M+H] + 1 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 3H), 7.50 (s, 1H), 7.27 (s, 1H), 6.52 (s, 1H), 6.34 (d, J = 13.3 Hz, 2H), 5.77 (d, J = 19.3 Hz, 1H), 5.44 (s, 2H), 5.37 (d, J = 19.3 Hz, 1H), 5.05 (s, 1H), 3.19 - 3.02 (m, 2H), 2.46 (s, 1H), 2.20 - 2.03 (m, 1H), 1.94 - 1.81 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

[0427] Second dissolution isomer: PBX-7012, 28 mg, 41% yield U_AN_ACID: m / z 448.2 [M+H] + 1 1H NMR (400 MHz, DMSO-d6) δ 8.57 (d, J = 4.7 Hz, 3H), 7.51 (s, 1H), 7.27 (s, 1H), 6.52 (s, 1H), 6.34 (d, J = 12.2 Hz, 2H), 5.76 (d, J = 19.4 Hz, 1H), 5.44 (s, 2H), 5.37 (d, J = 19.4 Hz, 1H), 5.08 (s, 1H), 3.16 - 2.98 (m, 2H), 2.46 (s, 1H), 2.18 - 2.06 (m, 1H), 1.94 - 1.80 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0428] Production Example 2: Synthesis of active camptothecin derivatives (PBX-7014 and PBX-7015) of Chemical Formula 4 or Chemical Formula 4-1 This example describes the synthesis of compounds PBX-7014 and PBX-7015 starting from two separated diastereoisomers of exatecan-hybrid compounds (PBX-7011 and PBX-7012).

[0429]

Chemical formula

[0430] 2-1: Synthesis of PBX-7014

Chem.

[0431] A stock solution of glycolic acid activated by the following procedure was prepared: Glycolic acid (17 mg, 0.224 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (25.7 mg, 0.223 mmol) and EDC (42.8 mg, 0.223 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. Next, 0.4 mL of the activated acid solution was added to a suspension of (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-11,14-dione (40 mg, 0.089 mmol) and triethylamine (0.025 ml, 0.179 mmol) in N,N-dimethylformamide (2.5 mL). The mixture was stirred at room temperature for 3 hours. 0.05 mL of the newly prepared activated acid solution was added. Next, the reaction mixture was stirred at room temperature for an additional 2 hours. The reaction mixture was evaporated to dryness. The crude product was purified by column chromatography (0 - 8% methanol in chloroform). This afforded a yellow solid containing the PBX-7014 product and residual succinimide. The product was further purified by acidic workup MPLC (Luna5-40), and after lyophilization of the product fraction, a bright yellow solid was obtained. Yield: 20 mg, 50% U_AN_ACID: m / z 506.2 [M+H] + 11H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 8.9 Hz, 1H), 7.40 (s, 1H), 7.23 (s, 1H), 6.49 (s, 1H), 6.28 (d, J = 4.6 Hz, 2H), 5.61 - 5.45 (m, 2H), 5.45 - 5.35 (m, 2H), 5.19 - 5.06 (m, 2H), 3.95 (s, 2H), 3.13 - 2.96 (m, 2H), 2.21 - 2.02 (m, 2H), 1.94 - 1.77 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0432] 2-2: Synthesis of PBX-7015

Chemical formula

[0433] A stock solution of the activated acid was prepared by the following procedure: Glycolic acid (17.00 mg, 0.223 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (25.7 mg, 0.223 mmol) and EDC (42.8 mg, 0.223 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. Next, in N,N-dimethylformamide (2.5 mL), (1R,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3’, A suspension of indolizino[1,2-b]quinoline-11,14-dione (25 mg, 0.056 mmol) and triethylamine (0.016 mL, 0.112 mmol) was added to 0.25 mL of the activated acid solution. The mixture was stirred overnight at room temperature. 0.03 mL of the freshly prepared activated acid solution was added. Next, the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was combined with a previously smaller batch and evaporated to dryness. The crude product was purified by column chromatography. This afforded a yellow solid containing the PBX-7015 product and residual succinimide. The product was purified by acidic workup MPLC (Luna 5-40). The product fractions were lyophilized to afford a bright yellow solid. Yield: 15 mg, 38% U_AN_ACID:506.2[M+H] + 1 H NMR (400 MHz, DMSO) δ 8.44 (d, J = 9.0 Hz, 1H), 7.41 (s, 1H), 7.24 (s, 1H), 6.48 (s, 1H), 6.29 (d, J = 2.3 Hz, 2H), 5.61 - 5.44 (m, 2H), 5.44 - 5.36 (m, 2H), 5.20 - 5.07 (m, 2H), 3.96 (s, 2H), 3.10 - 2.96 (m, 2H), 2.20 - 2.06 (m, 2H), 1.95 - 1.79 (m, J = 7.3 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0434] Production Example 3: Synthesis of the active camptothecin derivative (PBX-7016) of Chemical Formula 5 This example describes the synthesis of compound PBX-7016 starting from the exatecan-hybrid compound (PBX-7011).

[0435]

Chemical Formula

[0436] A stock solution of the activated D-lactic acid was prepared by the following procedure. D-Lactic acid (22 mg, 0.244 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (27 mg, 0.235 mmol) and EDC (38.6 mg, 0.201 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. Next, 0.3 mL of the activated acid solution was added to a solution of (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-11,14-dione (36 mg, 0.080 mmol) and triethylamine (0.022 mL, 0.161 mmol) in N,N-dimethylformamide (2.5 mL). The mixture was stirred at room temperature for 6 hours. 0.05 mL of the prepared activated acid solution was added. Next, the reaction mixture was stirred at room temperature overnight. The reaction mixture was directly purified by acidic fractionation MPLC (Luna10-50), and after freeze-drying the product fraction, a bright yellow solid was obtained. Yield: 22 mg, 52% U_AN_ACID: m / z 520.2 [M+H] + 1 1H NMR (400 MHz, DMSO) δ 8.43 (d, J = 9.1 Hz, 1H), 7.41 (s, 1H), 7.23 (s, 1H), 6.50 (s, 1H), 6.29 (d, J = 2.1 Hz, 2H), 5.62 - 5.58 (m, 1H), 5.58 - 5.51 (m, 1H), 5.41 (s, 2H), 5.21 - 5.01 (m, 2H), 4.17 - 4.07 (m, 1H), 3.14 - 2.95 (m, 2H), 2.19 - 2.04 (m, 2H), 1.92 - 1.78 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H). As described above, since PBX-7016 of Chemical Formula 5 can be synthesized from PBX-7011 of Chemical Formula 3, in the same way, PBX-7017 of Chemical Formula 5-1 can be synthesized from PBX-7012 of Chemical Formula 3-1.

[0437] Production Example 4: Synthesis of the active camptothecin derivative (PBX-7024) of Chemical Formula 9

Chem.

[0438] The (2S)-2-cyclopropyl-2-hydroxyacetic acid was coupled to the PBX-7011 compound to produce PBX-7024 in high yield. (2S)-2-Cyclopropyl-2-hydroxyacetic acid (26 mg, 0.244 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (26 mg, 0.226 mmol) and EDC (42 mg, 0.219 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. Next, 0.6 mL of the activated acid solution was added to a solution of (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-11,14 -dione (40 mg, 0.089 mmol) and DIPEA (0.047 mL, 0.268 mmol) in N,N-dimethylformamide (2.5 mL). The mixture was stirred at room temperature overnight. The reaction mixture was directly purified by acidic fractionation using MPLC (Luna 10-50), and after lyophilization of the product fraction, a bright white solid was obtained. Yield: 32 mg, 65% U_AN_ACID: m / z 520.2 [M+H] + 1 H NMR (400 MHz, DMSO-d6) δ 8.33 (d, J = 8.7 Hz, 1H), 7.39 (s, 1H), 7.23 (s, 1H), 6.48 (s, 1H), 6.28 (d, J = 4.9 Hz, 2H), 5.50 (q, J = 6.7 Hz, 1H), 5.40 (s, 3H), 5.23 - 5.06 (m, 2H), 3.62 (d, J = 6.3 Hz, 1H), 3.03 (q, J = 6.2 Hz, 2H), 2.21 - 2.02 (m, 2H), 1.92 - 1.79 (m, 2H), 1.18 - 1.08 (m, 1H), 0.87 (t, J = 7.3 Hz, 3H), 0.47 - 0.30 (m, 4H).

[0439] Example 1: Analysis of the binding mode of camptothecin derivatives to TOP1 / DNA [Table 3]

[0440] According to the binding mode prediction results (docking scores) through molecular dynamics simulations, all camptothecin derivatives in Table 2 basically bind through strong π-π stacking between DNA bases (Figures 4 and 5). The ester of the lactone forms hydrogen bonds with Arg488 of TOP1, and the ethyl and OH groups form hydrogen bonds with Asp533 of TOP1 (Figure 1), playing a role in supporting the good binding of camptothecin derivatives between the TOP1 protein and DNA.

[0441] The SN-38 drug is stabilized by forming hydrogen bonds with Glu356 and Arg364 of TOP1 (Figure 4). The exatecan drug forms hydrogen bonds with peripheral DNA bases and is further stabilized (Figure 4).

[0442] The -OCH2O- (methylenedioxo) pentagonal ring of the FL118 drug is weaker than hydrogen bonds (actually different from cytotoxicity) and shows a worse docking score than the SN-38 drug and the exatecan drug. However, such a binding binds perfectly between DNA bases and also helps with solvation (Figure 4).

[0443] As shown in Figure 5, the compounds of Chemical Formula 3 (PBX-7011) and Chemical Formula 3-1 (PBX-7012) designed by mixing the functional groups of the FL118 drug and the exatecan drug according to the present invention are in a relationship of partial stereoisomers, so they share the same binding pose and are predicted to bind stably to TOP1. In particular, the compound of Chemical Formula 3 (PBX-7011) was calculated to have a higher docking score than the SN-38 drug.

[0444] The compound of Formula 3 (PBX-7011) having chirality like an exatecan drug showed slightly better binding than the compound of Formula 3-1 (PBX-7012).

[0445] In addition, in relation to the TOP1 mutation (E418K) that showed resistance to trodelvy (payload: SN-38), as shown in Figure 6, the E418K mutation is at a distance from the drug binding site.

[0446] As shown in Figures 7 and 8, as a result of proceeding with energy minimization after the E418K mutation, it was confirmed by MD simulation that the newly introduced Lys418 changed a part of the drug binding site to a relatively polar environment and was also continuously ionically bonded to the surrounding Glu356. When a part of the binding site becomes polar due to the mutation, substances having a polar functional group like the SN-38 drug are observed by MD simulation to move away from the existing binding site a little in order to have an ionic interaction with Lys418 and Glu356. This can weaken the interaction between the lactone, which is the common inhibitory activity principle of TOP1 inhibitors in the same system, and Asp533 and Lys532.

[0447] When the SN-38 drug was docked to the mutant TOP1 structure, the SN-38 drug detached from the original binding site and moved to the side, but acquired new hydrogen bonding (H-bonding) etc. and showed a good binding docking score. The docking score has limitations in explaining the phenomenon that mutant TOP1 shows resistance to SN-38.

[0448] As shown in Figures 7 and 8, when analyzing the interaction between the SN-38 drug and TOP1 during the MD simulation, it was confirmed that the SN-38 interaction (89%) with Asp533 and Arg488 decreased to 10% or less due to the mutation, while the interaction with Asn352 and Glu356 increased from 40% to 80% level.

[0449] After the TOP1 mutation, when docking proceeds, the SN-38 drug moves to the newly deformed binding site, binds in a new form, and shows a score similar to the existing one (Table 2).

[0450] On the other hand, as shown in Table 2, the exatecan drug, FL118 drug, compound of Chemical Formula 3 (PBX-7011), and compound of Chemical Formula 3-1 (PBX-7012) that do not have a polar functional group (-OH) at the 10th carbon of the A-ring were confirmed to have a worse relative docking score with mutant TOP1 due to changes in part of the environment of the new binding site. Nevertheless, it was confirmed by MD simulation that the interaction between the existing lactone and Asp533 and Lys532 is maintained in the exatecan drug, FL118 drug, compound of Chemical Formula 3 (PBX-7011), and compound of Chemical Formula 3-1 (PBX-7012).

[0451] As shown in Figure 9, even when TOP1 is mutated, the compound of Chemical Formula 3 (PBX-7011) maintains the existing binding mode (especially the lactone and -OH group) as it is.

[0452] Conclusively, the E418K mutation does not directly interact with the TOP1 inhibitor. To explain the situation where the FL118 drug highly suppresses even the TOP1 mutation showing resistance to the SN-38 drug due to E418K, it can be considered that the new binding of SN-38 to the binding site deformed by the mutation proceeds in a form different from the docking result, rather, the interaction between the lactone and Asp533 and Lys532 weakens, and it is a binding that does not suppress the action of TOP1.

[0453] Under such a hypothesis, the exatecan drug, FL118 drug, compound of Chemical Formula 3 (PBX-7011), and compound of Chemical Formula 3-1 (PBX-7012) seem to have a continuous action with Asp533 and Lys532 of mutant TOP1 and show a little resistance.

[0454] Example 2 For FL118, exatecan, SN-38, Dxd, exatecan-lactate, PBX-7011, PBX-7014, PBX-7016, and PBX-7024, the following anti-apoptosis protein expression inhibition analysis and in vitro cell survival assay were performed.

[0455] 2-1: Confirmation of the expression inhibitory activity of cancer-associated survival genes of DDX5, survivin, Mcl-1, XIAP, and cIAP2 through Western blot

[0456] (1) Protein extraction 200,000 FaDu cell lines were seeded per well in a 6-well plate and incubated at a constant temperature (37 °C, 5% CO2). After 24 hours, drugs (FL118 drug, SN-38 drug, exatecan drug, PBX-7011, PBX-7014, PBX-7016) were treated in each well at concentrations of 0 nM, 10 nM, and 100 nM. They were incubated at a constant temperature (37 °C, 5% CO2) for 24 hours. 100 μl of RIPA buffer in which a protease inhibitor cocktail was dissolved was treated in the well. The plate was stabbed on ice and incubated at a constant temperature for 2 hours on an orbital shaker. The RIPA buffer containing the lysed cells was transferred to an ep tube and centrifuged (16,000 rcf, 20 min, 4 °C). Then, only the supernatant was transferred to a new ep tube. The protein concentration was confirmed through protein measurement.

[0457] (2) Protein separation through electrophoresis The protein sample and 4x SDS-PAGE loading buffer were mixed at a ratio of 3:1, boiled at 95 °C for 10 minutes, and then cooled. The samples were loaded into the wells of the gel so that the amount of protein was the same. Electrophoresis was performed on the gel at 60 V.

[0458] (3) Transfer of the protein from the gel to the membrane Trans-Blot (registered trademark) Turbo TMSeven activated filter papers, a PVDF membrane, a gel, and seven filter papers were sequentially placed in the cassette of the transfer system. After closing the lid and inserting it into the machine, the protocol was run.

[0459] (4) Antibody Incubation The transferred membrane was immersed in the blocking buffer and incubated at a constant temperature (RT, 1 h). Next, it was incubated with the primary antibody solution at a constant temperature (4 °C, overnight). It was washed with TBST buffer for 3 minutes (repeated 3 times). It was incubated with the secondary antibody solution conjugated with HRP at a constant temperature (RT, 1 h). It was washed with TBST buffer for 3 minutes (repeated 3 times).

[0460] (5) Imaging and Result Analysis The membrane was immersed in the ECL substrate for about 3 - 5 minutes, and then the signal was confirmed using the ChemiDoc TM MP imaging system. The light emitted by the oxidation of the ECL luminol by the HRP conjugated to the secondary antibody was detected and shown as an image. Since the thickness of the band is proportional to the amount of protein, the amount of protein can be compared through the thickness of the band.

[0461] As shown in Figures 10 - 12, it was confirmed how the expression level of the protein changed by the treatment with drugs (FL118 drug, SN-38 drug, exatecan drug, PBX-7011, PBX-7014, PBX-7016). GAPDH is an enzyme involved in glycolysis, an essential metabolic process in cells, and is constantly expressed in cells. It is a gene whose expression level does not change much and is an indicator to know whether the same amount of protein was loaded in the sample.

[0462] In the same way, instead of the FaDu cell line that does not express ABCG2, A549 that overexpresses ABCG2 was seeded, 4 μg of protein was loaded, and the Western blot was performed (Figures 13 and 14).

[0463] Investigation: Evaluation of PBX-7011, PBX-7014, and PBX-7016 As a result of confirming the degree of inhibition of the expression of anti-apoptotic proteins through Western blot in the FaDu cell line, PBX-7011, PBX-7014, and PBX-7016 showed that the expression of survivin, cIAP2, Mcl-1, and XIAP all decreased with increasing concentration. When compared with existing drugs FL118, SN-38, and exatecan, it was shown that they were at a similar level to FL118 and exatecan drugs and were even better than SN-38.

[0464] In Example 2-1, it showed a dual inhibitory effect of decomposing DDX5 while suppressing type I topoisomerase (topoisomerase 1), and it was confirmed that it had the heterogeneous characteristic of DDX5 decomposition and the resulting anti-cancer effect.

[0465] Specifically, after treating two cell lines (FaDu, A549) with drugs at a certain concentration (0, 10, 100 nM) and confirming the expression levels of each protein (DDX, survivin, Mcl-1, XIAP) by Western blot, the DDX5 decomposition by FL118 was superior to other substances, and then came 7011, 7016, and 7014 in that order.

[0466] Not only DDX5, but also the tendency of other anti-apoptotic proteins downstream of it was similar to this.

[0467] In short, PBX-7014 and PBX-7016 show a dual MoA that not only inhibits type I topoisomerase but also acts as a degrader of DDX5 (p68), a cancer protein that regulates survivin, Mcl-1, XIAP, etc.

[0468] Among the evaluation results, PBX-7014, and PBX-7016, PBX-7016 inhibits DDX5 in a concentration-dependent manner, and as a result, it is observed that it inhibits survivin, Mcl-1, and XIAP. Thus, PBX-7016 not only acts as a topoisomerase I inhibitor but also as a DDX5 degrader of FL118. It was also confirmed that PBX-7016 exhibits superior activity compared to PBX-7014 (Figs. 11 to 14).

[0469] 2-2: Cell viability assay for FaDu cell line / A549 cell line 3000 FaDu cell line (cancer cells that do not express ABCG2) or A549 cell line (cancer cells that overexpress ABCG2) were seeded per well in a 96-well plate and incubated at a constant temperature (37 °C, 5% CO2). After 24 hours, 100 μl of the drug at 9 concentrations (serially diluted 1 / 5 each from 1000 nM) was added to the cells. At this time, the Dxd drug, PBX-7014, PBX-7016, and PBX-7024 were treated. At this time, a control group without drug treatment (drug concentration 0) was also prepared. The cells were incubated at a constant temperature (37 °C, 5% CO2) for 3 or 6 days. 100 μl of CellTiter-Glo reagent (using CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega, G7571)) was added to each well and pipetted. After incubation at a constant temperature (RT) for 10 minutes, luminescence was measured. Assuming the luminescence value when the drug concentration is 0 is 100%, the concentration showing 50% of the luminescence value is the IC50 value.

[0470] Two cell lines (FaDu, A549) were treated with two compounds (PBX-7016, PBX-7024) and reference compounds (Dxd, SN-38, exatecan, FL118), and cell viability was observed through 3-day incubation.

[0471] As shown in Fig. 15, not only the FaDu cell line that does not express ABCG2, but also A In the cancer cell line A549 that overexpresses BCG2, PBX-7024 also exhibits a potent cell-killing effect. That is, it has an IC 50 equal to or higher than that of Dxd, which is the payload used in existing enhertu.

[0472] In the A549 cancer cell line that overexpresses ABCG2, as shown in Figure 15, camptothecin compounds containing Dxd show high IC 50 values, while it can be confirmed that PBX-7016 and PBX-7024 still maintain strong efficacy.

[0473] That is, when using the new camptothecin compounds PBX-7016 and PBX-7024 according to the present invention, different from ADCs using existing camptothecin compounds such as SN-38 and DXd, it has the effect of overcoming the resistance mechanism due to overexpression of ABCG2.

[0474] Example 3. New PBX series compounds as candidates for ADC payloads: In vitro cell viability assay Cell viability assays were performed on MDA-MB-453 (HER2++) cell line and FaDu (HER2+) cell line in the same manner as in Example 2-2.

[0475] Two cell lines (MDA-MB-453, FaDu) were treated with two compounds (PBX-7014, PBX-7016, PBX-7018, PBX-7020, PBX-7022) and one reference compound (Dxd), and cell viability was observed through 3-day incubation. The results are shown in Figure 16.

[0476] As shown in Figure 16, in the evaluation results, PBX-7016, PBX-7018, PBX-7020, and PBX-7022 have an IC 50 equal to or higher than that of Dxd. Also, in the FaDu cell line that does not express ABCG2, it has an IC 50It was confirmed to have a value.

[0477] Example 4. PBX-7016 IV and IP Pharmacokinetic Profiles in ODSCID Mice The experimental method is as shown in Table 3 below. The IV plasma concentration-time data of PBX-7016 are shown in Table 4, and the IP plasma concentration-time data of PBX-7016 are shown in Table 5.

[0478] [Table 4]

[0479] [Table 5]

[0480] [Table 6]

[0481] Through the results of Table 4 and Table 5, it is confirmed that the PBX-7016 compound shows high safety when administered in vivo alone in the form of the PBX-7016 compound rather than in the form of a prodrug or ADC by confirming that clearance occurs in the blood very quickly when administered intravascularly or intraperitoneally to mice. Since the PBX-7016 compound is released outside the body within a very short time when administered IV, it can act as a safety device that can reduce the systemic exposure of the drug even when the drug is released prematurely earlier than usual when it enters the body.

[0482] Also, this experiment suggests that the PBX-7016 compound used as the payload alone is a safe payload with rapid clearance of the PBX-7016 drug in the blood as the PK of the PBX-7016 compound used as the payload. That is, it suggests that after the PBX-7016 compound exerts a bystander killing effect at the tumor site, it is safely released outside the body within a very short time even when circulating in the blood.

[0483] Example 5. Carboxylate to Lactone conversion rate of PBX-7016 at pH 6.0 Example 5 (Figs. 17 and 18) is an experiment related to the activation of the lactone moiety of camptothecin, known as an active factor of the Top1 inhibitor.

[0484] 5-1. Conversion from carboxylate to lactone form at pH 6.0 PBX-7016 powder was prepared by dissolving it in DMSO at 5 mM. Since PBX-7016 dissolved in DMSO exists in the lactone form, it was diluted 1 / 10 with 0.1 N NaOH and incubated in a 25°C water bath for 30 minutes to convert it to the carboxylate form. The carboxylate form of PBX-7016 was diluted to 3 μM using 4°C PBS (pH 6.0) containing 10% DMSO. At this time, the same equivalent amount of HCl as the 0.1 N NaOH used was added to minimize the pH change. After dilution, it was centrifuged at 4°C for 2 minutes at 13000 RCF, and the supernatant was used as the sample.

[0485] When the PBX-7016 lactone form was generated, two solvents, Solvent A: 100 mM acetate buffer pH 5.5 and Solvent B: ACN, were used to separate it by RP-HPLC. The column used was a Cortecs C18 2.7 μm, 4.6 x 50 mm column, and the analysis proceeded under the following conditions: 1) 20 minutes: 5% B, 2) sample injection, 3) 2 minutes: 5% B, 4) 10 minutes: 5% B → 50% B, 5) 1 minute: 50% B.

[0486] To measure the change over time, the sample chamber was maintained at 37°C and set so that the sample was injected at each measurement time. The measurement times were 0, 20, 40, 60, 90, 120, 180, 240, 300, 360, 480, 600, 720, 840, 960, 1200, 1440 minutes.

[0487] The content was determined by the peak areas of lactone and carboxylate respectively. Since the extinction coefficients of lactone and carboxylate are different, the lactone area was determined by multiplying the carboxylate area by the following formula. Lactone EC 390 / Carboxylate EC 390 =0.918

[0488] The same experiment as in Example 5-1 was conducted, except that FL118, SN38, DxD, exatecan, PBX-7014, and PBX-7024 were used instead of PBX-7016.

[0489] The experimental results of the conversion from carboxylate to lactone form at pH 6.0 are shown in Figure 18. As shown in Figure 18, it was confirmed that various camptothecin derivatives changed from carboxylate to the active lactone form that inhibits topoisomerase I at a similar rate and equivalent level of amount at pH 6.0. Through this, it is generally predicted that they exhibit equivalent levels of activity in the tumor microenvironment with a pH of 6.0.

[0490] 5-2. Conversion from lactone to carboxylate form at pH 7.4 PBX-7016 powder was dissolved in DMSO at 5 mM to prepare a solution, which was diluted with 4°C PBS (pH 7.4) containing 10% DMSO to prepare 3 μM PBX-7016. Then, this was centrifuged at 4°C for 2 minutes under the condition of 13,000 RCF, and the supernatant was used as the sample. PBX-7016 in the dissolved state in DMSO existed in the lactone form.

[0491] When the carboxylate form of PBX-7016 was generated, in order to separate it by RP-HPLC, two solvents were used: Solvent A: 100 mM acetate buffer pH 5.5 and Solvent B: ACN. The column used was a Cortecs C18 2.7 μm, 4.6 x 50 mm column, and the analysis was carried out under the following conditions. 1) 20 minutes: 5% B, 2) sample injection, 3) 2 minutes: 5% B, 4) 10 minutes: 5% B → 50% B, 5) 1 minute: 50% B.

[0492] To measure the change over time, the sample chamber was maintained at 37 °C and set so that the sample was injected every measurement time. The measurement times were 0, 20, 40, 60, 90, 120, 180, 240, 300, 360, 480, 600, 720 minutes.

[0493] The content was determined by the peak area of each of lactone and carboxylate. Since the extinction coefficients of lactone and carboxylate are different, the lactone area was determined by multiplying the carboxylate area by the following formula. Lactone EC 390 / Carboxylate EC 390 = 0.918

[0494] The same experiment as in Example 5-2 was conducted except that FL118, SN38, DxD, exatecan, PBX-7014, and PBX-7024 were used instead of PBX-7016.

[0495] As shown in FIG. 17, which is the experimental result of the conversion from the lactone form to the carboxylate form at pH 7.4, the PBX-7014, PBX-7016, and PBX-7024 compounds represented by Chemical Formula 1 at pH 7.4, which is the pH of blood or extracellular fluid, are not only faster but also higher It was confirmed that the conversion from the lactone form (active form) to the carboxylate form (inactive form) that is inactive as a TOP1 decontaminant occurs at a higher rate.

[0496] In particular, the degree of formation (63 - 86%) and rate (0.4 - 0.8% / min) of the carboxylate form, which is inactive as a TOP1 poison at blood pH 7.4, vary significantly depending on the type of camptothecin derivative. PBX - 7014 and PBX - 7016 formed more than 86% of the carboxylate form and were relatively rapidly converted from the lactone form to the carboxylate form at a level of 0.8% per minute. The competitive drug, DXd, formed a carboxylate form at a level of 76.5% and was converted from the lactone form to the carboxylate form at a level of 0.6% per minute.

[0497] Through this, it is predicted that the PBX - 7014 and PBX - 7016 compounds exist as inactive carboxylates rather than the active - form lactones that exhibit cytotoxicity relatively rapidly and at a high rate in the blood compared to the reference compound, and show high safety in normal cells.

[0498] Production Example 5: Synthesis of 25 - 4 and 25 - 6 from PBX - 7014 and PBX - 7016 and Production of Their ADC (DAR7 - 8) (Trastuzumab - 25 - 4 and Trastuzumab - 25 - 6) Linker - payloads 25 - 4 (Chemical Formula 10) and 25 - 6 (Chemical Formula 11), which contain two compounds (PBX - 7014, PBX - 7016) and introduce GGFG, an enzymatically cleavable linker system, were synthesized.

[0499]

Chem.

[0500]

Chem.

[0501] The molecular structure of 25 - 4 is GGFG - PBX - 7014, and the molecular structure of 25 - 6 is GGFG - PBX - 7016. That is, they each use the same GGFG linker as Enhertu (registered trademark).

[0502] Furthermore, using the same GGFG linker and trastuzumab antibody as Enhertu (registered trademark), the ADCs of PBX-7014 and PBX-7016 were manufactured with these as payloads.

[0503] Linker-payload compounds of Chemical Formula 10 and Chemical Formula 11 were conjugated with trastuzumab, which is a Her2-targeting antibody, to synthesize ADCs (Tra-25-4 and Tra-25-6, all with DAR 8).

[0504] Trastuzumab-25-4 (trastuzumab-7014) was manufactured in the following manner. The prepared trastuzumab was buffer-exchanged into the reaction buffer (150 mM NaCl, 50 mM histidine pH 6.0) using a PD-10 desalting column, and then 825 μM TCEP was treated with 27.5 μM of the antibody at 25 °C for 2 hours to create the thiol sites necessary for the reaction from the disulfide bonds of the antibody.

[0505] Thereafter, excess TCEP was removed using a PD-10 desalting column, and 61.9 μM of the 25-4 drug linker (Chemical Formula 10) and 13.8 μM reduced trastuzumab were reacted at 25 °C for 1 hour to proceed with the primary conjugation reaction.

[0506] In the case of trastuzumab-25-6 (trastuzumab-7016), it was manufactured through the same process, and the same concentration of the 25-6 drug linker (Chemical Formula 11) was used instead of the 61.9 μM 25-4 drug linker (Chemical Formula 10).

[0507] Thereafter, each ADC was purified using SEC, and it was confirmed that they were purified as monomers without aggregation (Figure 19). It was confirmed that the drug was conjugated through the band shift of the light and heavy chains via SDS-PAGE (Figure 20a).

[0508] Production Example 6: Synthesis of 25-6 from PBX-7016 and Production of Its ADC (DAR 7-8) (Nimotuzumab-25-6 and Sacituzumab-25-6) Nimotuzumab-25-6, Sacituzumab-25-6, and Cetuximab-25-6 were synthesized in the same manner as in Production Example 5, except that nimotuzumab, sacituzumab, or cetuximab was used instead of trastuzumab.

[0509] Subsequently, each ADC was purified using SEC, and it was confirmed that the monomer was purified without aggregation. It was confirmed that the drug was conjugated through the band shift of the light chain and heavy chain via SDS-PAGE (Figure 20b, Figure 20c, and Figure 20d).

[0510] Example 6. Anti-HER2 ADC with PBX-Payload: Binding Affinity Analysis The coating buffer was prepared by diluting the target antigen to 0.66 μg / ml using ELISA plate coating buffer (R&D systems, #DY006). The prepared coating buffer was used at 100 μL per well for coating. The plate cover was closed and the process was carried out overnight (12 - 18 hours) while maintaining at 2 - 8°C. The plate used was Corning, #3590. More than 200 μL of TBS It was washed three times using Tween-20 buffer (Thermo, #28360). When washing, first remove the liquid with a pipette, then gently tap it on a paper towel to remove the excess buffer. Blocking was carried out at room temperature for 1 hour using 200 μL of assay buffer. Remove the liquid with a pipette and gently tap it on a paper towel to remove the excess buffer. After preparing the control antibody and ADC to be measured at 1 μg / ml in assay buffer, more than the required amount was prepared at the following concentrations through 1 / 5 serial dilution. For example, if samples are prepared by diluting at the following concentrations according to the replication standard [240 μl assay buffer + 60 μl sample of the previous concentration]: 1 μg / ml, 0.4 μg / ml, 0.08 μg / ml, 0.016 μg / ml, 0.0032 μg / ml, 0.00064 μg / ml, 0.000128 μg / ml. The diluted control antibody and ADC were bound in duplicates of 100 μL per well coated. Incubate for 1 hour at room temperature. It was washed three times using 100 μl of TBS Tween-20 buffer (Thermo, #28360). When washing, first remove the liquid with a pipette, then gently tap it on a paper towel to remove the excess buffer. The detection antibody was prepared by diluting it in assay buffer with reference to the manufacturer's manual. In the case of human antibodies, it was prepared by diluting goat anti-human Fc-HRP (Novex, #A18817) at 1 / 2000 as the detection antibody. After adding 100 μL of the diluted detection antibody to each well, incubate for 1 hour at room temperature. It was washed three times using 100 μl of TBS Tween-20 buffer (Thermo, #28360). When washing, first remove the liquid with a pipette, then gently tap it on a paper towel to remove the excess buffer. React for 30 minutes at room temperature using 100 μL of TMB substrate solution (Thermo, #N301). Stop the reaction using 100 μL of stop solution (Thermo, N600). Stop the reaction and measure the absorbance at 450 nm. It was completed within 30 minutes after adding the stop solution. When bubbles are present, transfer 100 - 150 μl to a new plate for measurement.Through this, trastuzumab, ADC conjugated with trastuzumab and PBX-7014, and ADC conjugated with trastuzumab and PBX-7016 were measured, and the results shown in Fig. 21 were obtained. As shown in Fig. 21, when the binding strength of ADC conjugated with PBX-7014 or PBX-7016 to trastuzumab against the Her2 antigen was carried out at various concentrations compared to the binding strength of trastuzumab against the Her2 antigen, it was confirmed that there was no difference in the binding strength.

[0511] Example 7. Anti-HER2 ADC with PBX-payload: In vitro efficacy and target selectivity For ADCs with Dxd, PBX-7014, and PBX-7016 as payloads respectively, in the same manner as in Example 2-2, the following MDA-MB-453 (HER2++) cell line, FaDu (HER2+) cell line, and MDA-MB-468 (HER2−) An in vitro cell viability assay was performed on the cell lines.

[0512] At this time, Herceptin (Herceptin, ingredient name: trastuzumab), a HER2-targeted therapeutic agent, Dxd drug, PBX-7014 (Production Example 2), PBX-7016 (Production Example 3), its ADCs Tra-25-4, Tra-25-6 (Production Example 5), and Tra-Dxd (Enhertu (registered trademark)) were treated.

[0513] As a reference, Enhertu (Tra-Dxd, DAR 8) was treated with three cell lines, MDA-MB-453 (Her2++), FaDu (Her2+), and MDA-MB-468 (Her2−) according to the Her2 expression level, and cell survival was observed through 3-day incubation (Fig. 22).

[0514] As shown in Fig. 22, it was confirmed that the anti-Her2 ADC with PBX-payload has excellent target selectivity.

[0515] Investigation: Evaluation of PBX-7014 and PBX-7016 as ADC Payloads As shown in Figure 22, in MDA-MB-453 which is Her2++, the ADC (Tra-25-6) with PBX-7016 as the payload has an IC that is approximately 5 times better than that of Trastuzumab deruxtecan (Tra-Dxd). 50 It was confirmed to have equivalent IC values in MDA-MB-468, a negative cell line. By confirming that the linker-payload systems of Chemical Formula 10 and Chemical Formula 11 have equivalent IC values when developed as ADCs, it was confirmed that not only do they operate by differentiating between positive / negative cell lines, but also show superior efficacy compared to the reference Trastuzumab deruxtecan. 50 showed excellent efficacy.

[0516] Investigation: Novelty and Excellence of PBX-7016 PBX-7016, a camptothecin-based compound newly designed and synthesized by Chemical Formula 1 according to the present invention, is a compound newly derived from FL118 which has an MD-CPT skeleton. Just as FL118 acts not only as a topoisomerase I inhibitor but also as a DDX5 degrader, it newly presents a new topoisomerase I inhibitor that degrades DDX5 (Figures 11 - 14).

[0517] Also, when simply comparing cell viability, PBX-7016 shows IC values at a level 1.5 - 2 times that of Dxd. Surprisingly, when used as an ADC payload, it was confirmed that the ADC containing PBX-7016 has cell viability that is approximately 5 times better than the ADC containing Dxd (Trastuzumab deruxtecan) (Figure 22). 50

[0518] Example 8. Anti-HER2 ADC with PBX-Payload: In Vivo Efficacy 8-1. Cell Culture ​​JIMT-1 breast cancer cells were cultured in DMEM containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin under the conditions of 5% CO2 and 37 °C, and subcultured in a routine manner twice a week by trypsin-EDTA treatment. Cells growing in the exponential growth phase were harvested and counted for tumor inoculation.

[0519] 8-2. BALB / c nude mice Both SCID mice and BALB / c nude mice are used as animal models for studying the immune system and testing treatment methods for various diseases.

[0520] Both SCID mice and BALB / c nude mice have damaged immune systems, but the basic mechanisms of immune deficiency are different.

[0521] SCID mice are genetically engineered mice lacking functional T cells and B cells, and their immune systems are severely damaged. These are used to study immune system development and function and to test potential treatment methods for diseases that affect the immune system, such as autoimmune disorders and certain types of cancer.

[0522] On the other hand, BALB / c nude mice are mice without a thymus and unable to produce T cells. These also have a defect in the hairless gene and are born without hair. This mouse is used to study the role of T cells in the immune response and to test potential treatment methods for diseases that affect T cell function, such as certain types of cancer.

[0523] 8-3. Tumor inoculation and drug treatment JIMT-1 tumor cells (5x10 6 ) in 0.2 mL of DPBS with Matrigel were subcutaneously inoculated into the right anterior flank of each BALB / c nude mouse. When the average tumor volume reached approximately 215 mm 3 , after randomly grouping the animals, treatment for the drug efficacy study was started as follows.

[0524] Treatment: I.V., Q1W Vehicle group: Vehicle, 0 mg / kg, 5 μL / g, i.v., twice (day 0, day 6) ) Isotype-Dxd(3) group: CTP63-Dxd, 3 mg / kg, 5 μL / g, i.v., twice (day 0, day 6) Isotype-Dxd(10) group: CTP63-Dxd, 10 mg / kg, 5 μL / g, i.v., twice (day 0, day 6) Isotype-PBX7016(3) group; CTP63-PBX7016, 3 mg / kg, 5 μL / g, i.v., twice (day 0, day 6) Isotype-PBX7016(10) group: CTP63-PBX7016, 10 mg / kg, 5 μL / g, i.v., twice (day 0, day 6) Isotype-PBX7014(3) group: CTP63-PBX7014, 3 mg / kg, 5 μL / g, i.v., twice (day 0, day 6) Isotype-PBX7014(10) group: CTP63-PBX7014, 10 mg / kg, 5 μL / g, i.v., twice (day 0, day 6) Trastuzumab-PBX7014(3) group: CTP6-PBX7014, 3 mg / kg, 5 μL / g, i.v., twice (day 0, day 6) Trastuzumab-PBX7014(10) group: CTP6-PBX7014, 10 mg / kg, 5 μL / g, i.v., twice (day 0, day 6) Trastuzumab-PBX7016(3) group: CTP6-PBX7016, 3 mg / kg, 5 μL / g, i.v., twice (day 0, day 6) Trastuzumab-PBX7016(10) group: CTP6-PBX7016, 10 mg / kg, 5 μL / g, i.v., twice (day 0, day 6) Enhertu(3) group: Enhertu, 3 mg / kg, 5 μL / g, i.v., twice (day 0, day 6) Enhertu(10) group: Enhertu, 10 mg / kg, 5 μL / g, i.v., twice ( day 0, day 6)

[0525] 8-4. Tumor measurement and endpoints The time point when it was possible to confirm whether tumor growth might be delayed or the mouse was treatable was set as the primary endpoint. The two-dimensional size of the tumor was measured twice a week using calipers, and the volume was calculated using the formula (V = 0.5ab 2 , where a and b are the major and minor diameters of the tumor respectively) and expressed in mm 3 .

[0526] TGI (Tumor growth inhibition) was calculated for each group. TGI (%) = [1 - (T i - T0) / (V i - V0)] × 100 (T i is the average tumor volume of the treatment group on a specific day, T0 is the average tumor volume of the treatment group on the first day of treatment, V i is the average tumor volume of the vehicle control group on the same day as T i , and V0 is the average tumor volume of the vehicle group on the first day of treatment).

[0527] Animals with a weight loss of 15% or more or a tumor volume of 3000 mm 3 or more were euthanized at the humane endpoint.

[0528] 8-5. Statistical analysis The results were presented as mean and standard error (mean ± SEM). The data were analyzed using a two-way RM ANOVA Dunnett's multiple comparison test with Graphpad Prism 6.0 software, and p < 0.05 was considered statistically significant.

[0529] Table 6 below shows the change in tumor volume (mm 3 ) over time (effect vs. time).

[0530]

Table 7

[0531] Figure 23, which graphically shows Table 6, shows the results of a potency test for ADCs with various camptothecin-based drugs as payloads in mice transplanted with the JIMT-1 cell line, i.e., an in vivo xenograft model.

[0532] 8-6. Body weight of the JIMT-1 tumor model The body weight of the JIMT-1 tumor model mice was regularly monitored (Figure 24).

[0533] Example 9: Anti-HER2 ADC with PBX-payload: Study on the bystander killing effect The bystander effect is one of the factors that significantly affect the efficacy of ADC drugs. To confirm the bystander effect, analysis was performed by FACS under the condition of 40 nM ADC treatment.

[0534] In order to verify the efficacy and utility of the ADC drug using PBX-7016, the lead compound of the present invention, as a payload in an antibody-drug conjugate (ADC) in which a payload-linker is conjugated to the trastuzumab antibody targeting the HER2 protein, the bystander effect was confirmed in vitro through a flow cytometric analysis experiment. In this example, the bystander effect was defined as follows: the HER2-positive cell line MDA-MB-453 was treated with ADC, which induced cell death, and the drug (payload) released during the process of cell death induced toxicity in the surrounding HER2-negative cell line, leading to cell death.

[0535] In Production Example 5, to evaluate the in vitro bystander killing effect of the produced ADC, the presence or absence of the bystander effect by ADC treatment was evaluated under the condition of co-culturing a HER2 expression-negative cell line and a HER2 expression-positive cell line in a 6-well scale cell culture plate according to the ratio.

[0536] Specifically, GFP-MDA-MB-468 cells (3x10 5 cells), which were engineered to express GFP (Green fluorescent protein) in the HER2-negative cell line MDA-MB-468, and MDA-MB-453 cells (1x10 5 cells), a HER2-positive cell line, were mixed at a ratio of 3:1 and co-cultured for 24 hours. Then, trastuzumab antibody and all five ADC samples (trastuzumab-DXd, isotype IgG-DXd, trastuzumab-25-6, isotype IgG-25-6, and enhertu) were each treated at a concentration of 40 nM. After culturing the cells for 6 days, GFP fluorescence measurement and cell count of GFP-MDA-MB-468 cells, which are HER2-negative cell lines among the living cells, were confirmed through flow cytometry analysis.

[0537] Meanwhile, MDA-MB-468 cells (3x10 5 cells), a HER2-negative cell line, and MDA-MB-453 cells (1x10 5 cells), a HER2-positive cell line, were mixed at a ratio of 3:1 and co-cultured for 24 hours in a 6-well cell culture plate. Then, trastuzumab antibody and all five ADC samples (trastuzumab-DXd, isotype IgG-DXd, trastuzumab-25-6, isotype IgG-25-6, and enhertu) were each treated at a concentration of 40 nM. After culturing the cells for 6 days, cells were stained using an anti-HER2-FITC antibody, which was labeled with FITC (Fluorescein isothiocyanate) fluorescent dye to recognize the HER2 protein and show fluorescence. After that, FITC fluorescence measurement and cell count of MDA-MB-453 cells, which are HER2-positive cell lines among the living cells, were confirmed through flow cytometry analysis.

[0538] For flow cytometry analysis, the cells present in each well of a 6-well cell culture plate were collected and diluted with buffer (400 μl), and then GFP or FITC fluorescence was measured for each sample at the same time (1 minute each) and the same flow rate using an FL1 laser.

[0539] As shown in FIGS. 25 and 26, as a result of confirmation through flow cytometry analysis, trastuzumab-25-6, an ADC based on PBX-7016, not only killed HER2-expressing positive cells but also simultaneously showed the death of HER2-expressing negative cells when compared with trastuzumab-Dxd, an ADC based on DXd, and Enhertu, and was judged to show a bystander effect at a level equivalent to that of the existing DXd-based Enhertu. In contrast, isotype IgG-DXd and isotype IgG-25-6, which are negative control ADCs, were confirmed to not significantly induce the death of HER2-expressing negative cells compared with the untreated condition without ADC treatment.

[0540] In short, the anti-HER2 ADC using PBX-7016 showed a bystander effect similar to that of Enhertu.

[0541] Example 10: In Vitro Cytotoxicity Test of Cetuximab-25-6 and Sacituzumab-25-6 Cetuximab-25-6 (FIG. 27a) was treated against the MDA-MB-468 (EGFR positive) cell line and the SW480 (EGFR negative) cell line, and sacituzumab-25-6 (FIG. 27b) was treated against the FaDu (Trop2+++) cell line and the Calu-6 (Trop2--) cell line in the same manner as in Example 2-2, and a cell viability assay was performed through 6 days of incubation. The results are shown in FIG. 27.

[0542] FIG. 27 shows that even in the case of ADCs such as cetuximab-25-6 and sacituzumab-25-6, a significant portion of the mAb can be localized to the target cell population through high-affinity mAb binding to cell membrane proteins, and the chemical conjugation of the PBX-7016 payload with the anti-cancer mAb suggests that it increases the selectivity by which the PBX-7016 payload is delivered to cancer cells and increases the therapeutic index of the payload.

[0543] In particular, the results of the comparative evaluation of in vitro cell viability (administering the ADC for 6 days) for sacituzumab-25-6 and PBX-7016 against the FaDu (Trop2+++) cell line and the Calu-6 (Trop2--) cell line (FIG. 27b) show that PBX-7016, designed to be released from the in vivo target site as an example of the active camptothecin derivative (a) represented by Chemical Formula 1, can penetrate the cell membrane of non-target cells and move into the cells to exert a cell death mechanism, that is, the PBX-7016-based ADC can exert a bystander effect.

Claims

1. (a) an active camptothecin derivative that binds to DDX5, which acts as an oncoprotein in cells, and induces cell death through DDX5 proteolysis; (b) a prodrug, preferably an antibody-drug conjugate (ADC), of the active camptothecin derivative (a) designed to release it in vivo. , or (c) a method for producing a ligand-containing complex that targets the DDX5 protein, wherein the active camptothecin derivative (a) has its type I topoisomerase inhibitory ability inactivated via linker linkage, A first step of selecting the active camptothecin derivative (a) from the group consisting of the compound of chemical formula 5, the compound of chemical formula 6, the compound of chemical formula 7, the compound of chemical formula 9 and its isomers; and A method for producing a product, comprising an optional step of selecting a prodrug (b) of the active camptothecin derivative (a) selected in the first step, which is designed to release the active camptothecin derivative (a) in vivo. 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】

2. The isomer of the active camptothecin derivative is represented by the following chemical formula 5-1 The manufacturing method according to claim 1. 【Transformation 5】

3. The method for producing the active camptothecin derivative (a) or its prodrug (b) is for administration to a group of patients in whom DDX5 acts as an oncoprotein within cells, as described in claim 1.

4. The method for producing the product according to claim 1, further comprising the step of identifying a patient group to whom the active camptothecin derivative (a) or its prodrug (b) should be administered, by quantitatively or qualitatively analyzing intracellular DDX5 protein using a ligand-containing complex (c) that targets the DDX5 protein, wherein the type I topoisomerase inhibitory ability of the active camptothecin derivative (a) has been inactivated via linker linkage in a tissue biopsy or liquid biopsy.

5. Compounds represented by the following chemical formulas 5, 5-1, 6, 7, or 9, or their pharmaceutically acceptable salts: 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】

6. A pharmaceutical composition for use in the prevention or treatment of cancer, comprising a compound represented by the following chemical formulas: 5, 5-1, 6, 7, or 9; a pharmaceutically acceptable salt thereof; a solvate thereof; or a prodrug thereof, preferably an antibody-drug conjugate (ADC): 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】

7. A carrier-drug conjugate comprising an active camptothecin derivative represented by the following chemical formulas 5, 5-1, 6, 7, and 9, a pharmaceutically acceptable salt thereof, or a solvate thereof. 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】 【Chemistry 20】

8. The carrier-drug conjugate according to claim 7, characterized in that the carrier is an antibody, peptide, repebody, or aptamer.

9. The carrier drug complex according to claim 8, characterized in that the carrier drug complex is in a form in which the carrier is complexed with a camptothecin derivative represented by chemical formula 5, chemical formula 6, chemical formula 7, or chemical formula 9 via a linker.

10. The carrier drug complex according to claim 9, characterized in that the linker contains GGFG.

11. The carrier contains 4-1BB, 5T4, integrin, activin, amyloid beta, angiopoietin (angiopoietin 1 or 2), angiopoietin-like substance 3, B cell maturation antigen (BCMA), B cell activating factor (BAFF), B7-H3, and complement 5. 5), CCR4, CCR5, CCL11, CD2, CD3, CD4, CD6, CD11a, CD16A, CD19, CD20, CD22, CD25, CD27, CD28, CD30, CD32B, CD33, CD38, CD40, CD45, CD46, CD 47, CD52, CD56, CD62, CD70, CD73, CD74, CD79b, CD80, CD105, CD123, CD154, CD166, CD262, CD278, CD319, CD326, Carcinoembryonic antigen Antigen (CEA), CGRP, Claudin-18, c-Met, CSF-1, CSF-1 receptor, CTLA4, DLL3, EGF receptor, hemophilia factor, Fc receptor, FGF23, folate receptor, GD2, glucocorticoid-induced TNF receptor (GITR), glypican 3, GM-CSF, HER2, HER3, TROP2, hepatocyte growth factor Factor, HGF), interferon receptor, interferon gamma, IgE, IGF-1 receptor, interleukin 1, interleukin 2 receptor, interleukin 4, interleukin 4 receptor, interleukin 5, interleukin 5 receptor, interleukin 6, interleukin 6 receptor, interleukin 8, interleukin 12 / 23, interleukin 13, interleukin 17A, interleukin 17 receptor A, interleukin 23, interleukin 31 receptor, interleuk 36 receptor, lymphocyte activation gene 3 (Lymphocyte-activation gene 3 (LAG3), lysyl oxidase homolog 2 (LOXL2), mesothelin, mucin-1, mucin-16, netin-4, nerve growth factor (NGF), OX40, protein convertase subtilisin / kexin type 9 (PCSK9), PD-1, PD-L1, phospholipase C, RANKL (receptor activator of nuclear Factors kappa B ligand), tyrosine-protein kinase transmembrane receptor (ROR1), sialic acid-binding ig-like lectin 15 (Siglec-15), transforming growth factor beta (TGFβ), TIGIT (T-cell immunoglobulin and ITIM domain), T-cell immunoglobulin and mucin domain-containing substance 3 (T-cell immunoglobulin and mucin-domain containing-3, Tim-3), tissue factor (Tissue factor), tissue factor pathway inhibitor (Tissue factor pathway inhibitor) inhibitor, TFPI), TORP-2, tumor necrosis factor (TNF), thymic stromal lymphopoietin (TSLB), colony stimulating factor 1 receptor (Colony stimulating factor 1 receptor, CSF1R), vascular endothelial growth factor (stimulating factor 1 receptor, CSF1R), vascular endothelial growth factor factor,The carrier drug conjugate according to claim 8, characterized in that it is an antibody, peptide, repebody, or aptamer that specifically binds to one or more substances (antigens) selected from the group consisting of VEGF, VEGF receptor, and vWF (von Willebrand Factor).

12. The carrier-drug conjugate according to claim 8, characterized in that the carrier is an antibody.

13. The antibodies mentioned above are urelumab, utomirumab, bebuterovimab, aducanumab, bapineuzumab, crenezumab, donanemab, gantenerumab, lecanemab, and soranezumab. lanezumab, nesvacumab, evinacumab, enoblituzumab, omburtamab, belimumab, ianalumab, tabalumab, vertilimmumab, mogamulizumab, Leronlimab, Siplizumab, Foralumab, Muromonab-CD3, Otelixizumab, Teplizumab, Ibalizumab, Tregalizumab, Zanolimumab, Itolizumab (Itolizumab), Efalizumab, Inebilizumab, Tafasitamab, Tositumomab, Ocrelizumab, Ofatumumab, Rituximab, Ublituximab, Vertuzumab umab), epratuzumab, basiliximab, daclizumab, varlilumab, rulizumab, iratumumab, lintuzumab, daratumumab, felzartamab, isatuximab, mezagitamab, bleselumab, dase Tuzumab, Iscalimab, Lucatumumab, Mitazalimab, Sotigalimab, Dapirolizumab, Apamistamab, Ligufalimab, Magrolimab, Alemtuzumab, Crizanlizumab, Inclacumab Cusatuzumab, Oleculumab, Miratuzumab, Galiximab, Carotuximab, Adecatumumab, Eptinezumab, Erenumab, Fremanezumab, Galcanezumab, Zolbetuximab, Onartuzumab Eculizumab, Pozelimab, Ravulizumab, Lacnotuzumab, Axatilimab, Cabilizumab, Emactuzumab, Ipilimumab, Quavonlimab, Tremelimumab, Zalfrelimab, CetuximabDepatuxizumab, Futuximab, Imgatuzumab, Matuzumab, Modotuximab, Necitumumab, Nimotuzumab, Panitumumab, Tomzotuximab, Zalutumumab, Batoclimab, Nipocalimab, Ro Rozanolixizumab, Burosumab, Farletuzumab, Dinutuximab, Naxitamab, Ragifilimab, Gimsilumab, Lenzilumab, Mavrilimumab, Namilumab, Otilimaimb, Pronmarlimab, Codlitz Codrituzumab, Margetuximab, Pertuzumab, Trastuzumab, Datopotamab, Patritumab, Seribantumab, Dunogotuzumab, Ficlatuzumab, Rilotumumab, Aromfilimab, Aniflorumab lumab), emapalumab, ligerizumab, omalizumab, cyclostomab, dalotuzumab, figitumumab, ganitumab, teprotumumab, bermekimab, canakinumab, gevokizumab, briakinumab,Ustekinumab, Anrukinzumab, ab), sendakimab, lebrikizumab, tralokinumab, brodalumab, bimekizumab, ixekizumab, secukinumab, brazikumab, guselkumab, mirikizumab, risankizumab, tildrakizumab ab), Nemolizumab, Imsidolimab, Spesolimab, Pascolizumab, Dupilumab, Depemokimab, Mepolizumab, Reslizumab, Benralizumab, Clazakizumab, Olokizumab, Siltuximab, Sirukumab, Ziltivekimab, Levirimab, Sarilumab, Satralizumab, Tocilizumab, Abituzumab, Favezerimab, Fianlimab, Ieramirimab, Relatlimab, Simtuzumab, Avagovomab vomab), olegovomab, tanezumab, ivuxolimab, locatinlimab, tavolimab, telazololimab, vonlerolizumab, alirocumab, vococizumab, ebronucimab, evolocumab, flubocimab,Ongericimab, Taforecimab, Dostarlimab, Balstilimab, Camrelizumab, Cemiprimab, Geptanolimab, Nivolumab, Pembrolizumab, Penpulimab, Pidilizumab, Progolimab ), Retifanlimab, Sasanlimab, Serplulimab, Sintilimab, Spartalizumab, Tislerizumab, Tripalimab, Ezabenlizumab, Zimberelimab, Atezolizumab, Avelumab, Cosibel imab), Sugemalimab, Durvalumab, Envafolimab, Subratoxumab, Denosumab, Zirovertamab, Elotuzumab, Domvanalimab, Etigilimab, Ociperlimab, Tiragolumab, Vibostol imab), surzebiclimab, cobolimab, sabatolimab, concizumab, marstacimab, adalimumab, golimumab, infliximab, certolizumab, conatumumab, tigatuzumab, tezeperumab,Gatipotuzumab, kabilizumab (C, abilizumab, bevacizumab, brolucizumab, ranibizumab, olimbacimab, iclucumab, ramucirumab, caplacizumab, abrilumab, etrolizumab, vedolizumab, intetumumab, natalizumab lizumab), obrindatamab, elranatamab, linvoseltamab, teclistamab, epcoritamab, globitamab, mosunetuzumab, odronextamab, flotetuzumab, vibecotamab, catumaxomab, Cibisatamab, Talquetamab, Ubamatamab, Emfizatamab, Blinatumomab, Amivantamab, Emicizumab, Xenocutuzumab, Zanidatamab, Tibrizumab, Naptumomab, Belantamab, Pibe Pivekimab, Praluzatamab, Coltuximab, Denintuzumab, Loncastuximab, Ibritumomab, Inotuzumab, Epratuzumab, Moxetumomab, Brentuximab, Gemtuzumab, Vadastuximab,Lorbotuzumab, Polatuzumab, Tusamitamab, Terisotuzumab, Robalpituzumab, Depatuxizumab, Farletuzumab, Mirbetuximab, Disitamab, Anetumab, Enfortumab, Sacituzumab The carrier drug conjugate according to claim 12, characterized in that it is one or more drugs selected from the group consisting of vobarilizumab, cadnilimab, budalimab, teboterimab, ivonesimab, erfonrilimab, ozoralizumab, faricimab, vanucizumab, and navicixizumab.

14. A pharmaceutical composition for use in the prevention or treatment of cancer, comprising a carrier-drug complex according to any one of claims 7 to 13.

15. The aforementioned cancers include caustic myxoma, intrahepatic biliary tract cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphoblastic leukemia, basal cell carcinoma, epithelial ovarian cancer, ovarian germ cell tumor, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, sinus and nasal cavity cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, childhood brain cancer, childhood lymphoma, childhood leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, heart cancer, duodenal cancer, and malignant cancers. The pharmaceutical composition according to claim 14, characterized in that it is one or more selected from the group consisting of soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoid, gastrointestinal stromal tumor, Wilms' tumor, breast cancer, triple-negative breast cancer (TNBC), sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal tumor, rectal cancer, rectal carcinoid, vaginal cancer, spinal cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, hematological cancer, and thymic cancer.

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