Camptothecin derivatives and their prodrugs that bind to DDX5 protein

Abstract

The present invention relates to (a) an activated camptothecin derivative represented by chemical formula 1, which is designed to bind to DDX5 protein and E3 ligase; (b) a prodrug thereof designed to release the activated camptothecin derivative (a) from a target site in the body; or (c) a ligand-containing complex targeting DDX5 protein, in which the type I topoisomerase inhibitory ability of the activated camptothecin derivative (a) or the FL118 compound represented by chemical formula 2 has been inactivated via a linker linkage. The active camptothecin derivatives designed according to the present invention can bind to DDX5 protein in cells and induce cell death through degradation of DDX5 protein.

Description

[Technical field]

[0001] The present invention relates to (a) an activated camptothecin derivative represented by the following chemical formula 1, which is designed to bind to DDX5 protein and E3 ligase; (b) a prodrug thereof designed to release the activated camptothecin derivative (a) from a target site in the body; or (c) a ligand-containing complex targeting DDX5 protein, in which the type I topoisomerase inhibitory ability of the activated camptothecin derivative (a) or the FL118 compound represented by chemical formula 2 has been inactivated via a linker bond.

[0002] The active camptothecin derivatives designed according to the present invention can bind to DDX5 protein in cells and induce cell death through degradation of DDX5 protein. [Background technology]

[0003] Cancer Genes are genes that exist to regulate the normal functions of cells, but when they are mutated or abnormally regulated, they can cause cancer. One of the normal cell functions is the cell death function, and there are cell death promoters and cell death inhibitors associated with this function.

[0004] For example, there are two types of cancer regulatory genes: (i) oncogenes that actively promote cell division when they mutate, and (ii) tumor suppressor genes that do not suppress cell division when they mutate. Examples of oncogenes include Src, EGFR, HER2, RAS, APC, and BCL-2, while examples of tumor suppressor genes include p53, BRCA, Rb, PTEN, and BAX.

[0005] Cancer is a group of diseases in which the cell cycle of growth and division is deregulated. Cancer can occur when programmed cell death (e.g., apoptosis) mechanisms are impaired, as shown in Figure 29.

[0006] Cancer arises due to the occurrence of mutations in genes whose protein products are involved in regulating the cell cycle. Many cancers are associated with the overexpression of certain genes or the abnormal activity of their mutant protein products (Oncogenes).

[0007] Proteins encoded by viral oncogenes resemble cellular proteins that have important regulatory functions. These cellular homologues are called proto-oncogenes or normal cellular oncogenes. Mutations in proto-oncogenes actively promote cell proliferation. Mutant cH-ras proteins have mutations that impair their ability to hydrolyze GTP. This keeps the mutant protein in an active signaling mode, stimulating cell division. Mutant versions of c-ras have been found in many types of tumors.

[0008] A single mutation does not generally cause cancer: various genes that generally regulate cell growth are mutated before a cancerous condition occurs.

[0009] HER2-positive breast cancer, in which HER2 is abnormally expressed in breast epithelial cells, accounts for approximately one-quarter of all breast cancer patients, and has a higher recurrence rate and worse prognosis when treated with chemotherapy compared to female hormone-related breast cancer. Herceptin (chemical name: trastuzumab), a HER2-targeted therapy, treats breast cancer by reducing the number of HER2 on the cell surface and helping immune cells kill HER2-positive breast cancer cells.

[0010] HER2-positive breast cancer is a type of breast cancer that harbors amplification or overexpression of the HER2 (ErbB2) oncogene, a member of the ErbB receptor. Trastuzumab (Herceptin) has increased the therapeutic efficacy of HER2-positive breast cancer, but HER2-positive breast cancer is more aggressive than other types of breast cancer, and more than half of patients are refractory to existing targeted therapies or develop resistance during treatment.

[0011] Ligands for ErbB receptors attached to the cell membrane include NRG1 and HB-EGF, and activated ErbB ligands bind to these receptors (EGFR, HER3), catalyzing phosphorylation (P) through receptor binding (EGFR-HER2, HER2-HER3), inducing intracellular growth and proliferation signals. Such excessive activation of ErbB receptors results in resistance to anti-HER2 therapeutic agents.

[0012] In addition, dysregulated transcription factors can decrease the sensitivity of cancer cells to chemotherapy and radiotherapy. Drug resistance in cancer therapy can be intrinsic or acquired drug resistance. Intrinsic drug resistance in cancer therapy can be caused by abnormally expressed transcription factors that are crucial regulators of cell proliferation 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 induced by anticancer drugs, indicating intrinsic drug resistance.

[0013] 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 radioresistance. In addition, upregulation of 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 therapy. Activation of transcription factor STAT3 also promoted drug resistance to cancer therapy. Feedback activation of STAT3 by chemotherapy and radiotherapy has been revealed to be a molecular mechanism of cancer therapy resistance. Therefore, co-targeting dysregulated transcription factors and their primary targets may be a promising access method to overcome drug resistance in cancer therapy.

[0014] The STAT (signal transducer and activator of transcription) family of transcription factors includes STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6. STAT proteins can be activated by JAK kinases and regulate interferon signaling upon stimulation with growth factors, cytokines, interferons or oncogenes. The DNA-binding domain of STATs is an immunoglobin-like structure that mediates binding to specific DNA target sequences. Phosphorylation of STAT3 by JAK kinases during activation activates dimerization. The dimers then translocate to the nuclear compartment, bind to sequences in target promoters and induce the expression of target genes. STAT3 target genes include Survivin, bcl-xl, mcl-1, cyclin D1, MMP2, MMP9, VEGF, My c, Sox2, etc. STAT proteins regulate many biological processes including cell growth, cell death, differentiation, and immunity. Within the STAT system, STAT3 and STAT5 have been implicated in cancer progression.

[0015] Dysregulated STAT3 and activated STAT5 are considered oncogenes, which increase angiogenesis and improve cancer cell survival. A study using many samples from the cBioPortal database showed that STAT3 is more frequently overexpressed in lung, ovarian, gastric, hematopoietic and brain cancers than in normal tissues, and STAT3 overexpression was associated with poor overall survival. Phosphorylation of STAT3 by JAK kinases is the first step in STAT3 activation. Yet another study of 90 glioblastoma patients found that high p-STAT3 values ​​were significantly associated with poor progression-free and overall survival. Multivariate survival analysis suggested that high p-STAT3 levels could serve as an important prognostic indicator for poor progression-free and overall survival.

[0016] Type I topoisomerase I inhibitors (irinotecan, topotecan, etc.) have an anti-cancer mechanism whose efficacy and safety have been verified in clinical trials, and their excellent anti-cancer efficacy has been verified in various refractory solid cancers such as colorectal cancer, lung cancer, breast cancer, and ovarian cancer.

[0017] In relation to this, camptothecin derivatives, which are low molecular weight compounds that inhibit type I topoisomerase (topoisomerase-1) and exhibit antitumor activity, are known.

[0018] Camptothecin (CPT) has low solubility in water, and in preparation 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 exhibited by the compound (hemorrhagic cystitis, gastrointestinal toxicity, e.g., nausea, vomiting, diarrhea, and bone marrow suppression, especially leukopenia and hypothrombocytopenia).

[0019] Subsequently, numerous CPT congeners have been synthesized to obtain compounds with lower toxicity and higher aqueous solubility. Two drugs are irinotecan (CPT-11) and topotecan.

[0020] Irinotecan (CPT-11), jointly developed by Dallchi and Yakult in Japan, was the world's first camptothecin-based anticancer drug in 1994. Its effectiveness against lung cancer (small cell and non-small cell lung cancer) was proven and it was launched in Europe and Japan, and its effectiveness against colon cancer and breast cancer was further proven in 1995. In addition, topotecan, developed by Glaxo Smith Kline, was approved by the US FDA in April 1995 as effective against metastatic ovarian cancer and launched. Recently, CKD-602 (belotecan), a new camptothecin derivative developed in Korea, has successfully overcome the toxicity caused by its poor solubility as a water-soluble drug in addition to the effect of a strong type I topoisomerase inhibitor.

[0021] All camptothecin derivatives identified so far contain the parent structure with five rings essential for cytotoxicity (Figure 1). In terms of molecular structure, the E-ring and the A- and B-ring sites have been identified as important sites. Camptothecin contains an alpha-hydroxylactone structure or a 6-membered lactone structure in the E-ring, which is essential for cytotoxicity. Among these, the lactone group and alpha-hydroxyl group located at carbon 20 of the E-ring are important for stabilizing type I topoisomerase-DNA by-products, and it has been demonstrated that modifications of the A- and B-rings can increase water solubility and activity. Modifications on the first ring, for example, increase the solubility in water and provide greater tolerance for the drugs mentioned above. It has been demonstrated to be tolerable.

[0022] CKD-602 also attempted to substitute the B-ring site at carbon 7 to increase water solubility and anticancer effect. Lee et al. stated that CKD-602 has superior anticancer effect compared to camptothecin and topotecan in a wide range of cancer cell lines. They also confirmed that CKD-602 is a relatively safe drug with a maximum tolerated dose (MTD) of 25mg / kg in the L1210 leukemia nude mouse model. The commonly known side effects of camptothecin drugs can be broadly classified into hematological side effects and non-hematological side effects. Hematological side effects include neutropenia accompanied by fever, sepsis, and bleeding, while non-hematological side effects include skin side effects such as nausea, vomiting, and hair loss, as well as toxicity to the digestive tract, kidneys, and nervous system. In an animal study of CKD-602, Kim et al. found no abnormal drug reactions, except for increased gastric secretion, when camptothecin-based drugs were administered at doses 10 times higher than the clinically applicable dose. In addition, a recent Korean clinical study reported only reversible and controllable side effects such as neutropenia and leukopenia, rather than serious systemic toxicity, proving the drug's relatively stable side effects. However, to date, it has been used in a limited manner for the treatment of patients who have failed or cannot undergo standard chemotherapy, i.e., refractory or recurrent ovarian and colorectal cancers that have recurred or worsened after standard treatment and are unlikely to benefit from further anti-cancer chemotherapy or surgery, refractory or recurrent limited disease small cell lung cancers that have failed first-line chemotherapy, and extensive disease small cell lung cancers.

[0023] SN-38 has IC in the nanomolar range in a variety of cell lines. 50It is a potent topoisomerase I inhibitor with therapeutic value. It is the active form of irinotecan, a precursor drug used in the treatment of colorectal cancer, and also shows activity in lung, breast and brain cancers. Trop-2-SN-38 ADC has been successfully developed in a number of cancer types, including TNBC, bladder and gastric cancers, but there are still many issues to be addressed, including overexpression of drug efflux transporters, type I topoisomerase, and other cancer types. 1) Problems of resistance due to epigenetic silencing and increased anti-apoptotic proteins remain.

[0024] In addition, 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 killing effect on surrounding cells, which makes it advantageous for the treatment of heterogeneous tumors. However, it has a short half-life that can reduce off-target effects. DXD was conjugated to the cysteine ​​residue of an anti-HER2 antibody with a maleimide linker, and the homogeneous DAR value reached 8. Despite the high DAR value, DXD was so stable that only 2.1% was released from plasma for 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 therapy at least twice in the past.

[0025] Camptothecin-based derivatives such as SN-38 and Dxd (derivatives of the clinical-stage topoisomerase I inhibitor exatecan) have recently been successfully developed as novel payloads for antibody-drug conjugates (ADCs). As a result, two ADCs using camptothecin-based payloads have been developed: Trodelvy (sacituzumab govitecan) and Dxd, which contain an SN-38 payload. Enhertu (trastuzumab deruxtecan), which contains the xd payload, was recently approved by the FDA. Such camptothecin-based payloads are ADCs (DAR = 2 or 4) of existing ultra-toxic payloads such as MMAE and MMAF. Although it has intermediate cytotoxicity compared to , the excellent safety profile allows the use of higher drug-antibody ratios (DAR) such as 8. In addition, linker systems with faster drug release profiles such as CL-2A or GGFG linker systems can be used with such camptothecin-based payloads. With the combination of such components, new ADCs with wider therapeutic ranges have been developed.

[0026] In short, since camptothecin compounds act as topoisomerase I inhibitors and their potential use as anticancer drugs was confirmed, various compounds with similar structures have been synthesized and researched. Among them, since topotecan was approved by the FDA in 1996 and irinotecan in 2002, various compounds were synthesized, but they did not show excellent effects and development momentum was lost. However, in 2019, Enhertz, an ADC with Dxd, a new camptothecin derivative, as a payload, and Trodelvy, an ADC with SN-38, an in vivo active metabolite of irinotecan, as a payload, were approved by the FDA, rekindling research activity.

[0027] Antibody-drug conjugates (ADCs) using camptothecin derivatives such as Trodelvy and Enhertz as payloads have been successful in selectively delivering camptothecin compounds that exhibit strong anticancer efficacy only to cancer cells, and in providing excellent anticancer efficacy without severe systemic side effects, but still show various shortcomings, the most representative of which is the occurrence of refractoriness due to overexpression of the ABCG2 drug efflux pump. Many camptothecin derivatives are rapidly excreted outside the cells by ABCG2, which causes a problem that the therapeutic efficacy is seriously reduced in cancer cells that overexpress ABCG2 compared to cancer cells that do not express ABCG2 or express it at a low normal level. Such overexpression of ABCG2 can be the main cause of reduced therapeutic efficacy not only in small molecule camptothecin compounds such as irinotecan and topotecan, but also in ADCs such as Trodelvy and Enhertz that use camptothecin compounds as payloads.

[0028] In contrast to Kadcyla's DM1, Enhertz's DXd has high membrane permeability, and as a result, research results have shown that the payload released from inside the cell is delivered to cells that do not express HER2 and are adjacent to the target cell. This means that Enhertz has a potential bystander effect due to the characteristics of its payload, and can provide clinical benefits to patients who are refractory to Herceptin or Kadcyla. Summary of the Invention [Problem to be solved by the invention]

[0029] The present invention seeks to provide camptothecin derivatives designed to bind to DDX5 protein and E3 ligase as drug candidates for killing target cells.

[0030] The present inventors have synthesized the FL118 compound (Figure 2), a camptothecin-based drug with a dual mechanism of action (MoA) that degrades the oncoprotein DDX5 (p68) along with excellent type I topoisomerase inhibitory activity, and have been conducting extensive research into its physicochemical properties. However, the FL118 compound has limitations in its formulation development due to its limited physicochemical properties.

[0031] Based on the structure of FL118, which is differentiated from the SN38 drug, the inventors intend to design and synthesize a camptothecin derivative with a new structure that exhibits a dual mechanism of action (dual MoA) in view of the advantages of FL118, namely type I topoisomerase inhibition and DDX5 degradation.

[0032] Thus, the present invention provides a method for the detection of oncoproteins (oncoproteins) with type I topoisomerase inhibitory capacity. The present invention aims to newly design and provide an active camptothecin derivative having a dual mechanism of action (dual MoA) that degrades the DDX5 coprotein and / or an antibody-drug conjugate (ADC) that is a prodrug thereof designed to release the active camptothecin derivative in vivo.

[0033] The present invention also aims to provide an anti-cancer mechanism as a new modality for killing cancer cells and / or surrounding cells before intrinsic or acquired drug resistance occurs, by inducing cell death through degradation of DDX5 protein by binding to DDX5 protein in cells together with a bystander effect. [Means for solving the problem]

[0034] A first aspect of the present invention provides a ligand-containing complex targeting DDX5 protein, which comprises: (a) an activated camptothecin derivative represented by the following chemical formula 1, designed to bind to DDX5 protein and E3 ligase; or (b) a prodrug thereof designed to release the activated camptothecin derivative (a) from a target site in the body; or (c) the activated camptothecin derivative (a) or the FL118 compound represented by chemical formula 2, in which the type I topoisomerase inhibitory ability is inactivated via a linker bond.

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[0036] 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 (n=values ​​of 1 to 2); Y1, Y2 and Y3 can each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0037] 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.

[0038] An active camptothecin derivative can be designed to exert a bystander effect or control the degree of the bystander effect in cancer tissue by adjusting cell membrane permeability as desired through modification of X1, (X2)n, X3, Y1, Y2 and / or Y3 in Chemical Formula 1.

[0039] Preferably, the active camptothecin derivative (a) designed to bind to DDX5 protein and E3 ligase is / can act as a molecular glue degrader and a binder that binds to E3 ligase; is / can kill target cells expressing DDX5 protein through a molecular glue degrader mechanism (MoA); and may have a mechanism of action (MoA) that degrades the oncoprotein DDX5 along with type I topoisomerase inhibitory ability.

[0040] A second aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer or a composition for diagnosing cancer, which comprises the active camptothecin derivative (a) of the first aspect, a prodrug thereof (b), or a ligand-containing complex (c) that targets DDX5 protein.

[0041] For example, it may be administered to treat HER2-positive cancer, breast cancer, lung cancer, or colon cancer.

[0042] Preferably, the pharmaceutical composition for the prevention or treatment of cancer may be used as a first line therapeutic agent after cancer diagnosis or may be administered to individuals who (over)express DDX5 in cancer tissues.

[0043] A third aspect of the present invention provides a method for producing (a) an activated camptothecin derivative designed to bind to DDX5 protein and E3 ligase, (b) a prodrug thereof designed to release the activated camptothecin derivative (a) from a target site in the body, or (c) a ligand-containing complex targeting DDX5 protein, in which the type I topoisomerase inhibitory ability of the activated camptothecin derivative (a) or the FL118 compound of chemical formula 2 has been inactivated via a linker linkage, the method comprising the steps of designing the activated camptothecin derivative (a) to contain the camptothecin-based skeleton represented by chemical formula 1 as a parent nucleus, selecting a compound designed in this way, or synthesizing a compound designed in this way.

[0044] For example, the step of designing an active camptothecin derivative (a) to include a camptothecin-based skeleton represented by Chemical Formula 1 as a parent core or selecting a compound designed in this manner can be carried out by: (1) Selecting / selecting an active camptothecin derivative designed to have a mechanism of action (MoA) for inhibiting type I topoisomerase and / or a mechanism of action (MoA) for degrading the oncoprotein DDX5 from a compound library containing a camptothecin-based skeleton represented by chemical formula 1 as a parent nucleus, (2) It may be a step of confirming through in vitro experiments and / or in vivo experiments whether a compound having a camptothecin-based skeleton represented by Chemical Formula 1 as a parent nucleus inhibits type I topoisomerase and / or degrades oncoprotein DDX5.

[0045] The fourth aspect of the present invention provides: (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 thereof designed to release the active camptothecin derivative (a) in vivo; or (c) a prodrug of the active camptothecin derivative (a) that has a type I topoisomerase inhibitory ability by linking the prodrug with a linker. The present invention provides a method for producing a ligand-containing complex targeting DDX5 protein inactivated via a cytosol, the method comprising the steps of: a first step of selecting the active camptothecin derivative (a) from the group consisting of compounds of the following formulas: 3, 4, 5, 6, 7, 8, and 9, and isomers thereof; and optionally a second step of selecting a prodrug (b) thereof designed to release the active camptothecin derivative (a) selected in the first step in vivo.

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[0053] The manufacturing method of the third or fourth aspect may further include a step of identifying a patient group to which the active camptothecin derivative (a) or its prodrug (b) is to be administered by quantitatively or qualitatively analyzing intracellular DDX5 protein in a tissue or liquid biopsy using the FL118 compound of chemical formula 2 or a ligand-containing complex (c) targeting DDX5 protein in which the type I topoisomerase inhibitory ability of the active camptothecin derivative (a) has been inactivated via a non-cleavable linker linkage.

[0054] A 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 pharma- ceutically acceptable salt thereof.

[0055] A sixth aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising 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 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 pharma- ceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof.

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[0066] A seventh aspect of the present invention provides a pharmaceutical composition for killing cells, comprising: (a) an active camptothecin derivative represented by the above chemical formula 1, which is designed to bind to DDX5 protein and E3 ligase; or (b) a prodrug thereof, which is designed to release the active camptothecin derivative (a) from a target site in the body.

[0067] An eighth aspect of the present invention provides an anti-cancer dosage form, characterized in that (a) an active camptothecin derivative represented by the above chemical formula 1, which is designed to bind to DDX5 protein and E3 ligase; or (b) a prodrug thereof, which is designed to release the active camptothecin derivative (a) from a target site in the body, is administered in a dosage that can stimulate antigen-presenting cells (APCs) through cell death of cancer cells and induce an anti-tumor immune response.

[0068] In a ninth aspect of the present invention, there is provided a method for producing an anticancer drug based on an active camptothecin derivative (a), in which the bystander effect and / or ADME profile of the active camptothecin derivative (a) is controlled so that T cell exhaustion does not occur due to chronic antigen exposure, the active camptothecin derivative (a)-based anticancer drug being an anticancer dosage form containing (a) an active camptothecin derivative designed to bind to DDX5 protein and E3 ligase as a molecular glue degrader; or (b) a prodrug thereof designed to release the active camptothecin derivative (a) from a target site in the body; the method for producing the anticancer drug comprising the steps of designing the active camptothecin derivative (a) or its prodrug (b) to contain the camptothecin-based skeleton represented by Chemical Formula 1 as a parent nucleus, selecting a compound designed in this way, or synthesizing a compound designed in this way.

[0069] The present invention will now be described. In this specification, drug refers to any substance that is used for diagnosis, cure, mitigation, treatment or prevention of disease (excluding food or equipment), or that is used to affect body structure or function.For example, any chemical or biological substance that affects the body and body metabolism.Preferably, as long as it can bind to DDX5 protein, it belongs to the category of drug.

[0070] For example, the drug of the present invention can be an active camptothecin derivative or a prodrug thereof, an active camptothecin derivative as a payload for a prodrug such as targeted drug delivery (e.g., a drug-carrier complex), or a camptothecin derivative or a prodrug thereof as a diagnostic agent.

[0071] For a drug to act effectively in the body, the drug concentration at the target site in the body must be maintained within the therapeutic range for a certain period of time (Example 8, Table 6, Figure 23). If an excessive amount of drug is present in the body, it will be toxic, and if the amount at the target site is too small, no therapeutic effect will be observed.

[0072] Drug efficacy means that the drug remains in the body without being broken down for the expected time to be effective for the target indication (Example 8, Table 6, Figure 23). The slower the metabolic rate, the longer the blood concentration will be maintained, and therefore the longer the duration of efficacy.

[0073] Furthermore, the in vivo efficacy and in vivo side effects of anticancer drugs are closely related to their absorption, distribution, metabolism, and excretion (ADME) properties. The ADME of a drug determines its pharmacokinetics, which describes how the drug moves through the body and interacts with tissues and organs.

[0074] Drug absorption can affect efficacy and side effects. Poorly absorbed drugs may not reach therapeutic concentrations in target tissues and may have reduced efficacy, whereas well absorbed drugs may increase systemic exposure and cause non-targeted side effects.

[0075] Drug distribution can also affect efficacy and side effects. Drugs that are poorly distributed in target tissues may be ineffective, while drugs that distribute widely in non-target tissues may induce toxicity in those tissues. Drugs that bind highly to plasma proteins may have reduced distribution to target tissues, resulting in reduced efficacy.

[0076] Drug distribution within the body can be influenced by physicochemical properties including size, charge and lipophilicity. The ability of a drug to penetrate tumor tissue can also be influenced by factors such as the tumor microenvironment, including blood flow and cell density. Some anticancer drugs can accumulate in certain tissues and induce toxic effects.

[0077] Drug metabolism can also affect both efficacy and side effects. Rapidly metabolized drugs can have reduced efficacy due to their short half-life, while slowly metabolized drugs can become toxic due to accumulation. Some metabolites are also more toxic than the parent drug and can cause side effects.

[0078] The rate and route of excretion can also affect both the efficacy and side effects of a drug. Drugs that are eliminated quickly can have a short half-life and reduced efficacy, while drugs that are eliminated slowly can accumulate and induce toxicity. Also, some drugs are excreted unchanged or as active metabolites, which can lead to prolonged or increased exposure and increased risk of side effects.

[0079] Therefore, it is essential to analyze the ADME properties of anticancer drugs to optimize their efficacy and minimize their side effects in vivo. That is, evaluating and optimizing the ADME profile can improve the therapeutic index of such drugs and provide better outcomes for cancer patients. do.

[0080] Anti-cancer formulation or anti-cancer composition refers to a medication or therapy designed to treat cancer or prevent the growth and spread of cancer cells in the body. Anti-cancer formulations may include combinations of drugs, chemical compounds and / or biological agents that target cancer cells by interfering with their ability to grow and divide.

[0081] Anti-cancer drug formulations can reflect a complex process involving various stages, including drug discovery, pre-clinical development, clinical trials, and regulatory approval. The goal is to develop safe and effective treatments that can be administered to patients with minimal side effects.

[0082] The development of anti-cancer drug forms involves designing drugs or drug combinations that can identify and selectively target cancer cells while sparing healthy cells. This can be achieved by targeting specific proteins or enzymes that are overexpressed in cancer cells or by disrupting the cell cycle, DNA replication or other cellular processes essential for the growth and survival of cancer cells.

[0083] Once promising drug candidates are identified, they undergo preclinical development, in which the drug is tested in laboratory and animal models to evaluate safety and efficacy. If a drug shows potential in preclinical studies, it can proceed to clinical trials, where it is tested in humans to determine safety, dosage and efficacy.

[0084] During clinical trials, drug dosage forms are optimized so that they can be safely administered to patients and achieve the desired therapeutic effect, which may include adjusting the drug's dose, route of administration, or dosage form to improve efficacy and minimize side effects.

[0085] The present invention provides, as a drug candidate for killing target cells, (a) an active camptothecin derivative represented by the following chemical formula 1, which is designed to bind to DDX5 protein and E3 ligase; or (b) a prodrug thereof, which is designed to release the active camptothecin derivative (a) from a target site in the body.

[0086] [ka]

[0087] 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 (n=values ​​of 1 to 2); Y1, Y2 and Y3 can each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0088] 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 nH 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 , R3 =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 - , 0≦s≦20, 0≦t≦2(s+u)+1, 0≦u≦2s, 0≦w≦2s, 0≦x≦2s, 0≦y≦2s, 0≦z≦2 s), -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 groups, -hydrazones, -ONO2, -PO(OH)2, -C=NNH2, -HC=CH-, -C=C-, -C≡C- and hydrocarbons having 2 or more carbon atoms:

[0089] The activated camptothecin derivative (a) designed by the present invention to bind to DDX5 protein and E3 ligase can kill target cells expressing DDX5 protein through a molecular glue degrader mechanism (MoA).

[0090] The target cells may be cancer cells or senescent cells, including cells that do not perform the characteristic functions specific to an organ.

[0091] Preferably, the active camptothecin derivative (a) designed by the present invention to bind to DDX5 protein and E3 ligase may have a multiple mechanism of action (MoA) to degrade the oncoprotein DDX5 along with type I topoisomerase inhibitory ability.

[0092] For example, the present invention can expose tumor antigens through cell death of cancer cells in tumor tissues, and induce anti-tumor immune responses through stimulation of antigen-presenting cells (APCs) such as dendritic cells and macrophages.

[0093] In addition, the present invention provides a variety of active camptothecin derivatives represented by Chemical Formula 1, which are designed to bind to DDX5 protein and E3 ligase; or (b) prodrugs thereof, which are designed to release the active camptothecin derivative (a) from target sites in the body, as drug candidates for killing cancer cells. This makes it possible to design a variety of anticancer dosage forms, doses, and dosages based on the active camptothecin derivative (a) in order to control the bystander killing effect and / or ADME profile of the active camptothecin derivative (a) so that T cell exhaustion does not occur due to chronic antigen exposure.

[0094] By utilizing camptothecin derivatives that exert the effect of type I topoisomerase inhibitors, and in conjunction with the R1 and R2 modifications (Group A) on the A ring that have been proven to allow greater tolerance than when camptothecin-based drugs are designed, as in general formula 1, the active camptothecin derivative of the present invention is designed identically to the FL118 compound of chemical formula 2 (Example 1), and is characterized by exerting an anti-cancer mechanism that degrades the oncoprotein DDX5 (Example 2).

[0095] [ka]

[0096] [ka]

[0097] 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).

[0098] [ka]

[0099] [ka]

[0100] [ka]

[0101] In general formula 1 or formula 2, the group C site binds to type I topoisomerase and the group A site binds to DNA and stabilizes the covalent bond of the topoisomerase-DNA complex, preventing / preventing further ligation of the cleaved DNA fragments. In Example 1, the group A site in general formula 1 or chemical formula 2 can bind to DDX5, and the group C site can bind to E3 ligase to induce degradation of DDX5 (FIGS. 11 to 14).

[0102] Topoisomerase I inhibitors have an anticancer mechanism whose efficacy and safety have been verified in clinical trials, and camptothecin-based drugs such as exatecan, exatecan derivative Dxd, and SN-38 have been developed as payloads for ADCs.

[0103] Exatecan is a substance that has been confirmed to have 5 to 10 times stronger cytotoxicity than the drug SN-38. Unlike irinotecan, exatecan does not require enzyme activation. It also has stronger type I topoisomerase inhibitory activity than SN-38, the active ingredient of irinotecan, and topotecan, which is used in the same clinical trial, and has stronger cytotoxic activity against various cancer cells in vitro. It was particularly effective against cancer cells that are resistant to SN-38, etc. due to the expression of P-glycoprotein. It also showed strong antitumor effects in a mouse model of human tumors subcutaneously transplanted, and clinical trials were conducted.

[0104] The FL118 drug of formula 2 is a hydrophobic small molecule 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 (Figures 1 and 2). Even when administered alone, it has differentiated anticancer efficacy and excellent safety compared to SN-38 in various cancer cells and animal models, and has a wide therapeutic range. It is an anticancer drug that has secured a therapeutic window.

[0105] The present inventors have synthesized the camptothecin-based compound FL118 drug and continued to conduct extensive research into its physicochemical properties, but the development of dosage forms for FL118 is limited due to its limited physicochemical properties.

[0106] The inventors have confirmed that when used as a payload for an antibody-drug conjugate (ADC), the FL118 drug has stronger cytotoxicity than an ADC using SN-38 as the payload, and has a level of cytotoxicity equivalent to that of the exatecan drug.

[0107] In addition, the drug FL118 has a dual mechanism of action that not only inhibits type I topoisomerase but also degrades the oncoprotein DDX5 (p68). It is a camptothecin-based compound that has the 5'-aminobutyric acid (ABA) moiety.

[0108] Surprisingly, we found that the drug Exatecan, like the drug FL118, not only strongly inhibits the expression of anti-apoptotic proteins (survivin, cIAP2, XIAP, etc.), which are the main cause of resistance to other anticancer drugs, at low concentrations, but also blocks the drug resistance mechanism (Figure 10).

[0109] Based on this, the present invention is characterized by designing, from the FL118 compound, (a) a novel active camptothecin derivative having a dual mechanism of action (dual MoA) for degrading oncoprotein DDX5 (p68) together with excellent type I topoisomerase inhibitory ability, (b) a prodrug thereof designed to release the active camptothecin derivative (a) in vivo, and (c) a ligand-containing complex targeting DDX5 protein in which the type I topoisomerase inhibitory ability of the active camptothecin derivative (a) is inactivated via a linker linkage, as shown in FIG. 1. do.

[0110] Specifically, the synthetic design concept of an active camptothecin derivative (a) having a dual mechanism of action (MoA) for degrading oncoprotein DDX5 as well as type I topoisomerase inhibitory ability is as shown in FIG. 1, where the structure of group C, which is a type I topoisomerase inhibitory region, is maintained based on SAR (Structure Activity Relationship), and group A, which is a binding site for DDX5 degradation, is also maintained to be the same structure as FL118. By adjusting the structure (R3 and R4) of group B in general formula 1 as shown in chemical formula 1, it is possible to design a structure that can not only improve the physicochemical properties of the drug but also regulate the cytotoxicity and / or cell membrane permeability of the drug and regulate the bystander effect (Example 9, FIG. 25 and FIG. 26).

[0111] When comparing cell viability simply, the newly designed and synthesized PBX-7016 (chemical formula 5) of the present invention had an IC of 1.5 to 2 times higher than that of Dxd drugs. 50 Surprisingly, when used as an ADC payload, the ADC containing PBX-7016 exhibited approximately 5-fold superior cell survival-related cytotoxicity effects compared to the ADC containing Dxd (Enhertu) (Example 7, FIG. 22). The compound represented by formula 5 (PBX-7016) and its isomer, the compound represented by formula 5-1 (PBX-7017), are partial stereoisomers of each other.

[0112] Therefore, in the present invention, it is preferable to design an active camptothecin derivative that can adjust cell membrane permeability as desired through R3 and / or R4 modifications in general formula 1 (e.g., PBX-7014, PBX-7016, PBX-7018, PBX-7020, PBX-7022, PBX-7024) and exert an appropriate bystander effect in cancer tissue.

[0113] As shown in Figure 1, the present invention has modified R3 and R4 of group B in general formula 1 to design and synthesize a compound with a new structure, such as PBX-7011, which is similar to the structure of Exatecan (Preparation Example 1). As shown in Figure 2, it was confirmed that PBX-7011 of formula 3, newly designed by the present invention, has relatively higher hydrophilicity than FL118 of formula 2 through the calculated LogP (or cLogP) and tPSA values.

[0114] Furthermore, by adjusting group B of general formula 1 to the newly designed PBX-7011 structure, new structural compounds PBX-7014 and PBX-7016 were designed and synthesized (Preparation Example 2 and Preparation Example 3), and PBX-7014 and PBX-7016 were calculated to show similar hydrophilicity values ​​to Dxd derived from exatecan (Figures 1 and 2, Figure 27).

[0115] FIG. 27 compares the solubility and topological polar surface area (tPSA) of FL118, exatecan, SN-38, Dxd, exatecan-lactate, PBX-7011 / PBX-7012, PBX-7014 / PBX-7015, and PBX-7016 / PBX-7017.

[0116] The relative hydrophilic / hydrophobic nature of a drug has important effects on its solubility, absorption, distribution, metabolism, and excretion (ADME); how easily a drug crosses cell membranes; and is important in receptor interactions.

[0117] The hydrophobicity of a drug is indicated by its partition coefficient (p).

[0118] Hydrophobicity is indicated by a large p-value, whereas hydrophilicity (polarity) is indicated by a small p-value. In practice, the log P value is used as a measure to evaluate hydrophobicity. clogP means that the log P value of a given compound is calculated through appropriate software. The smaller the cLogP value, the higher the polarity.

[0119] The polar surface area (PSA) or topological polar surface area (TPSA) of a molecule is defined as the surface sum for all polar atoms or molecules, primarily oxygen and nitrogen, including attached hydrogen atoms. tPSA is a medicinal chemistry measurement commonly used to optimize the cell penetration ability of drugs. 2 Molecules with larger polar surface areas tend not to penetrate cell membranes. For a molecule to cross the blood-brain barrier and act on a receptor in the central nervous system, it generally needs a minimum of 90 Å. 2 A tPSA of less than 0.01 is required.

[0120] PBX-7016 / PBX-7017 (chemical formula 5 and chemical formula 5-1) of the present invention are obtained by introducing a metabolically unstable functional group, a methyl group, into PBX-7014 / PBX-7015 (chemical formula 4 and chemical formula 4-1) in order to reduce the drug's lifespan. Drugs that are extremely metabolically stable and metabolized very slowly are necessary to ensure an appropriate residence time, as they may cause toxicity and serious side effects due to accumulation.

[0121] The newly designed active camptothecin derivative (a) of the present invention is a hydrophobic small molecule designed by modifying R3 and R4 of general formula 1 so that it can permeate the cell membrane, and therefore when it is delivered to a 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 cell, and is released into the extracellular fluid, and then continuously penetrates the cell membrane of surrounding cells and moves into the cell, exerting a cell killing mechanism (Example 9, Figures 25 and 26). Therefore, the active camptothecin derivative of the present invention can convert / transform a solid cancer with high cell density and aggregation into a scattered cancer with low cell density, or convert a cold tumor with low immune activity into a hot tumor with high immune activity. In addition, it has an advantage in the treatment of heterogeneous tumors due to its high killing effect on surrounding cells.

[0122] In summary, the present invention not only enables the design of various camptothecin derivatives and their prodrugs (FIG. 3) with new structures that exert a dual mechanism of action (dual MoA) in terms of type I topoisomerase inhibition and DDX5 protein degradation as shown in FIG. 1, FIG. 2, FIG. 27 and FIG. 28, but also enables the regulation of cell membrane permeability, which is related to the bystander effect, by controlling hydrophilicity in a variety of ways when designing camptothecin derivatives.

[0123] [FL118 Drug] FL118 of Chemical Formula 2 can act as a molecular glue degrader that directly binds DDX5 and ubiquitination regulators and degrades DDX5.

[0124] FL118, which acts as a molecular glue, directly binds to the tumor protein DDX5, a multifunctional master regulator, via the proteasome degradation pathway, and dephosphorylates and degrades it without reducing DDX5 mRNA. Silencing of DDX5 promotes the expression of survivin, Mcl-1, XIAP, cIAP2, and IL-1. We show that it is a master regulator that controls the expression of various oncogenic proteins, including c-Myc and mutant Kras.

[0125] FL118 indirectly regulates DDX5 downstream targets and inhibits cancer initiation, development, metastasis, recurrence and treatment resistance with high potency, as demonstrated in studies using human colorectal cancer / pancreatic adenocarcinoma cells and tumor models.

[0126] Genetic manipulation of DDX5 in PDAC cells affects tumor growth. KO PDAC cells are resistant to FL118 treatment. Human tumor animal model studies showed that FL118 was highly effective in eliminating human PDAC and CRC tumors with high DDX5 expression, whereas FL118 was less effective in PDAC and CRC tumors with low DDX5 expression.

[0127] DDX5 protein is a direct target of FL118 drug and can serve as a biomarker for predicting PDAC and CRC tumor sensitivity to FL118.

[0128] In addition, the FL118 drug has a similar or higher level of Top1 inhibitory efficacy to SN-38 in cancer cells, and is 5 to 20 times more potent than SN-38 in various cancer cell lines, i.e., has a low IC 50 It showed cytotoxicity in terms of IC values, and in evaluation of 140 cell lines derived from various cancer types, it showed IC values ​​of <100 nM for the majority of cancer cells. 50 FL118 showed very strong anti-cancer efficacy, demonstrating excellent safety through GLP toxicity tests in rats and beagle dogs, and showed superior efficacy compared to SN-38 in various cancer cell line xenograft models. Camptothecin-based anti-cancer drugs show excellent anti-cancer responses in the early stages when administered to patients, but strong resistance to these drugs appears through epigenetic silencing of the Top1 gene and Top2 dependency of cancer cells. Meanwhile, FL118 showed strong efficacy in xenograft models of cancer cell lines in which Top1 is not expressed through epigenetic silencing or knockout.

[0129] In addition, the FL118 drug is a triple-targeted anticancer drug that directly targets type I topoisomerase, a well-established anticancer target, while simultaneously inhibiting Bcl family members such as survivin, which are resistance proteins involved in the resistance mechanism, and suppresses the action of efflux pumps.

[0130] Specifically, camptothecin-based anticancer drugs such as SN-38 show resistance due to the overexpression of the ABCG2 transporter, which causes the drug to be excreted outside the cell, but the FL118 drug is not affected by the ABCG2 transporter, making it possible to overcome this resistance. The FL118 drug can block the expression of resistance by strongly suppressing, at low concentrations, the expression of anti-apoptotic proteins (survivin, cIAP2, XIAP, etc.), which are the main cause of resistance to other anticancer drugs (Figure 10).

[0131] Therefore, FL118 drug is not excreted outside the cell by the efflux pump ABCG2, and can block resistance caused by various anti-apoptotic proteins, thereby overcoming the various resistance mechanisms of SN-38 / exatecan.

[0132] When administered in vivo at the same dose as SN-38, the drug FL118 showed stronger tumor regression efficacy than SN-38 in colon cancer, head and neck cancer, pancreatic cancer, etc.

[0133] The FL118 drug retained strong anticancer efficacy even when administered with FL118 after induction of SN-38 resistance in tumor xenografts. The potential of the FL118 drug to overcome various resistance mechanisms to SN-38 / exatecan was confirmed in vivo.

[0134] Furthermore, the FL118 drug has an optimal PK / safety profile for the application of targeted drug delivery (e.g., drug-carrier complexes). When the FL118 drug is administered alone, it is rapidly metabolized / excreted in the blood and exhibits only low concentrations, but it is rapidly accumulated in cancer tissues immediately after administration and maintains high concentrations for a long time. For example, it ensures maximum selectivity between tumor tissues and normal tissues when applied as an ADC. This is also the case with the activated camptothecin derivative (a) represented by chemical formula 1 (Examples 6 and 8).

[0135] In summary, the FL118 drug: (i) In addition to the well-validated anti-cancer target Top 1, it selectively inhibits / degrades other anti-cancer targets DDX5, UBE2T and USP2a, exerting potent anti-cancer effects; (ii) showed cellular cytotoxicity 5-10 times stronger than that of SN-38 and equal or greater than that of exatecan; (iii) unlike SN-38 and exatecan, it is not a substrate for ABCG2 and P-GP, thus circumventing the resistance mechanisms mediated by overexpression of these transporter proteins; (iv) demonstrated potency in animal models of cancer where epigenetic silencing of Top 1 / overexpression of Top 2 occurs, a key mechanism for the development of resistance to Top 1 inhibitors; (v) It essentially blocks the overexpression of anti-apoptotic proteins, a common resistance mechanism in many cancers, thereby circumventing resistance; (vi) Biomarkers (DDX5, K-ras, p53) that can predict anti-cancer responses in patients and companion diagnostic techniques have already been established. With the availability of custom-made biomarkers, personalized treatment through companion diagnostics is possible, and this is particularly effective in treating p53 / K-ras mutated cancer cells, which have poor prognosis.

[0136] KRAS is one of three genes related to the production of RAS proteins, which are involved in cell proliferation signaling. It is known that mutations occur in approximately 20% or more of cancers overall, although this varies depending on the type of cancer. However, despite the scientific evidence that it may have anti-cancer effects, it was treated as an undruggable target. This is because the surface of the RAS protein is smooth and no pockets suitable for binding compounds have been found.

[0137] [Anti-cancer mechanism as a selective inhibitor of type I topoisomerase (topoisomerase-1)] Camptothecin is a selective inhibitor of type I topoisomerase-1, an isomerase involved in DNA replication and recombination (Figure 2). After it was found to exhibit strong cytotoxicity in vitro, development through clinical trials was initiated at the National Cancer Institute (NCI) and other institutions. However, due to the limitation of its extremely poor solubility, development was halted due to the associated various side effects such as bone marrow suppression and hemorrhagic cystitis. However, since 1990, the unique mechanism of action of camptothecin, namely, its inhibition of DNA type I topoisomerase, which is different from the mechanism of inhibition of DNA type II topoisomerase (topoisomerase-2), has been widely recognized. It was confirmed that this compound selectively inhibits the enzyme and exhibits antitumor effects.

[0138] DNA topoisomerases are nuclear enzymes that temporarily break DNA or unwind the double helix when cells need access to genetic material for replication or transcription. They also participate in a variety of cellular activities such as chromosome condensation and recombination, and DNA repair. The genetic code for topoisomerases is fairly conserved across species.

[0139] The level of type I topoisomerase, the drug target of camptothecin, was observed to increase in various malignant tumors. The drug does not inhibit the free enzyme, but stabilizes the covalent bond of the topo-DNA complex, preventing further ligation of the cleaved DNA fragments. Thus, the sensitivity of cells to such topoisomerase-targeted drugs is related to the level of enzyme present in the nucleus. The drug prevents DNA recombination, making it impossible for transcription to proceed. The more type I topoisomerase is present, 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 leads to further sensitivity to type II topoisomerase inhibitors. Such results are supported by the antagonistic relationship between type I and type II topoisomerase. Unlike type II topoisomerase, type I topoisomerase is not closely related to proliferation in normal tissues. It is found in greater quantities in solid cancers in the "S" phase, where cell division is active, such as colorectal cancer, including lymphoma, ovarian cancer, and esophageal cancer, than in surrounding normal tissues. It is known that type I topoisomerase can actually induce cell death by blocking gene replication and transcription in tumor cells during the "S" phase of the cell cycle.

[0140] [Anti-cancer mechanism as a molecular glue degrader that binds to DDX5 2] The ubiquitin-proteasome system (UPS) is an important pathway for intracellular protein degradation that regulates a wide range of cellular processes. Ubiquitin is a small protein that is covalently attached to 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 that regulates protein degradation, signaling and trafficking.

[0141] Ubiquitin ligases are responsible for substrate specificity in the ubiquitination pathway. In this regard, the active camptothecin derivative represented by Chemical Formula 1 according to the present invention is designed to bind to DDX5 protein and E3 ligase.

[0142] DDX5 (also called p68) is a multifunctional master regulator that acts on the following mechanisms: (1) the biological process of coactivating the transcription of many oncogenes through direct interaction with various transcription factors (e.g., c-Myc) at oncogenic gene promoters; (2) the biological process of regulating miRNA and pre-RNA splicing (e.g., U1, U2, U3, ... snRNP); and (3) ribosome biogenesis (e.g., 32S rRNA, pre-ribosome).

[0143] According to the present invention, the active camptothecin derivative represented by Chemical Formula 1 binds to DDX5 protein without decreasing DDX5 mRNA, and functionally degrades it via dephosphorylation and proteasome degradation pathway. This is because the active camptothecin derivative represented by Chemical Formula 1 acts as a regulator of DDX5 and ubiquitin-involved protein stability / degradation. ors), suggesting that it can adhere to all surfaces and act as a "molecular adhesion breaker."

[0144] DDX5 downstream protein targets are all known to be involved in cancer initiation, progression, metastasis, recurrence and resistance to treatment. Therefore, when DDX5 downstream targets are indirectly blocked through the degradation of DDX5 protein by the activated camptothecin derivative represented by Chemical Formula 1 according to the present invention, the activated camptothecin derivative represented by Chemical Formula 1 can exhibit high antitumor efficacy.

[0145] [DDX5 as a drug target and / or biomarker] Transcription factors are a group of proteins that bind to DNA strands called promoters or enhancers through their DNA-binding domains and initiate the transcription of genes with the cooperation of RNA polymerase and other cofactors. They play an important role in regulating transcription during embryogenesis and development. The physiological state of transcription factors in other cell types is also important for maintaining cellular homeostasis. Thus, deregulation of transcription factors leads to cancer cell development and tumor progression. Depending on their function 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 dysregulated transcription factors may be able to predict the therapeutic outcome of cancer patients, and targeting dysregulated transcription factors may be an inspiring strategy for cancer treatment. Dysregulated key transcription factors can induce poor clinical outcomes in cancer patients. Therefore, predicting prognosis and drug response and targeting dysregulated transcription factors is helpful for designing new drugs and therapeutic strategies for cancer treatment.

[0146] It is well known that transcription is the first step of gene expression in cells during embryogenesis, development and homeostasis. During the transcription process, RNA transcripts carrying 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 genes to be transcribed into RNA transcripts and control the transcription. Transcription factors have a DNA-binding domain and can bind to specific DNA sequences, called promoters or enhancers of genes, and initiate the transcription of genes with the cooperation of activators, mediators, co-factors and RNA polymerase.

[0147] Transcription factors also control the rate of transcription from DNA to mRNA. Consequently, the physiological state of transcription factors in different types of cells is important for maintaining cellular homeostasis. Changes in the physiological state of intracellular transcription factors can cause transcriptional disorder and translation of specific genes, resulting in pathological changes in cells. Thus, dysregulated transcription factors can induce many human diseases. One common disease with dysregulated transcription factors is cancer.

[0148] Homeostatic regulation of human cells generally prevents inappropriate proliferation and inhibits the growth of cells that grow abnormally outside of their normal niche. Transcriptional regulation is a core process of homeostasis, so if the levels or activities of transcription factors are not regulated, cells can escape homeostatic control, inducing the development and progression of human cancers. Indeed, many chemotherapeutics can activate tumor suppressor transcription factors.

[0149] Dysregulated transcription factors play important roles in tumorigenesis 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 mediate the development of cancer cells, cancer stem cells, and EMT cells. These drugs favor cell survival and induce intrinsic or acquired drug resistance and tumor progression during cancer therapy.

[0150] DDX5(p68) is a well-known multifunctional DEAD-box RNA helicase and a transcription cofactor. Therefore, when the physiological state of DDX5 causes deregulation of transcription factors and cancer develops, selective degradation of the transcription cofactor DDX5(p68) can treat cancer. Similarly, selective degradation of the transcription cofactor DDX5(p68) can prevent cancer.

[0151] DDX5 has been recognized as a potential biomarker and target for the treatment of various cancer types. Through years of research, our understanding of the functional diversity of DDX5 in various cancer types has been greatly expanded, and knowledge of its Mechanism of Action (MoA) has been expanded. This provides a rationale for the development of new cancer therapeutics using DDX5 as a biomarker and therapeutic target. However, although many published studies have revealed that DDX5 is both a carcinogenic target and a cancer therapy resistance biomarker, some studies have reported that DDX5 can act as a tumor suppressor in certain scenarios. The multiple functions of DDX5 make it an excellent independent carcinogenic biomarker and target for targeted cancer therapy.

[0152] The transformation of normal cells into cancerous cells occurs due to the deregulation of different metabolic pathways that involve a complex network of protein-protein interactions. Cellular enzymes and DDX5 play important roles in maintaining normal cellular metabolism, but their deregulation can accelerate tumor transformation. DDX5 interacts with hundreds of different cellular proteins in total, and depending on the specific pathway involved, both proteins can act as tumor suppressors or oncogenes.

[0153] DEAD-box RNA helicases are involved in various metabolic pathways ranging from transcription and translation to cell proliferation, innate immunity and stress response. Taking into account these diverse roles, their deregulation or their mutations have been linked to diverse pathological conditions including cancer. However, in some cases, loss of function of a given DEAD-box helicase promotes tumor transformation, indicating a tumor suppressor role, whereas in other situations, overexpression of the same enzyme acts as a typical oncogene, favoring cancer progression. The role of DDX5 as an oncogene and tumor suppressor has been documented in various cancer types. Understanding the interplay of different cellular contexts defined by the molecular interaction network of DDX5 in different tumors helps to explain the cancer-specific roles played by these proteins as clearly opposing roles as cancer inducers or suppressors.

[0154] We show that DDX5 frequently acts as an oncogene but can also have tumor suppressor functions in certain cancer types (Table 1).

[0155] 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

[0156] [Table 1]

[0157] The active camptothecin derivative represented by Chemical Formula 1 according to the present invention can avoid drug resistance, unresponsiveness to targeted therapy and / or resistance during therapy because the DDX5 protein is the drug target. In addition, the active camptothecin derivative can turn off the transcription induction of anti-apoptotic genes. Furthermore, the active camptothecin derivative can degrade the transcription cofactor DDX5 protein, thereby maintaining or improving the sensitivity of cancer cells to chemotherapy and radiotherapy.

[0158] [Camptothecin derivatives and their isomers represented by chemical formulas 1, 3 to 9] According to the present invention, the activated camptothecin derivatives represented by Chemical Formula 1, in which R1 and R2 are designed to be the same as those of the FL118 compound, for example, the compounds represented by Chemical Formula 3, Chemical Formula 4, Chemical Formula 5, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8, Chemical Formula 9 and their isomers degrade DDX5, which is a transcriptional cofactor and an oncoprotein, thereby strongly suppressing the expression of anti-apoptotic proteins (survivin, cIAP2, XIAP, etc.) in a concentration-dependent manner (FIGS. 11 to 14), and also kill the cells into which the compounds are introduced through cell survival experiments (FIGS. 15 and 16).

[0159] According to the present invention, the camptothecin derivatives represented by Chemical Formula 1, for example, Chemical Formulae 3 to 9, have a pentacyclic structure having a lactone in the E-ring, which is essential for cytotoxicity, like camptothecin shown in FIG. 1, and are designed to maintain the lactone group and alpha hydroxyl group located at carbon 20 of the E-ring, which are important for stabilizing topoisomerase I-DNA by-products. The FL118 drug structure maintains the advantages of the above-mentioned FL118 drug, that is, R1 and R2 (-OCHO-(methylenedioxo) pentagonal ring) in General Formula 1, while the structural feature of the exatecan drug (R3 and R4 in General Formula 1) is such that the various orientations of the continuous carbon-carbon single bonds of the 6-membered rings extended from the A- and B-rings form a dynamic equilibrium, thereby weakening or suppressing the stacking of the aromatic rings formed by the A- and B-rings, compared to SN-38, which induces aggregation due to π-π stacking of the aromatic rings formed by the A- and B-rings.

[0160] In addition, the compound represented by Chemical Formula 3 or Chemical Formula 3-1 is a compound designed to combine the fact that the molecular bond can be rotated with respect to the carbon-carbon single bond in the 6-membered ring extended from the A- and B-rings, and that -NH2, which has a large degree of freedom, is exposed to water (H2O) and takes on a (+) charge or hydrogen bonds with water to increase water dispersibility.

[0161] In addition, the compound represented by Chemical Formula 4 or Chemical Formula 4-1 is a compound designed to combine the following: 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, which has a large degree of freedom, is exposed to water (H2O), and rotates like a propeller while forming a hydrogen bond with water, thereby increasing water dispersibility.

[0162] In addition, the compound represented by Chemical Formula 5 or Chemical Formula 5-1 is a compound designed to combine the fact 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 CH3CH(OH)CONH- of the lactic acid form of -NH2, which has a large degree of freedom, is exposed to water (H2O) and rotates like a propeller while hydrogen bonding with water to increase water dispersibility.

[0163] The compound represented by Chemical Formula 5 or Chemical Formula 5-1 of the present invention is a compound represented by Chemical Formula 4 or Chemical Formula 4-1 in which a methyl group, which is a metabolically unstable functional group, is introduced to shorten the drug's lifespan. Drugs that are extremely metabolically stable and metabolized very slowly are necessary to ensure an appropriate residence time because they may cause toxicity and serious side effects due to accumulation.

[0164] The DXd payload used in Enhertz was originally made from the exatecan compound, which is hardly affected by ABCG2, but was strongly affected by ABCG2 due to the presence of the glycolic acid (α-hydroxyacetic acid) functional group used to convert exatecan to DXd. However, the glycolic acid functional group plays a very important role in the excellent safety / efficacy profile of Enhertz, and removing it not only poses difficulties in ADC production, but also leads to poor performance of ADC in animal models and clinical trials (emergence of safety issues or reduced efficacy). Thus, there is a high demand for new camptothecin derivatives that can be easily used in ADC production and are not affected by ABCG2. The compound represented by formula 9 (PBX-7024) is a compound derived from the compound represented by formula 3 (PBX-7011), and is a new camptothecin compound that is not affected by ABCG2 and can be easily used in ADC production.

[0165] To confirm the anti-cancer efficacy of PBX-7024, efficacy evaluation was performed in FaDu cancer cells, which do not express ABCG2, and A549 cancer cells, which overexpress ABCG2. In the A549 cancer cell line, which overexpresses ABCG2, camptothecin compounds including DXd showed high IC 50 It can be seen that, whereas PBX-7016 and PBX-7024 still maintain strong efficacy.

[0166] In other words, when the new camptothecin compounds PBX-7016 and PBX-7024 according to the present invention are used, they have the effect of overcoming the resistance mechanism caused by overexpression of ABCG2, unlike ADCs using existing camptothecin compounds such as SN-38 and DXd.

[0167] Figures 27 and 28 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'.

[0168] The compound of formula 3 and the compound of formula 3-1 are in a partial stereoisomer relationship with each other. Similarly, the compound of formula 4 and the compound of formula 4-1, and the compound of formula 5 and the compound of formula 5-1 are also in a partial stereoisomer relationship with each other. Therefore, compounds that are in a partial stereoisomer relationship with the compound of formula 6, the compound of formula 7, the compound of formula 8, or the compound of formula 9 also belong to the scope of the present invention.

[0169] As illustrated in Preparation 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.

[0170] As illustrated in Preparation 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).

[0171] As used herein, the camptothecin-based drug of formula 3, formula 3-1, formula 4, formula 4-1, formula 5, formula 5-1, formula 6, formula 7, formula 8 or formula 9 according to the present invention includes not only the compound of formula 3, formula 3-1, formula 4, formula 4-1, formula 5, formula 5-1, formula 6, formula 7, formula 8 or formula 9, but also pharma- ceutically acceptable salts thereof, solvates thereof, and prodrugs thereof.

[0172] In this specification, pharma- ceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, including, for example, salts of inorganic ions including sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc., salts of inorganic acids such as hydrochloric acid, phosphoric acid, and sulfuric acid, 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, and acetylsalicylic acid, and salts of amino acids such as lysine, arginine, and guanidine. In addition, there are salts of organic ions such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium, and benzethonium that can be used in pharmaceutical reactions, purification, and separation processes. However, the types of salts referred to in the present invention are not limited by these listed salts.

[0173] "Solvates" refer to compounds of formula 3, 3-1, 4, 4-1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 59, 60, 61, 62, 63, 64, 65, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 109, 109, 110, 111, 112, 113, 1 -1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, and pharma- ceutically acceptable salts thereof. When the solvent is water, the solvate is a hydrate.

[0174] [Advantages of molecular glue degrader drug modality] Intracellular proteins in the body are naturally degraded within a few hours to a few days after performing their function. Every cell in the body has a purification function called the ubiquitin proteasome system (UPS) that breaks down proteins. In this process, ubiquitin acts as a marker that indicates proteins to be degraded, and proteasomes recognize the ubiquitin label and act as a grinder that destroys the protein. In other words, multiple molecules of ubiquitin are attached to the sides of proteins that have completed their function like a label, and a substance called proteasome selects only proteins that have this label and breaks them down like a grinder. E3 ligase is an enzyme that initiates the in vivo protein degradation system and is responsible for substrate specificity in the ubiquitination pathway.

[0175] Molecular glue degraders, or molecular glues, are compounds that act as glue to attach target proteins and specific enzymes (E3 ligases) in the human body to each other. One of the advantages of molecular glues is their catalytic role, which means that after degrading the target, they can be further separated and degrade other target proteins.

[0176] When E3 ligase enzyme is attached to a tumor protein via molecular glue, the tumor protein is degraded, and other tumor proteins are successively degraded until the targeted tumor protein is gone, preventing the proliferation of cancer cells. Therefore, molecular glue for tumor proteins overcomes drug resistance, a problem with targeted anticancer drugs, and has a high therapeutic effect even at low doses.

[0177] In addition to the FL118 drug, the active camptothecin derivative designed by the present invention with formula 1 is a molecular glue degrader that binds to DDX5 protein and E3 ligase, i.e., a molecular glue that activates the degradation of the tumor protein DDX5 (Figures 11 to 14).

[0178] Molecular glue degraders can act not only as warheads that bind to target proteins, but also as binders for E3 ligases.

[0179] Therefore, not only FL118 but also the active camptothecin derivatives designed according to the present invention represented by Chemical Formula 1 are molecular adhesives that activate the degradation of the tumor protein DDX5, and therefore can be used as ligands that target or bind to DDX5 protein.

[0180] Unlike kinase inhibitors, molecular glue degraders are "proximity-driven" and "event-driven" in that their ability to induce degradation depends on the formation of a temporary "target protein-molecular glue-E3 ligase" ternary complex. After degradation has occurred, the molecular glue dissociates and forms another ternary complex with the target protein, allowing several rounds of degradation to proceed until the target protein is depleted.

[0181] Most drug target proteins develop resistance by adapting and evolving to the drug. However, molecular glues, small molecules that act as glue to attach tumor proteins and E3 ligases to each other, are resistant to resistance and are therefore a suitable modality for cancer therapy.

[0182] Each time a gene mutation occurs, the shape of the drug target protein changes slightly. It would be ideal if one drug could block the activity of all mutants, but this is not possible due to selectivity issues. In order to block the activity of all mutants with one drug, the drug target protein is degraded and removed from the beginning.

[0183] Therefore, the active camptothecin derivative represented by Chemical Formula 1 according to the present invention is a drug that can precisely bind to the DDX5 tumor protein, and can avoid the resistance problem of targeted therapeutics through a molecular glue access method that quickly and selectively degrades the protein.

[0184] 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, DDX5 tumor protein, is determined by its physicochemical properties and which binds very strongly to the target protein, DDX5 tumor protein, and cannot return to its original state.

[0185] The active camptothecin derivatives designed according to the present invention based on chemical formula 1, including FL118, have high affinity with DDX5 and are not easily peeled off, unlike the existing SN38 drug, and degrade DDX5 through the function of molecular glue, thereby irreversibly inhibiting the signal transduction of cancer cells related to DDX5. In addition, the physicochemical properties of the active camptothecin derivatives allow the desired regulation of the cell membrane permeability of the drug, thereby exerting a bystander effect or controlling the degree of the effect, thereby having a high killing effect on surrounding cells, which is advantageous in the treatment of heterogeneous tumors, as well as suppressing cancer progression for a long period of time, reducing the risk of resistance development, and increasing the treatment response rate.

[0186] [Cell death and drug resistance to anticancer drugs] 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 the degradation of DDX5 protein (FIG. 29).

[0187] Tumor growth is determined by two factors: first, cell proliferation and second, cell death. When the cell cycle of tumor cells is arrested by cytotoxic agents, the tumor cells die by apoptosis.

[0188] Cell death plays a vital role in maintaining tissue homeostasis and morphology, regulating cell number, removing damaged or abnormal cells, and as a defense mechanism against infection. Living organisms maintain normal life by precisely regulating the processes of cell growth, differentiation, and death.

[0189] Apoptosis is described as a complex process that induces cell death through the expression of various genes and the activation of proteins that are the products of their expression. Apoptosis is an intracellular physiological, active suicide mechanism that plays an essential role in the development, differentiation, and homeostasis of multicellular organisms. Apoptosis occurs in various types of cells due to various factors, and unlike cell necrosis, there is no release of lysosomal enzymes, apoptotic bodies are found, cells shrink, nuclear condensation, and characteristic ladder DNA segments are found.

[0190] Apoptosis is part of the normal developmental program in animals. It is important in the prevention of cancer. The proteolytic enzyme system caspases is involved in cell death and cleaves many target proteins. If cell death is impaired, the cells that should be killed can survive, grow, and form clones that can potentially become cancerous.

[0191] In cancer therapy, essential drug resistance may be caused by abnormally expressed transcription factors that are crucial regulators of cell proliferation and cell death. Thus, the active camptothecin derivative of the present invention can induce DDX5 proteolysis and cell death through its mechanism of action as a molecular glue degrader that binds to DDX5, a transcription factor cofactor and oncoprotein. In this case, DDX5 proteolysis can downregulate the transcription of anti-apoptotic genes. Thus, unlike other targeted therapeutic agents, drug resistance caused by abnormally expressed cell proliferation and / or cell death-related transcription factors does not occur well / does not occur, or acquired drug resistance induced through activation of transcription factors that are not regulated by chemotherapy and / or radiotherapy can be suppressed.

[0192] In other words, the anti-cancer mechanism of the active camptothecin derivatives of the present invention can circumvent the molecular mechanism of cancer treatment resistance, so that the problem of drug resistance can be avoided. When resistance occurs, the therapeutic effect is reduced compared to before, so the active camptothecin derivatives of the present invention are preferred as standard therapeutic agents or first-line therapeutic agents after cancer diagnosis.

[0193] In summary, the active camptothecin derivative represented by Chemical Formula 1 according to the present invention can be a targeted anticancer drug that acts on DDX5 protein, which plays an important role in the growth, survival, proliferation, metastasis and / or metabolism of cancer cells.

[0194] [ADME profile of antibody-drug conjugates (ADCs)] The in vivo efficacy of anticancer drugs can be influenced by their absorption, distribution, metabolism, and excretion (ADME) properties. The ADME profile of a drug can affect its ability to reach and act on cancer cells. Thus, the ADME properties of an anticancer drug can have a significant impact on its in vivo efficacy.

[0195] The ability of a drug to penetrate tumor tissue can also be affected by factors such as the tumor microenvironment, including blood flow and cell density.

[0196] Therefore, understanding the ADME properties of anticancer drugs and optimizing drug / drug modality selection and dose / regimens can improve the therapeutic index of such anticancer drugs and provide better outcomes for cancer patients.

[0197] Antibodies (Abs) and antibody-drug conjugates (ADCs) have different lifetimes and ADME profiles due to their different structures and mechanisms of action.

[0198] Antibodies are large proteins produced naturally by the immune system in response to foreign substances (antigens). Their size and complex structure give them a long circulating half-life (weeks to months) and protect them from degradation and elimination. Antibodies are distributed throughout the body, including tissues, and can interact with target antigens with high specificity and affinity. Antibodies are primarily cleared by the reticuloendothelial system (RES), including the liver and spleen, and by catabolism in the kidneys and other organs.

[0199] ADCs consist of an antibody (usually a monoclonal antibody) conjugated to a cytotoxic drug molecule. The antibody component provides specificity and targeting to the tumor or diseased tissue, whereas the drug component provides cytotoxic activity to kill the target cells. ADCs have a shorter half-life than antibodies, typically reaching several days to a week. This is because ADCs are internalized into the target cells, leading to lysosomal degradation of the ADC and release of the drug payload. ADCs are primarily eliminated by the RES, although the drug payload can also undergo metabolism and excretion via the liver and kidney (Example 4).

[0200] Antibodies are generally administered by subcutaneous or intravenous injection and absorbed into the bloodstream. They can be distributed throughout the body, including tissues, but are generally restricted to the extracellular space due to their size. ADCs are also administered by injection and absorbed into the bloodstream, but after targeting to the tumor or diseased tissue by the antibody component and possibly receptor-mediated endocytosis, the drug payload released from the ADC can penetrate cells and tissues. Furthermore, the metabolism and excretion of ADCs vary depending on the specific structure and the specific drug or antibody used.

[0201] Antibody-drug conjugates (ADCs) using camptothecin-based payloads have attracted great interest as a new approach to treat various cancers, especially solid tumors. Among these ADCs, new ADCs (e.g., Enhertz) containing newly synthesized camptothecin-based payloads (e.g., DXd) optimized for ADC modalities have recently attracted special attention due to successful clinical results.

[0202] The success of such ADCs is due in part to the fact that the new camptothecins have features such as (1) superior cell growth inhibition; (2) a better safety profile than existing ADC payloads; (3) optimized bystander effects; and (4) excellent physicochemical properties suitable for generating high DAR ADCs. However, despite the remarkable success of ADCs utilizing camptothecin-based payloads such as Enhertz or DS-1062a, there are clear requirements that remain to be met, including improved safety profiles (minimizing interstitial lung disease (ILD) and neutropenia) and the development of new ADCs with multiple MoA payloads to address cancer heterogeneity.

[0203] To address these unmet needs, the present invention provides various active camptothecin derivatives of Chemical Formula 1 as candidate drugs, which are designed to bind to DDX5 protein and E3 ligase as molecular glue degraders, and further synthesizes new camptothecin-based payloads (PBX series compounds) represented by Chemical Formula 1 (Preparation Examples 1 to 4) and evaluates them (Examples 2 to 9).

[0204] The new PBX series compounds represented by formula 1 are FL118-based derivatives of formula 2, which are potent dual Top1 / anti-cell death pathway inhibitors with excellent safety profiles, exhibiting potent ex-vivo cytotoxicity comparable to Dxd, excellent safety profiles in preliminary toxicity studies in mice, and rapid clearance in PK studies (Example 4, Tables 4 and 5), minimizing systemic side effects when the linker is prematurely cleaved when used in ADC payloads.

[0205] Two lead compounds, PBX-7014 and PBX-7016, were selected for further evaluation with ADC payloads linked to various HER2 antibodies, including trastuzumab, from the PBX series of compounds.

[0206] Trastuzumab-7014 and trastuzumab-7016 (DAR 7-8) were easily synthesized in the same manner by linking PBX-7014 or PBX-7016 to trastuzumab with existing enzyme-cleavable linkers for comparative evaluation with Enhertz (dxd-trastuzumab) without aggregation issues (Preparation Example 5, Figures 19 and 20).

[0207] These two new ADCs showed superior cytotoxicity to DS-8201a (anti-HER2-DXd) in Her2-high cell lines (MDA-MB-453) and Her2-low / mid cell lines (FaDu), whereas they showed similar levels of cytotoxicity to DS-8201a in Her2-negative cell lines (MDA-MB-468).

[0208] Not only did trastuzumab-7014 and trastuzumab-7016 show clear dose-dependent anticancer efficacy similar or even superior to that of DS-8201 at the same dose in the NCI-N87CDX model with high Her2 expression, but trastuzumab-7016 also showed a bystander effect similar to that of Enherz (Example 9, Figures 25 and 26).

[0209] Trastuzumab-7014 and trastuzumab-7016 exerted a tumor regression effect in tumor tissue for more than 20 days after administration through a bystander effect, and also induced a tumor regressing effect by inhibiting one methyl group. Each compound exerted different ADMEs, and showed not only dose-dependent differences in anti-cancer efficacy (conc. vs. effect) but also differences in anti-cancer efficacy over time (effect vs. time) in targeted tumor tissues (Example 8, Figures 23 and 24).

[0210] Increasing the hydrophobicity of the linker and drug can lead to problems such as increased aggregation of the ADC and thus a decreased therapeutic index of the drug. Surprisingly, trastuzumab-7016 has a hydrophobicity that is increased by one methyl group compared to trastuzumab-7014. The dose-dependent anti-cancer efficacy (conc. vs. effect) and the time-dependent anti-cancer efficacy (effect vs. time) were excellent (Example 8, Figures 23 and 24).

[0211] However, in some cases, superior anticancer efficacy over time may also result in greater side effects (sustained T cell activation and accumulation of inhibitory signals due to chronic antigen exposure, resulting in T cell exhaustion, ILD, and neutropenia). For example, it is important to exceed a certain drug concentration (high) depending on the drug modality (small molecule vs. ADC) of the active camptothecin derivative of Chemical Formula 1, and in some cases, the time for which the drug concentration is maintained above a certain drug concentration (low) is important.

[0212] Therefore, it is a main feature of the present invention that the selection range of ADC payloads exhibiting appropriate anti-cancer efficacy, taking into consideration not only various side effects but also issues such as a reduced therapeutic index, can be expanded to a diverse group of candidates including the activated camptothecin derivative of Chemical Formula 1, which is designed to bind to DDX5 protein and E3 ligase as a molecular glue degrader.

[0213] Despite the remarkable success of ADCs utilizing camptothecin-based payloads such as ENHARTZ or DS-1062a, the present invention provides a variety of activated camptothecin derivatives represented by Chemical Formula 1 that are designed to bind to DDX5 protein and E3 ligase as candidates for multiple MoA payloads to improve safety profiles (minimize ILD and neutropenia) and address cancer heterogeneity. In order to precisely control the desired efficacy (e.g., anticancer, combination therapy) and side effects (carcinogenic inflammation, drug resistance, ILD, neutropenia), one or more appropriate drugs from the activated camptothecin derivatives of Chemical Formula 1 can be selected, and furthermore, an appropriate linker can be selected and an appropriate dose-dosage can be applied. .

[0214] [Cancer cell death and the resulting immune response] One aspect of the present invention provides an anticancer dosage form comprising (a) an activated camptothecin derivative represented by the above chemical formula 1, which is designed to bind to DDX5 protein and E3 ligase; or (b) a prodrug thereof, which is designed to release the activated camptothecin derivative (a) from a target site in the body, administered in a dosage that can stimulate antigen-presenting cells (APCs) through cell death of cancer cells and induce an anti-tumor immune response.

[0215] Here, the antigen presenting cells (APCs) can be dendritic cells and macrophages.

[0216] In addition, one aspect of the present invention is a method for producing an anticancer drug based on an active camptothecin derivative (a), in which the bystander killing effect and / or ADME profile of the active camptothecin derivative (a) is controlled so that T cell exhaustion does not occur due to chronic antigen exposure, The active camptothecin derivative (a)-based anticancer drug is (a) an active camptothecin derivative designed to bind to DDX5 protein and E3 ligase via a molecular glue degrader; or (b) an anticancer dosage form containing a prodrug thereof designed to release the active camptothecin derivative (a) from a target site in the body, The present invention provides a method for producing an anticancer agent, comprising the steps of designing an active camptothecin derivative (a) or its prodrug (b) to contain the camptothecin-based skeleton represented by the above chemical formula 1 as a parent nucleus, selecting a compound designed in this way, or synthesizing a compound designed in this way.

[0217] The signaling pathways that regulate cell death are a complex network of intracellular signaling pathways that are tightly regulated by diverse proteins and can be activated by diverse internal or external stimuli, such as DNA damage, oxidative stress, and growth factor deprivation. Dysregulation of such pathways can induce a variety of diseases, including cancer and neurodegenerative disorders.

[0218] The major signaling pathways regulating cell death all converge on the activation of caspases, a family of cysteine ​​proteases that cleave a variety of cellular substrates and ultimately lead to cell death. Caspase activation is tightly regulated by a variety of proteins, including those of the Bcl-2 family, which can promote or suppress apoptosis.

[0219] The Bcl-2 protein system contains a full complement of pro- and anti-apoptotic members that can promote or suppress cell suicide, respectively. The balance between pro- and anti-apoptotic Bcl-2 system members is important in determining cell fate and susceptibility to cell death.

[0220] Other signaling 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, leading to the transcription of pro-apoptotic genes such as Bax and Puma, while the PI3K / Akt pathway can phosphorylate and inactivate pro-apoptotic proteins, such as Bad and Bim, to suppress apoptosis.

[0221] Some forms of necroptosis may be a programmed cellular response to stimuli such as infection or DNA damage.

[0222] Immunogenic cell death is a process in which cancer cells are damaged and die, accompanied by changes in the cell surface, release of cancer antigens and damage-associated molecular patterns, etc. The released immune inducers can stimulate immune cells, such as dendritic cells and macrophages, to induce antitumor immune responses.

[0223] Apoptosis is a method by which damaged or aged cells die, and involves the transformation and degradation of various intracellular substances. During this process, immune-inducing substances such as cancer antigens and damage-associated molecular patterns (DAMPs), which induce the body's immune system against cancer cells, are damaged by proteolytic enzymes and oxidation, which can limit the effectiveness of immunotherapy against residual cancer or metastatic cancer after treatment.

[0224] Cell death caused by reactive oxygen species generated by chemical anticancer drugs is apoptosis, which is mainly mediated by the proteolytic enzyme caspase.

[0225] Necroptosis is a type of necrosis in which cells die by destroying the cell membrane. Unlike apoptosis, which involves degradative enzymes such as caspases, necroptosis does not involve protein degradation or oxidation, so it can minimize damage to cancer markers and immune inducers.

[0226] Damage-associated molecular patterns (DAMPs) are molecules that are released during cell death and can induce immune responses within cells, stimulating immune cells. Representative DAMPs include calreticulin, heat shock protein 70 / 90, HMG box protein 1, and ATP.

[0227] Inflammation is the body's natural response to infection or injury and involves the release of inflammatory cytokines and recruitment of immune cells to the affected area. However, if inflammation persists for a long time, it can damage tissue and increase the risk of developing cancer.

[0228] In the context of cancer, chronic antigen exposure can also lead to T cell exhaustion. Tumor cells express antigens that can be recognized by T cells, and continued exposure to these antigens can lead to T cell exhaustion, weakening the anti-tumor response and allowing tumors to grow and spread.

[0229] The relationship between T cell exhaustion, chronic antigen exposure and disease in general is complex and multifaceted.

[0230] Trastuzumab-7016, despite its increased hydrophobicity due to a difference of one methyl group from trastuzumab-7014, exhibited superior dose-dependent and time-dependent anticancer efficacy (Example 8, Figures 23 and 24), although lower concentrations of trastuzumab-7014 may be required for treatment due to side effects such as T cell exhaustion, ILD, and / or neutropenia.

[0231] Only by knowing the characteristics of a drug can a drug be used correctly. To understand the characteristics of a drug, the pharmacodynamic and pharmacokinetic parameters of the drug are helpful.

[0232] Pharmacodynamics describes the magnitude and nature of the effects (cell survival, clinical efficacy) that occur in a cell or in the body after a drug binds to a receptor in relation to drug concentration.

[0233] Pharmacokinetics (PK) is a method to determine the effect of a drug or drug modality. It shows how the drug concentration changes when it moves through other compartments of the body via ADME. Example 8, Table 6, Figures 23 and 24 show that the drug concentration of active camptothecin derivative (a) in tumor tissue over time can be predicted for various drugs / modalities.

[0234] From a pharmacokinetic point of view, the reasons for failure in new drug development are that toxic drugs can accumulate, useful drugs may be of no benefit because the doses required to establish treatment are too small, and drugs can be metabolized quickly.

[0235] Therefore, to use the medicine correctly, the appropriate drug must be selected and the appropriate dose-dosage must be applied.

[0236] Dose indicates the amount of drug administered at one time. Drug doses are typically prescribed by a healthcare provider based on factors such as the patient's age, weight, and health. Dosage, on the other hand, indicates how often and for how long a drug is administered. It is a measure of the total amount of drug given over a period of time and is typically expressed in daily or weekly amounts. Dose and dosage are important considerations for the safe and effective use of drugs.

[0237] From this perspective, the present invention provides various active camptothecin derivatives of Chemical Formula 1 that bind to DDX5 protein and E3 ligase as molecular glue degraders as various drug candidates, thereby enabling one or more appropriate drugs to be selected and their dosage and administration methods to be appropriately applied to precisely control the desired efficacy (e.g., anticancer, combination therapy) and side effects (chronic cancer antigen exposure, drug resistance, ILD).

[0238] For example, it is important for the active camptothecin derivative of Chemical Formula 1 to exceed a specific drug concentration (high) in tumor tissue depending on the drug modality (small molecule, ADC), and in some cases, the time for which the drug concentration is maintained at or above a specific drug concentration (low) in tumor tissue is important.

[0239] [Pro-drug] As used herein, prodrugs include, but are not limited to, pro-drugs that become biologically active or further activated after being placed in a predetermined physiological environment.

[0240] For example, it means a compound designed to chemically modify a physiologically active substance or a therapeutically active organic compound and liberate or release the parent compound under enzymatic or other conditions in the body. A prodrug is converted into the desired compound in the body after administration. A useful drug that has incompatible properties in terms of side effects, stability, solubility, absorption, duration of action, etc. is chemically modified to make it clinically usable.

[0241] According to the present invention, an activated camptothecin derivative represented by the following formula 1, e.g., a camptothecin drug represented by formula 3, formula 3-1, formula 4, formula 4-1, formula 5, formula 5-1, formula 6, formula 7, formula 8, or formula 9, designed to bind to DDX5 protein and E3 ligase, can be used as a payload for various carrier-drug conjugates. That is, various carrier-drug conjugates in which an activated camptothecin derivative is linked with a payload are a type of prodrug of an activated camptothecin derivative that releases the active camptothecin derivative in vivo. In this case, the carrier may be an antibody, a peptide, a receptor body, and / or an aptamer.

[0242] [Table 2]

[0243] For example, antibody-drug conjugates (ADCs) are a new drug platform that selectively delivers a payload with strong anti-cancer efficacy to cancer tissues using the high tissue selectivity of antibodies that specifically bind to specific antigens expressed on the surface of cancer cells. ADCs selectively deliver a powerful payload that kills cancer cells even at low pM concentrations to cancer tissues, minimizing systemic drug exposure and ensuring both anti-cancer efficacy and safety.

[0244] Most ADCs are introduced into cells via clathrin-coated pits. After ADCs enter cells, they are detached from clathrin and fuse with other intracellular vesicles, then proceed to 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 to the cytoplasm, where it binds to the drug's molecular target, causing the cell cycle of tumor cells to stop and cancer cells to die by apoptosis. Of these, a certain amount of the drug is passively diffused in the cells, actively transported, or released outside the cells via dead cells. If the released drug has cell membrane permeability, it may enter surrounding cells, causing the so-called bystander cell killing phenomenon.

[0245] In the case of HER2-positive cancer, the active camptothecin derivative (a) or its prodrug ADC, which targets DDX5 protein according to the present invention, may become a new treatment that can resolve resistance to anti-HER2 therapy.

[0246] The prodrug of the present invention may be an immunoconjugate comprising an antibody or a fragment containing the antigen-binding site thereof, or a pharma-ceutically acceptable salt thereof, which is designed to release the active camptothecin derivative (a) of the present invention in vivo.

[0247] The immunoconjugate of the present invention specifically binds to an antigen on a cancer cell, releases a drug inside or outside the cancer cell, and exhibits cytotoxicity, and therefore can be usefully used in the treatment or prevention of cancer.

[0248] Aptamer-Drug Conjugate (ApDC) is an ADC that introduces an aptamer instead of the antibody. 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 technology that introduces a target protein molecule into a compound library and obtains functional nucleic acids that bind to it.

[0249] Aptamers can bind to targets very strongly and selectively, and are also called chemical antibodies. Aptamers are about 20 kDa in size and are known to have superior cell permeability and low immunogenicity compared to antibodies.

[0250] Aptamers can be chemically synthesized, so precise design of the conjugation position and number of drugs during the production of aptamer-drug conjugates is possible. The production costs are lower than those of ADCs.

[0251] Aptamers are generally made of natural nucleic acids and are degraded by nucleases in the body, resulting in low in vivo stability. However, the ease of chemical modification of aptamers can be used to overcome the limitations on the stability of modified aptamers.

[0252] Peptide-drug conjugates (PDCs) are ADCs in which peptides have been introduced instead of antibodies. Peptides are composed of amino acids and have sizes ranging from 500 to 5,000 Da (Daltons). This is a very small size compared to antibodies, which are over 150 kDa (kilodaltons). Therefore, peptide-based PDCs have superior cell penetration ability compared to ADCs, and the possibility of immunogenicity is very low. In addition, peptides can be chemically synthesized. Therefore, PDCs not only have very low production costs, but also allow precise adjustment of the binding position and ratio of peptides and drugs.

[0253] In general, peptides have a short biological half-life because they are easily degraded by proteolytic enzymes. To overcome the limitations of peptide-based drug conjugates, strategies have been proposed that utilize modified peptides, such as cyclic peptides and peptides with unnatural amino acids.

[0254] Repebody is a type of artificial antibody that does not have an antibody skeleton but has the function of recognizing antigens like an antibody. Repebody specific to a target protein can be discovered through phage display.

[0255] Phage display is a technology that expresses a desired protein on the surface of a bacteriophage. Repebodies are about 30kDa in size, which is 20% the size of antibody drugs. Therefore, they are 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 Repebodies can be adjusted, thereby enhancing their structural stability. It is also considered that the production costs are relatively low compared to antibodies. Due to these advantages of Repebodies, there is growing interest in the development of Repebody-drug conjugates (Repebody-DCs) as a strategy to convert antibodies into lipobodies.

[0256] [Pharmaceutical composition for preventing or treating cancer] The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, a compound represented by the above-mentioned 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 pharma- ceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof.

[0257] According to the present invention, the active camptothecin derivative (a) represented by Chemical Formula 1 or its prodrug (b) inhibits DDX5 from functioning as an oncoprotein in cells. The compound may be included as an active ingredient in a pharmaceutical composition for administration to patient populations that are responsive to the compound.

[0258] For example, a patient group to which the active camptothecin derivative (a) or its prodrug (b) is to be administered can be identified by quantitatively or qualitatively analyzing intracellular DDX5 protein in a tissue or liquid biopsy using the FL118 compound of chemical formula 2 or a ligand-containing complex (c) that targets DDX5 protein in which the type I topoisomerase inhibitory ability of the active camptothecin derivative (a) has been inactivated, preferably via a non-cleavable linker linkage.

[0259] According to the present invention, the active camptothecin derivative (a) represented by Chemical Formula 1, for example, 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, is a camptothecin derivative with a new structure. Therefore, through this, a drug candidate substance having more preferable properties in terms of efficacy, toxicity, selectivity, duration of action, administration, handling, stability and / or producibility of Dxd drugs can be provided. In addition, the interaction between the target and the drug candidate substance, i.e., pharmacodynamic properties, can be optimized, and the access of the drug to the target can be optimized, i.e., pharmacokinetic ability can be improved.

[0260] In addition, one embodiment of the present invention provides a method for treating or preventing cancer, comprising administering a therapeutically effective amount of a compound represented by Formula 1, for example, Formula 3, Formula 3-1, Formula 4, Formula 4-1, Formula 5, Formula 5-1, Formula 6, Formula 7, Formula 8 or Formula 9, a pharma- ceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof to a subject in need thereof. The subject may be a mammal, including a human.

[0261] In the present invention, the cancer includes all cancers treatable 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 may be a solid cancer or a blood cancer. For example, 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 lymphocytic 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 gland cancer, paranasal 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, 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 cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsillar cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer, and thymic cancer. Moreover, the cancer includes not only primary cancer but also metastatic cancer.

[0262] As used herein, "patient," "subject," and "subject" refer to animals, such as mammals. In certain embodiments, the patient is a human. In other embodiments, the patient is a human, such as a dog, cat, livestock (e.g., horse, pig, or donkey), chimpanzee, or monkey. It is a non-human animal.

[0263] The term "therapeutically effective amount" used in the present invention refers to an amount of the compound represented by formula 3, formula 3-1, formula 4, formula 4-1, formula 5 or formula 5-1, its pharma- ceutically acceptable salt, its solvate, or its prodrug that is effective for treating or preventing cancer. Specifically, "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined by factors including the type and severity of the individual, age, sex, type of disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, co-used drugs, 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. It can also be administered singly or multiple times. It is important to administer an amount that can obtain maximum effect at a minimum amount without side effects by considering all the above factors, and 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 of the present invention and its pharma- ceutical acceptable salts show dose-dependent effects, so that the administration dose can be easily determined by those skilled in the art depending on various factors such as the condition, age, sex and comorbidities of the patient. The active ingredient of the pharmaceutical composition of the present invention has excellent safety and can be used at a dose higher than the determined administration dose.

[0264] According to one embodiment of the present invention, the present invention provides a use of the compound represented by Formula 3, Formula 3-1, Formula 4, Formula 4-1, Formula 5, Formula 5-1, Formula 6, Formula 7, Formula 8 or Formula 9, its pharma- ceutically acceptable salt, its solvate, or its prodrug for use in the manufacture of a medicament for use in the treatment or prevention of cancer. The compound represented by Formula 3, Formula 3-1, Formula 4, Formula 4-1, Formula 5, Formula 5-1, Formula 6, Formula 7, Formula 8 or Formula 9, its pharma- ceutically acceptable salt, its solvate, or its prodrug for the manufacture of a medicament may be mixed with an acceptable adjuvant, diluent, carrier, etc., and may be prepared into a composite preparation together with other active agents to have a synergistic effect of the active ingredients.

[0265] All the matters mentioned in the applications, compositions, and treatment methods of the present invention are applicable to the same unless they are inconsistent with each other.

[0266] As used herein, the anti-cancer effect or therapeutic effect of an anti-cancer drug can refer to the effect of reducing the severity of cancer, reducing the size of a tumor, or delaying or slowing the progression of cancer that occurs while a patient is suffering from a particular cancer.

[0267] For example, the anti-cancer effect of an anti-cancer drug can be cell survival (change in cell number or degree of cytotoxicity) of cancer cells after treatment of the anti-cancer drug in vitro and / or in vivo. For example, it can be indirectly confirmed through drug response examination in cell line or non-clinical animal model (xenograft). In addition, the anti-cancer effect of an anti-cancer drug can be directly confirmed in cancer patients, and related data can be extracted and used in a database. In addition, PK parameters and / or toxicity profile of animal models can be considered in parallel when designing dosing guidelines for anti-cancer drugs.

[0268] The anticancer effect of an anticancer drug is measured by the in-vitro data, i.e., the % maximum effect of the anticancer drug, e.g., IC 50 ,I C 60 ,I C 70 ,I C 80 and I.C. 90 This can be inferred from the above and can be confirmed in non-clinical animal models and clinical cancer patients through in vivo data such as the maximum drug concentration (Cmax) and / or area under the drug concentration-time curve (AUC).

[0269] The reactivity of an anticancer drug means clinical sensitivity in terms of the anticancer effect.

[0270] When referred to in connection with treatment with anti-cancer drugs, "sensitivity" and "hypersensitive" are relative terms that refer to the degree of effectiveness of a compound in alleviating or reducing the progression of the tumor or disease being treated.

[0271] An "effective patient anti-cancer effect / response" can be, for example, a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more inhibition in patient response as measured by any suitable means, e.g., gene expression, cell counts, analytical results, etc.

[0272] 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 ).

[0273] 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.

[0274] 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.

[0275] In some embodiments, if the patient's condition improves, administration of the compound is subsequently provided at the discretion of the physician; alternatively, the dose of drug administered can be temporarily reduced or temporarily suspended for any length of time (i.e., a "drug holiday"). The length of the drug holiday can vary from 2 days to 1 year, including, by way of example only, 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, dose reductions during drug holidays are 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0276] Once improvement of the patient's condition has occurred, maintenance doses may be administered, if necessary. The amount or frequency of administration, or both, can be reduced as a function of the symptoms to a level at which the ameliorated disease, disorder or condition is maintained, however, the patient may require intermittent treatment for a long period of time upon any recurrence of symptoms.

[0277] The amount of a given formulation that corresponds to such an amount will vary depending on factors such as the particular compound, the severity of the disease, and the identity (e.g., body weight) of the subject requiring treatment, but can nevertheless be routinely determined 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. In general, however, doses used for adult treatment can typically range from 0.02 to 5000 mg / day, or from about 1 to 1500 mg / day.

[0278] The dosage referred to herein may be provided in a single dose or in divided doses administered simultaneously, for example as two, three, four or more sub-doses.

[0279] In some embodiments, oral dosage form is a unit dosage form suitable for single administration of precise dosage.In unit dosage form, dosage form is divided into unit doses that contain appropriate amounts of one or more compounds.In some embodiments, unit dose is in the form of a package that contains separate amounts of dosage form.Non-limiting examples are packaged tablets or capsules, and powder vials or ampoules.Aqueous suspension compositions can be packaged in single-dose non-reclosable containers.Optionally, multiple-dose reclosable containers can be used, in which case it is typical to include a preservative in the composition.

[0280] In some embodiments, dosage forms for parenteral injection are provided in unit dosage form, including, but not limited to, ampoules, or in multi-dose containers, with an added preservative.

[0281] Typically, they are prepared in unit dose injectable form with a pharma- ceutically acceptable parenteral vehicle for parenteral administration, i.e., bolus, intravenous, and intratumoral injection, and are optionally mixed in lyophilized or aqueous solution form with a pharma- ceutically acceptable diluent, carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.).

[0282] [Ligands targeting DDX5 protein] Not only FL118, but also the activated camptothecin derivative represented by Chemical Formula 1 according to the present invention is a molecular adhesive that activates the degradation of tumor protein DDX5, and therefore can be used as a ligand that targets DDX5 protein or a ligand that binds to DDX5 protein. Therefore, it is possible to provide a ligand-containing complex that targets DDX5 protein, in which the type I topoisomerase inhibitory ability of the FL118 compound or the activated camptothecin derivative (a) is inactivated via a linker linkage.

[0283] The linker may be a non-cleavable linker, a non-limiting example of a non-cleavable linker is a thioether linker.

[0284] The FL118 compound or the active camptothecin derivative (a) represented by Chemical Formula 1 binds to the DDX5 protein, and the DDX5 protein is a direct target of the active camptothecin derivative (a).

[0285] Therefore, DDX5 can be used as a biomarker for predicting drug or tumour sensitivity.

[0286] Activity of the FL118 compound of formula 2 or of formula 1 in tissue or liquid biopsies By quantitatively or qualitatively analyzing intracellular DDX5 protein using a ligand-containing complex (c) that targets DDX5 protein, in which the type I topoisomerase inhibitory ability of an active camptothecin derivative (a) has been inactivated via a linker bond, it is possible to determine the patient group to which the active camptothecin derivative (a) or its prodrug (b) should be administered, or to predict the recurrence rate and / or prognosis during chemotherapy with an anticancer drug.

[0287] [Cancer diagnostic composition] The present invention provides a cancer diagnostic composition comprising a ligand-containing complex (c) targeting DDX5 protein, in which the type I topoisomerase inhibitory ability of the FL118 compound represented by chemical formula 2 or an active camptothecin derivative (a) represented by chemical formula 1 has been inactivated via a linker linkage.

[0288] The cancer diagnostic composition of the present invention can be used to determine the patient group to which the active camptothecin derivative (a) or its prodrug (b) should be administered, or to predict drug response, recurrence rate and / or prognosis during chemotherapy with an anticancer drug. Effect of the Invention

[0289] According to the present invention, the camptothecin derivative represented by Chemical Formula 1, for example, the camptothecin derivative represented by Chemical Formula 3 to Chemical Formula 9 or its isomers, is a compound with a new structure. Through this, it is possible to provide a drug candidate substance having more preferable properties in terms of efficacy, toxicity, selectivity, duration of action, administration, handling, stability and / or producibility of Dxd drugs. In addition, it is possible to optimize the interaction between the target and the drug candidate substance, i.e., the pharmacodynamic properties, and to optimize the access of the drug to the target, i.e., to improve the pharmacokinetic ability.

[0290] The present invention provides compounds of formula 1, e.g., compounds of formulas 3 to 9 or isomers thereof, that maintain the structure of the FL118 drug, which maintains the advantages of the FL118 drug, and can maximize the efficacy of regulating the main drug target through an additional mechanism of action of (i) simultaneously inhibiting / suppressing Bcl family members such as the resistance protein survivin, or (ii) inhibiting the action of an efflux pump.

[0291] Therefore, the compound of formula 1, for example, the compounds of formulas 3 to 9 or their isomers are designed based on a well-designed polypharmacology-based access method that obtains excellent therapeutic efficacy by simultaneously targeting various drug targets that need to be controlled together, unlike the existing drug development method in which one drug targets one drug and therapeutic efficacy is limited, as in the case of FL118 drug, and can treat intractable diseases for which existing therapeutic agents have not shown clear therapeutic effects. In addition, recurrent / resistant cancers cannot obtain sufficient efficacy by only regulating the activity of one drug target, and there is a problem that safety cannot be guaranteed when targeting an unspecified number of drug targets at the same time, but by utilizing two or more well-selected drug targets, as in the case of FL118 drug, synergy can be shown in terms of efficacy and sufficient safety can be ensured at the same time. [Brief description of the drawings]

[0292] [Figure 1] FIG. 1 shows the synthetic design concept of a new active camptothecin derivative (a) that has a dual mechanism of action (MoA) for degrading the oncoprotein DDX5, together with type I topoisomerase inhibitory ability, through the improvement of the FL118 structure. [Diagram 2] FIG. 2 shows the structural formula of camptothecin (CPT) and its binding to type I topoisomerase-1. It also shows the extension of the molecular design concept of FL118 and PBX-7011 from camptothecin, and the derivation of new structural compounds PBX-7014 and PBX-7016 extended from PBX-7011, along with the results of hydrophilicity calculations. [Diagram 3] FIG. 3 shows the synthesis of 25-4 and 25-6 from PBX-7014 and PBX-7016, as well as the process for synthesizing deruxtecan from Dxd. [Figure 4] FIG. 4 shows molecular dynamics simulations and docking scores analyzing the interactions between existing camptothecin-based drugs and TOP1. [Diagram 5] FIG. 5 shows molecular dynamics simulations and docking scores analyzing the interaction between the novel camptothecin-based drugs of the present invention and TOP1. [Figure 6] FIG. 6 is a molecular dynamics simulation analyzing the interaction of camptothecin with mutant TOP1(E418K). [Figure 7] FIG. 7 shows SN38-TOP1 interactions in normal TOP1. [Figure 8] FIG. 8 shows SN38-TOP1 interaction in E418K mutant TOP1. [Figure 9] FIG. 9 shows the compound of formula 1-1 (compound A)-TOP1 interaction in E418K mutant TOP1. [Figure 10] Figures 10 to 14 are Western blot results showing the degree of inhibition of anti-apoptotic proteins by various camptothecin drugs (FL118 drug, SN-38 drug, Exatecan drug, PBX-7011, PBX-7014, PBX-7016) in FaDu cell line and A549 cell line to verify Dual MoA (Topoisomerase I inhibition & DDX5 degradation) in Example 2-1. Figures 12 and 14 are graphs showing the concentration quantification results of the Western blot experiment (Figures 11 and 13) performed on FaDu cell line and A549 cell line. [Figure 11] Figures 10 to 14 are Western blot results showing the degree of inhibition of anti-apoptotic proteins by various camptothecin drugs (FL118 drug, SN-38 drug, Exatecan drug, PBX-7011, PBX-7014, PBX-7016) in FaDu cell line and A549 cell line to verify Dual MoA (Topoisomerase I inhibition & DDX5 degradation) in Example 2-1. Figures 12 and 14 are graphs showing the concentration quantification results of the Western blot experiment (Figures 11 and 13) performed on FaDu cell line and A549 cell line. [Figure 12] Figures 10 to 14 are Western blot results showing the degree of inhibition of anti-apoptotic proteins by various camptothecin drugs (FL118 drug, SN-38 drug, Exatecan drug, PBX-7011, PBX-7014, PBX-7016) in FaDu cell line and A549 cell line to verify Dual MoA (Topoisomerase I inhibition & DDX5 degradation) in Example 2-1. Figures 12 and 14 are graphs showing the concentration quantification results of the Western blot experiment (Figures 11 and 13) performed on FaDu cell line and A549 cell line. [Figure 13]Figures 10 to 14 are Western blot results showing the degree of inhibition of anti-apoptotic proteins by various camptothecin drugs (FL118 drug, SN-38 drug, Exatecan drug, PBX-7011, PBX-7014, PBX-7016) in FaDu cell line and A549 cell line to verify Dual MoA (Topoisomerase I inhibition & DDX5 degradation) in Example 2-1. Figures 12 and 14 are graphs showing the concentration quantification results of the Western blot experiment (Figures 11 and 13) performed on FaDu cell line and A549 cell line. [Figure 14] Figures 10 to 14 are Western blot results showing the degree of inhibition of anti-apoptotic proteins by various camptothecin drugs (FL118 drug, SN-38 drug, Exatecan drug, PBX-7011, PBX-7014, PBX-7016) in FaDu cell line and A549 cell line to verify Dual MoA (Topoisomerase I inhibition & DDX5 degradation) in Example 2-1. Figures 12 and 14 are graphs showing the concentration quantification results of the Western blot experiment (Figures 11 and 13) performed on FaDu cell line and A549 cell line. [Figure 15] FIG. 15 shows the results of comparative in vitro cell viability evaluation of various camptothecin-based drugs in FaDu cell line or A549 cell line. [Figure 16] FIG. 16 shows the results of comparative in vitro cell viability evaluation of various camptothecin-based drugs in MDA-MB-453 (HER2++) cell line and FaDu (HER2+) cell line. [Figure 17] FIG. 17 shows the results of carboxylate to lactone form conversion at pH 6.0. [Figure 18] FIG. 18 shows the results of lactone to carboxylate conversion at pH 7.4. [Figure 19] Figures 19 and 20 show the results of confirming ADC production using SEC and SDS PAGE. [Figure 20] Figures 19 and 20 show the results of confirming ADC production using SEC and SDS PAGE. [Figure 21] FIG. 21 shows the results of comparing the binding ability to Her2 of ADCs conjugated with trastuzumab, trastuzumab, PBX-7014, and PBX-7016. [Figure 22] FIG. 22 shows the results of a comparative in vitro cell viability assessment of various camptothecin-based drugs and ADCs using camptothecin-based drugs as payloads in MDA-MB-453 (HER2++) cell line, FaDu (HER2+) cell line, and MDA-MB-468 (HER2-) cell line. [Diagram 23] FIG. 23 shows the efficacy test results for ADCs carrying various camptothecin-based drugs as payloads in an in vivo xenograft model in mice using the JIMT-1 cell line. [Figure 24] FIG. 24 shows the results of mouse body weight change in response to ADCs carrying various camptothecin-based drugs as payloads in an in vivo xenograft model in mice using the JIMT-1 cell line. [Diagram 25] Figure 25 shows the results of co-culturing the MDA-MB-453 (HER2++) cell line with the GFP-expressing MDA-MB-468 (HER2-) cell line, and then counting the number of cells by FACS using an ADC with a camptothecin drug as the payload, based on the presence or absence of GFP expression. [Figure 26] Figure 26 shows the results of co-culturing MDA-MB-453 (HER2++) and MDA-MB-468 (HER2-) cell lines, and then counting the number of cells by FACS using an ADC with a camptothecin drug as the payload and a HER2-FITC antibody based on the presence or absence of HER2 expression. [Figure 27]FIG. 27 is a diagram comparing LogP, cLogP, and tPSA (topological polar surface area) of FL118, exatecan, SN-38, Dxd, compound A (PBX-7011), compound B (PBX-7012), compound C (PBX-7014), compound D (PBX-7015), compound E (PBX-7016), and compound F (PBX-7017). [Figure 28] FIG. 28 is a diagram comparing LogP, cLogP and tPSA (topological polar surface area) of various camptothecin-based drugs that can be derived from Chemical Formula 1, Compound G, Compound H, Compound I, Compound J, Compound G', Compound H', Compound I', and Compound J'. [Figure 29] FIG. 29 is a detailed classification of cell death associated with programmed cell death. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0293] The present invention will be described in more detail below with reference to examples. However, the following examples are merely intended to clearly illustrate the technical features of the present invention and are not intended to limit the scope of the present invention.

[0294] Preparation Example 1: Synthesis of active camptothecin derivatives (PBX-7011 and PBX-7012) of formula 3 or formula 3-1

[0295] [ka]

[0296] 1-1. Synthesis of compound 2 [ka]

[0297] To a solution of 5-nitrobenzo[d][1,3]dioxole (25 g, 150 mmol) in a dichloromethane (748 mL) flask was added silver triflate (57.7 g, 224 mmol) and iodine (57.0 g, 224 mmol). The solution was stirred in a dark room at room temperature under a N2 atmosphere.

[0298] The AgI was removed by filtration and the solid was washed with dichloromethane (100 mL). The solvent 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 and washed with 1M Na2SO 3 (5x200 mL) and brine (200 mL), dried over Na2SO4, treated with activated charcoal and filtered through celite. The solvent was evaporated under reduced pressure to give 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 give the product as a light 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 give an additional amount of product (10.2 g) as a dark brown-gray solid. Both batches were used as is without further purification. SC_ACID: m / z 294.2[M+H] +

[0299] 1-2. Synthesis of compound 3 [ka]

[0300] To 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 allowed to cool to room temperature and concentrated under reduced pressure. The resulting black solid was suspended in ethyl acetate (100 ml) and the solution poured off. The residue was washed with EtOAc (3×50 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 collect a brown solid. (4.75 g, 60% yield). The iron residues were washed thoroughly with ethyl acetate to recover additional product. The organic fraction was then 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] +

[0301] 1-3. Synthesis of compound 4 [ka]

[0302] To a solution of 7-iodobenzo[d][1,3]dioxol-5-amine (6.39 g, 24.29 mmol) in dichloromethane (49 ml) was added acetic anhydride (2.75 mL, 29.2 mmol) and triethylamine (4.06 mL, 29.2 mmol). The reaction mixture was stirred at room temperature overnight. 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 give a light grey solid (4.46 g). The filtrate was concentrated under reduced pressure and 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 product were triturated from ice-cold EtOAc (5-10 mL). The two batches were combined and further dried to give an off-white solid. Total yield: 5.0g, 66% SC_ACID: m / z 306.0[M+H] + 1 H NMR (400MHz, DMSO-d6) δ9.88(s, 1H), 7.39(d, J=1.9Hz, 1H), 7.17(d, J=1.9Hz, 1H), 6.04(s, 2H), 1.99(s, 3H).

[0303] 1-4. Synthesis of compound 5 [ka]

[0304] In a three-neck flask equipped with a condenser, add N-(7-iodobenzo[d][1,3]dioxol-5-yl)acetamide (5.06 g, 16.59 m) in acetonitrile (40 mL). A slurry of 1.25 mmol), but-3-enoic acid (1.69 mL, 19.90 mmol) and potassium carbonate (2.98 g, 21.56 mmol) was cooled to 0-5 °C. Water (13.33 mL) was added slowly to evolve gas. When gas evolution ceased, the mixture was degassed with Ar for 30 min. 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 an additional 30 min before being heated to reflux under Ar. The reaction was allowed to cool 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 concentrated solution. HCl until pH=1-2. The aqueous layer was extracted with EtOAc and the combined organic phase was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give 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 via flash chromatography (80 g Si, 0-100% EtOAc in heptane). The product fractions were concentrated under reduced pressure to give 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] +

[0305] 1-5. Synthesis of compound 6 [ka]

[0306] A suspension of 4-(6-acetamidobenzo[d][1,3]dioxol-4-yl)but-3-enoic acid (3.47 g, 13.18 mmol) in tetrahydrofuran (50 mL) / water (50 mL) was degassed with N2 for 15 min. Pd / C (2.97 g, 1.397 mmol) was added and the suspension was degassed with H2 for 5 min before heating under H2 (balloon) at 40 °C. After 24 h, the reaction mixture was added nitrogen and more Pd / C (2.97 g, 1.397 mmol). The reaction mixture was bubbled with hydrogen for 10 min and stirred under hydrogen at 45 °C overnight. The reaction was filtered through celite. The filter cake was washed with H2O (50 mL). EtOAc (50 mL) and organic solvents were removed from the filtrate under vacuum. The aqueous solution was acidified with concentrated HCl until pH=1 and a dark brown / green precipitate was collected by filtration through a sintered funnel. The aqueous filtrate was extracted. The combined organic phase was washed with brine, dried over Na2SO4, filtered and the solvent removed under vacuum to give a brown oil. Product recovery was low so the filter cake was added to the flask containing the solid from the MeOH flash with EtOAc (50 mL), water (200 ml) and NaOH (400 ml), the water / EtOAc flush. The aqueous layer was acidified with concentrated HCl until pH=1 and an off-white precipitate formed which was collected by filtration through a sintered funnel. After extraction of the aqueous filtrate with EtOAc (3x200 mL), LCMS showed that all the product was removed with the aqueous. The combined organic phase was washed with brine, dried over Na2SO4, filtered and the solvent removed under vacuum to give a light brown solid. All product batches were combined and purified by flash column chromatography (40 g Si in DCM, 0-10% MeOH). The product fractions were concentrated to give a light brown solid. Yield: 2.52 g, 72%. SC_ACID:266.2[M+H] + 1H NMR (400MHz, DMSO-d6) δ12.07(s, 1H), 9.78(s, 1H), 7.14(d, J=2.0Hz, 1H), 6.80(d, J=2.2Hz, 1H), 5.95(s, 2H), 2.50-2.46(m, 2H), 2.23(t, J=7.4Hz, 2H), 1.98(s, 3H), 1.84-1.70(m, 2H).

[0307] 1-6. Synthesis of compound 7 [ka]

[0308] A suspension of 4-(6-acetamidobenzo[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, turning into a dark solution over time. The reaction mixture was added dropwise to ice-cold saturated aqueous NaHCO3 solution (10 mL) and the aqueous solution was extracted with ethyl acetate (3x25 mL). The combined organic layers were dried over sat.NaHCO3 and brine with Na2SO4, filtered and the solvent removed in vacuo to give a pale pink solid. Yield: 140 mg, 100%. SC_ACID: m / z 248.2[M+H] + 1 H NMR (400MHz, DMSO-d6) δ12.34(s, 1H), 8.11(s, 1H), 6.13(s, 2H), 2.80(t, J=6.2Hz, 2H), 2.66-2.58(m, 2H), 2.12(s, 3H), 2.01-1.91(m, 2H).

[0309] 1-7. Synthesis of compound 8 [ka]

[0310] N-(6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide (50 mg) in tetrahydrofuran (1.2 mL). A suspension of 1,2-dichloromethane (1,2-dichloromethane, 0.202 mmol) 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 over celite and flushed with DCM. The filtrate was concentrated in vacuo to give a black oil. The crude product was purified using flash column chromatography (4 g Si, 0-4% MeOH in DCM). The product fractions were concentrated to give a grey solid (32 mg, 52%) in 80-90% purity. A sample of even higher purity can be obtained by purification using MPLC. SC_ACID: m / z 305.4[M+H] + 1 H NMR (400MHz, DMSO-d6) δ12.10(s, 1H), 8.21(d, J=8.0Hz, 1H), 8.12(s, 1H), 6.15(d, J=9.3Hz, 2H) ), 4.66-4.56(m, 1H), 2.93(dd, J=8.9, 4.0Hz, 2H), 2.14(s, 3H), 2.13-1.93(m, 2H), 1.91(s, 3H).

[0311] 1-8. Synthesis of compound 9 [ka]

[0312] 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 allowed to cool to 0-5 °C. Triethylamine (1.4 mL, 10.04 mmol) was added dropwise with stirring. The mixture was then 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%) in 86% purity. SC_ACID: m / z 263.0[M+H] + 1 H NMR (400MHz, DMSO) δ8.04(d, J=8.0Hz, 1H), 6.20(s, 1H), 5.94(d, J=6.0Hz, 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).

[0313] 1-9. Synthesis of compound 10 [ka]

[0314] (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 dissolved in 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 allowed to cool to room temperature. The suspension was diluted with 2 mL DCM and filtered. A black residue (210 mg) was obtained. The crude product was purified by column chromatography (12 g Si, 0-7% methanol in DCM) to yield the product (45 mg, 21%) as a brown solid. LCMS analysis showed two partial stereoisomers. SC_ACID: m / z 490.2[M+H] + 1 H NMR (400MHz, DMSO-d6) δ8.47(t, J=9.3Hz, 1H), 7.42(s, 1H), 7.24(s, 1H), 6.49(s, 1H), 6.29(d, J=5.2Hz, 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=7.1Hz, 3H).

[0315] 1-10.Synthesis of PBX-7011 and PBX-7012 [ka]

[0316] 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]ky 1.0 mL of (1-amino-1-yl)acetamide (73.5 mg, 0.150 mmol) The mixture was dissolved in 6N HCl and stirred at 90° C. for 8 h followed by overnight at room temperature. The reaction mixture was concentrated under reduced pressure and purified using two acidic preparative MPLC runs (Luna 2-30). The fractions containing the separated partial stereoisomers were acidified with 5 drops of 3N HCl and lyophilized to give the products as yellow solids. First eluting isomer: PBX-7011, 23 mg, 34% yield U_AN_ACID: m / z 448.4[M+H] + 1 H NMR (400MHz, DMSO-d6) δ8.54(s, 3H), 7.50(s, 1H), 7.27(s, 1H), 6.52(s, 1H), 6.34(d, J=13.3Hz, 2H), 5.77(d, J=19.3Hz, 1H), 5.44(s, 2 H), 5.37(d, J=19.3Hz, 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=7.3Hz, 3H). Second eluting isomer: PBX-7012, 28 mg, 41% yield U_AN_ACID: m / z 448.2[M+H] + 1 H NMR (400MHz, DMSO-d6) δ8.57(d, J=4.7Hz, 3H), 7.51(s, 1H), 7.27(s, 1H), 6.52(s, 1H), 6.34(d, J=12.2Hz, 2H), 5.76(d, J=19.4Hz, 1H), 5.44 (s, 2H), 5.37(d, J=19.4Hz, 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=7.3Hz, 3H).

[0317] Preparation Example 2: Synthesis of active camptothecin derivatives (PBX-7014 and PBX-7015) of formula 4 or formula 4-1 This example describes the synthesis of compounds PBX-7014 and PBX-7015 starting from two separated partial stereoisomers of exatecan-hybrid compounds (PBX-7011 and PBX-7012).

[0318] [ka]

[0319] 2-1: Synthesis of PBX-7014 [ka]

[0320] A stock solution of activated glycolic acid was prepared by the following procedure: Glycolic acid (17 mg, 0.224 mmol) was dissolved in 1 ml of N,N-dimethylformamide. The mixture was dissolved in 1 L of HOSu (25.7 mg, 0.223 mmol) and EDC (42.8 mg, 0.223 mmol) were added, and the reaction mixture was stirred at room temperature for 1 h. Then, 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 h. 0.05 mL of freshly prepared activated acid solution was added. Then, the reaction mixture was stirred at room temperature for another 2 h. The reaction mixture was evaporated to dryness. The crude product was purified by column chromatography (0-8% methanol in chloroform). This provided a yellow solid containing the PBX-7014 product and residual succinimide. The product was further purified by acidic preparative MPLC (Luna 5-40) to give a bright yellow solid after lyophilization of the product fractions. Yield: 20 mg, 50% U_AN_ACID: m / z 506.2[M+H] + 1 H NMR (400MHz, DMSO-d6) δ8.40(d, J=8.9Hz, 1H), 7.40(s, 1H), 7.23(s, 1H), 6.49(s, 1H), 6.28(d, J=4.6Hz, 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=7.3Hz, 3H).

[0321] 2-2: Synthesis of PBX-7015 [ka]

[0322] A stock solution of 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 h. Next, (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', To a suspension of 4':6,7]indolizino[1,2-b]quinoline-11,14-dione (25 mg, 0.056 mmol) and triethylamine (0.016 ml, 0.112 mmol) was added 0.25 mL of the activated acid solution. The mixture was stirred at room temperature overnight. 0.03 mL of freshly prepared activated acid solution was added. The reaction mixture was then stirred at room temperature for 2 hours. The reaction mixture was combined with the previous smaller batch and evaporated to dryness. The crude product was purified by column chromatography. This provided a yellow solid containing the PBX-7015 product and residual succinimide. The product was purified by acidic preparative MPLC (Luna 5-40). The product fractions were lyophilized to give a bright yellow solid. Yield: 15 mg, 38%. U_AN_ACID:506.2[M+H] + 1H NMR (400MHz, DMSO) δ8.44(d, J=9.0Hz, 1H), 7.41(s, 1H), 7.24(s, 1H), 6.48(s, 1H), 6.29(d, J=2.3Hz, 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=7.3Hz, 2H), 0.87(t, J=7.3Hz, 3H).

[0323] Preparation Example 3: Synthesis of active camptothecin derivative (PBX-7016) of formula 5 This example describes the synthesis of compound PBX-7016 starting from an exatecan-hybrid compound (PBX-7011). [ka]

[0324] A stock solution of 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 h. Then, 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. The reaction mixture was then stirred overnight at room temperature. The reaction mixture was directly purified by acid preparative MPLC (Luna 10-50) to give a bright yellow solid after lyophilization of the product fraction. Yield: 22mg, 52% U_AN_ACID: m / z 520.2[M+H] + 1 H NMR (400MHz, DMSO) δ8.43(d, J=9.1Hz, 1H), 7.41(s, 1H), 7.23(s, 1H), 6.50(s, 1H), 6.29(d, J= 2.1Hz, 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=6.8Hz, 3H), 0.87(t, J=7.3Hz, 3H).

[0325] As described above, PBX-7016 of Chemical Formula 5 can be synthesized from PBX-7011 of Chemical Formula 3, and PBX-7017 of Chemical Formula 5-1 can be synthesized from PBX-7012 of Chemical Formula 3-1 in the same manner.

[0326] Preparation Example 4: Synthesis of active camptothecin derivative (PBX-7016) of formula 9 [ka]

[0327] PBX-7024 was produced in high yield by coupling (2S)-2-cyclopropyl-2-hydroxyacetic acid to the PBX-7011 compound. (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 h. Then, (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 in N,N-dimethylformamide (2.5 mL) was added. To a solution of .ALPHA.-dione (40 mg, 0.089 mmol) and DIPEA (0.047 mL, 0.268 mmol) was added 0.6 mL of the activated acid solution. The mixture was stirred at room temperature overnight. The reaction mixture was directly purified by acidic preparative MPLC (Luna 10-50) to give a bright white solid after lyophilization of the product fraction. Yield: 32mg, 65% U_AN_ACID: m / z 520.2[M+H] + 1 H NMR (400MHz, DMSO-d6) δ8.33(d, J=8.7Hz, 1H), 7.39(s, 1H), 7.23(s, 1H), 6.48(s, 1H), 6.28(d, J=4.9Hz, 2H), 5.50(q, J=6.7Hz, 1H), 5.40(s, 3 H), 5.23-5.06(m, 2H), 3.62(d, J=6.3Hz, 1H), 3.03(q, J=6.2Hz, 2H), 2.21-2.02(m, 2H), 1.92-1.79(m, 2H), 1.18-1.08(m, 1H), 0.87(t, J=7.3H) z, 3H), 0.47-0.30(m, 4H).

[0328] Example 1: Analysis of the binding mode of camptothecin derivatives with TOP1 / DNA [Table 3]

[0329] According to the binding mode prediction results (docking scores) through molecular dynamics simulations, all camptothecin derivatives in Table 2 basically bind to DNA bases through strong π-π stacking (Figures 4 and 5), and the lactone ester forms hydrogen bonds with Arg488 of TOP1, and the ethyl and OH groups form hydrogen bonds with Asp533 of TOP1 (Figure 2), supporting the camptothecin derivatives to bind well between the TOP1 protein and DNA.

[0330] The SN-38 drug is stabilized by hydrogen bonding (H-bonding) with Glu356 and Arg364 of TOP1 (Figure 4). The exatecan drug is further stabilized by hydrogen bonding (H-bonding) with the surrounding DNA bases (Figure 4).

[0331] Although the -OCHO-(methylenedioxo) pentagonal ring of the FL118 drug is weaker than hydrogen bonds (unlike the actual cytotoxicity) and shows a worse docking score than the SN-38 and exatecan drugs, such bonds provide perfect binding between DNA bases and also aid in solvation (Figure 4).

[0332] As shown in Figure 5, the compound of formula 3 (PBX-7011) and the compound of 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 partial stereoisomers, and therefore are predicted to share the same binding pose and bind stably to TOP1. In particular, the compound of formula 3 (PBX-7011) was calculated to have a higher docking score than the SN-38 drug.

[0333] The compound of formula 3 (PBX-7011), which has chirality similar to that of the exatecan drug, showed slightly better binding than the compound of formula 3-1 (PBX-7012).

[0334] In addition, in relation to the TOP1 mutation (E418K) that conferred resistance to Trodelvy (payload: SN-38), as shown in Figure 6, the E418K mutation is distant from the drug binding site.

[0335] As shown in Figures 7 and 8, energy minimization after the E418K mutation confirmed by MD simulation that the newly introduced Lys418 changes part of the drug binding site to a relatively polar environment and also has persistent ionic bonds with the surrounding Glu356. When part of the binding site becomes polar due to the mutation, it was observed in the MD simulation that substances with polar functional groups, such as the SN-38 drug, move slightly away from the existing binding site to form ionic interactions with Lys418 and Glu356. This can weaken the interaction between lactone and Asp533 and Lys532, which is the inhibitory activity principle shared by TOP1 inhibitors of the same type.

[0336] When the SN-38 drug was docked to the mutant TOP1 structure, the SN-38 drug left the original binding site and moved to the side, but acquired new hydrogen bonds (H-bonding), etc., resulting in a good binding docking score. However, the docking score has limitations in explaining the phenomenon in which mutant TOP1 shows resistance to SN-38.

[0337] As shown in Figures 7 and 8 , analysis of the interaction of SN-38 drug with TOP1 during MD simulation confirmed that the mutations reduced the SN-38 interaction with Asp533 and Arg488 (89%) to below 10%, while the action with Asn352 and Glu356 increased from 40% to 80% levels.

[0338] After the TOP1 mutation, when docking proceeded, the SN-38 drug was transferred to the newly altered binding site and bound in a new form, showing a score similar to the existing one (Table 2).

[0339] On the other hand, as shown in Table 2, it was confirmed that the exatecan drug, FL118 drug, compound of formula 3 (PBX-7011), and compound of formula 3-1 (PBX-7012), which do not have a polar functional group (-OH) at the 10th carbon of the A-ring, had poor relative docking scores with mutant TOP1 due to changes in the partial environment of the new binding site. Nevertheless, MD simulation confirmed that the exatecan drug, FL118 drug, compound of formula 3 (PBX-7011), and compound of formula 3-1 (PBX-7012) maintained interactions between the existing lactone and Asp533 and Lys532.

[0340] As shown in FIG. 9, even after the TOP1 mutation, the compound of formula 3 (PBX-7011) still maintains the existing binding mode (especially the lactone and -OH groups).

[0341] In conclusion, the E418K mutation does not directly interact with TOP1 inhibitors, but to explain the situation in which the FL118 drug exerts a high level of inhibition on TOP1 mutations that are resistant to the SN-38 drug due to E418K, we can consider the hypothesis that the new binding of SN-38 to the binding site altered by the mutation proceeds to a form that is different from the docking result and that weakens the effects of lactone and Asp533 and Lys532, resulting in a bond that does not inhibit the action of TOP1.

[0342] Under this assumption, the drugs exatecan, FL118, the compound of formula 3 (PBX-7011) and the compound of formula 3-1 (PBX-7012) are expected to have a persistent effect on Asp533 and Lys532 of mutant TOP1 and to show some resistance.

[0343] Example 2 FL118, exatecan, SN-38, Dxd, exatecan-lactate, PBX-7011, PBX-7014, PBX-7016, and PBX-7024 were subjected to anti-apoptotic protein expression inhibition assays and in vitro cell viability assays as described below.

[0344] 2-1: Confirmation of the suppressive activity of DDX5, survivin, Mcl-1, XIAP, and cIAP2 on the expression of cancer-associated survival genes by Western blot

[0345] (1) Protein extraction 200,000 FaDu cell lines were seeded per well in a 6-well plate and incubated at 37℃, 5% CO2. After 24 hours, the wells were treated with drugs (FL118 drug, SN-38 drug, Exatecan drug, PBX-7011, PBX-7014, PBX-7016) at concentrations of 0nM, 10nM, and 100nM. The plates were incubated at 37℃, 5% CO2 for 24 hours. The wells were treated with 100ul of RIPA buffer containing a protease inhibitor cocktail. The plate was placed on ice and incubated on an orbital shaker for 2 hours. The RIPA buffer containing the lysed cells was transferred to an EP tube and centrifuged (16000rcf, 20min, 4℃). The supernatant was then transferred to a new EP tube. The protein concentration was confirmed through protein measurement.

[0346] (2) Protein separation through electrophoresis Protein samples were mixed with 4x SDS-PAGE loading buffer in a 3:1 ratio, boiled at 95°C for 10 min, and cooled. Samples were loaded into the gel wells so that the protein amounts were the same. Gel electrophoresis was performed at 60V.

[0347] (3) Transferring proteins from the gel to a membrane Trans-Blot® Turbo TM Seven activated filter papers, a PVDF membrane, a gel, and seven more filter papers were placed in the cassette of the transfer system in that order, the lid was closed, and the cassette was inserted into the machine, after which the protocol was run.

[0348] (4) Antibody incubation The transferred membrane was immersed in blocking buffer and incubated (RT, 1h), then incubated (4℃, overnight) in primary antibody solution, washed with TBST buffer for 3 min (repeated 3 times), incubated (RT, 1h) in HRP-conjugated secondary antibody solution, washed with TBST buffer for 3 min (repeated 3 times).

[0349] (5) Imaging and analysis of results The membrane was immersed in the ECL substrate for 3 to 5 minutes, and then the ChemiDoc TM The signal was confirmed using the MP imaging system. The HRP bound to the secondary antibody oxidized the luminol in the ECL, and the light emitted was detected and displayed in an image. The thickness of the band is proportional to the amount of protein, so the amount of protein can be compared through the thickness of the band.

[0350] As shown in Figures 10 and 12, the drug (FL118 drug, SN-38 drug, exatecan drug, PBX-7011, PBX-7014, PBX-7016) treatment We confirmed how the expression level of the protein changed. GAPDH is an enzyme involved in glycolysis, an essential metabolic process in cells. It is a gene that is constantly expressed in cells and its expression level does not change much, so it is an indicator of whether the same amount of protein was loaded onto the gel in each sample.

[0351] Using the same method, A549 cells overexpressing ABCG2 were seeded instead of the FaDu cell line that does not express ABCG2, 4 μg of protein was loaded, and Western blotting was performed (FIGS. 13 and 14).

[0352] Discussion: PBX-7011, PBX-7014 and PBX-7016 Evaluation The degree of inhibition of anti-apoptotic protein expression in the FaDu cell line was confirmed by Western blot. As a result, it was shown that PBX-7011, PBX-7014 and PBX-7016 all reduced the expression of survivin, cIAP2, Mcl-1 and XIAP with increasing concentration. When compared with the existing drugs FL118, SN-38 and exatecan, it was shown to be at a similar level to FL118 and exatecan, but even better than SN-38.

[0353] In Example 2-1, it was confirmed that the compound exhibits a dual inhibitory effect of degrading DDX5 while suppressing type I topoisomerase (topoisomerase 1), and thus has a unique characteristic of degrading DDX5 and an anticancer effect due to the same.

[0354] Specifically, two types of cell lines (FaDu, A549) were treated with drugs at fixed concentrations (0, 10, 100 nM), and the expression levels of each protein (DDX, survivin, Mcl-1, XIAP) were confirmed by Western blotting. The results showed that FL118 was superior in degrading DDX5 compared to other substances, followed by 7011, 7016, and 7014.

[0355] Similar trends were observed not only for DDX5 but also for other downstream anti-apoptotic proteins.

[0356] In summary, PBX-7014 and PBX-7016 display dual MoA that not only inhibit type I topoisomerases but also act as degraders of DDX5 (p68), an oncoprotein that regulates survivin, Mcl-1, XIAP, etc.

[0357] As a result of the evaluation, it was observed that, among PBX-7014 and PBX-7016, PBX-7016 inhibited DDX5 in a concentration-dependent manner, and as a result, inhibited survivin, Mcl-1, and XIAP. This not only demonstrated the role of PBX-7016 as a topoisomerase I inhibitor, but also as a DDX5 degrader of FL118, and confirmed that PBX-7016 worked better than PBX-7014 (Figures 11 to 14).

[0358] 2-2: Cell viability assay for FaDu cell line / A549 cell line 3000 FaDu cell lines (cancer cells that do not express ABCG2) or A549 cell lines (cancer cells that overexpress ABCG2) were seeded per well in a 96-well plate and incubated at constant temperature (37°C, 5% CO2). After 24 hours, the cells were treated with 100 ul of drugs at nine concentrations (serial dilutions of 1 / 5 from 1000 nM). Dxd drugs, PBX-7014, PBX-7016, and PBX-7024 were treated. A control group (drug concentration 0) was also prepared without drug treatment. The cells were incubated at constant temperature (37°C, 5% CO2) for 3 or 6 days. 100 ul of CellTiter-Glo reagent (CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay Kit (Promega, G7571) was used) was added to the wells and then pipetted. After 10 minutes of incubation at room temperature, luminescence was measured. The luminescence value when the drug concentration was 0 was set as 100%. The concentration showing 50% of the luminescence value is the IC50 value.

[0359] Two types of cell lines (FaDu, A549) were treated with two types of compounds (PBX-7014, PBX-7016, PBX-7024) and reference compounds (Dxd, SN-38, exatecan, FL118) and cell survival was observed throughout three days of incubation.

[0360] As shown in Figure 15, PBX-7024 exhibits strong cell killing efficacy not only in the FaDu cell line that does not express ABCG2, but also in the A549 cancer cell line that overexpresses ABCG2. In other words, PBX-7024 has an IC at a level equal to or higher than that of Dxd, the payload used in existing enhancers. 50 It was confirmed that the

[0361] In A549, a cancer cell line that overexpresses ABCG2, camptothecin compounds containing Dxd showed high IC 50 It can be seen that, whereas PBX-7016 and PBX-7024 still maintain strong efficacy.

[0362] In other words, when the new camptothecin compounds PBX-7016 and PBX-7024 according to the present invention are used, they have the effect of overcoming the resistance mechanism caused by overexpression of ABCG2, unlike ADCs using existing camptothecin compounds such as SN-38 and DXd.

[0363] Example 3. New PBX series compounds as potential ADC payloads: In vitro cell viability studies A cell viability assay was performed on the MDA-MB-453 (HER2++) cell line and the FaDu (HER2+) cell line in the same manner as in Example 2-2.

[0364] 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 observed cell survival through 3-day incubation. The results are shown in Figure 16.

[0365] As shown in Figure 16, the evaluation results showed that PBX-7016, PBX-7018, PBX-7020, and PBX-7022 were ICs with the same or higher level than Dxd. 50In addition, we confirmed that it has IC50 levels equal to or higher than Dxd in the FaDu cell line, which does not express ABCG2. 50 It was confirmed that it had a value.

[0366] Example 4. PBX-7016 IV and IP Pharmacokinetic Profile in OD SCID Mice The experimental method is as shown in Table 3 below. PBX-7016 IV plasma concentration-time data is shown in Table 4, and PBX-7016 IP plasma concentration-time data is shown in Table 5.

[0367] [Table 4]

[0368] [Table 5]

[0369] [Table 6]

[0370] From the results of Tables 4 and 5, it was confirmed that the PBX-7016 compound was cleared in the blood very quickly when administered intravenously or intraperitoneally to mice, and it is expected that the PBX-7016 compound alone, not in the form of a prodrug or ADC, will show high safety when administered in vivo. Since the PBX-7016 compound is released from the body in a very short time when administered IV, it can act as a safety device that can reduce systemic exposure to the drug even when the drug is released prematurely after entering the body.

[0371] This experiment also suggests that the PK of the PBX-7016 compound used as the payload alone is a safe payload, with clearance of the PBX-7016 drug occurring in the blood within a short period of time. In other words, after exerting its bystander killing effect at the tumor site, the PBX-7016 compound is safely released from the body within a very short period of time even when circulating in the blood.

[0372] 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 lacto portion of camptothecin, a known activator of Top1 inhibitors.

[0373] 5-1. Conversion of carboxylate to lactone at pH 6.0 PBX-7016 powder was prepared by dissolving in DMSO at 5 mM. PBX-7016 dissolved in DMSO exists in lactone form, so it was diluted 1 / 10 with 0.1N NaOH and incubated in a 25℃ water bath for 30 minutes to convert it to the carboxylate form. The carboxylate form of PBX-7016 was diluted to 3μM with 4℃ PBS (pH 6.0) containing 10% DMSO. In this case, an equivalent amount of HCl to the 0.1N NaOH used was added to minimize pH changes. After dilution, it was centrifuged at 4℃ for 2 minutes at 13,000RCF, and the supernatant was used as the sample.

[0374] When the PBX-7016 lactone form was produced, two solvents were used to separate it by RP-HPLC: Solvent A: 100 mM acetate buffer pH 5.5 and Solvent B: ACTH. N was used. 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 min: 5% B, 2) sample injection, 3) 2 min: 5%B, 4) 10 min: 5%B → 50%B, 5) 1 min: 50%B.

[0375] To measure the change over time, the sample chamber was maintained at 37°C and samples were injected at each measurement time, which were 0, 20, 40, 60, 90, 120, 180, 240, 300, 360, 480, 600, 720, 840, 960, 1200, and 1440 minutes.

[0376] The lactone and carboxylate contents were calculated from their peak areas. Since the extinction coefficients of lactone and carboxylate are different, the lactone area was calculated by multiplying the carboxylate area by the following formula: Lactone EC 390 / Carboxylate EC 390 =0.918

[0377] The experimental results of the conversion from carboxylate to lactone form at pH 6.0 are shown in Figure 17. As shown in Figure 17, various chemotactic compounds were confirmed to convert from carboxylate to active lactone form that inhibits Topoisomerase I at similar rates and in similar amounts at pH 6.0, and thus are expected to exhibit similar levels of activity in the tumor microenvironment, which generally has a pH of 6.0.

[0378] 5-2.Conversion of lactone to carboxylate at pH 7.4 PBX-7016 powder was dissolved in DMSO at 5 mM, and diluted with 10% DMSO in 4°C PBS (pH 7.4) to prepare 3 μM PBX-7016. This was then centrifuged at 4°C for 2 minutes at 13,000 RCF, and the supernatant was used as a sample. PBX-7016 dissolved in DMSO existed in the lactone form.

[0379] When the PBX-7016 carboxylate form was produced, two solvents were used to separate it by RP-HPLC: Solvent A: 100 mM acetate buffer pH 5.5 and Solvent B: ACN. The column used was a Cortecs C18 2.7 μm, 4.6x50 mm column, and the analysis proceeded under the following conditions: 1) 20 min: 5% B, 2) sample injection, 3) 2 min: 5% B, 4) 10 min: 5% B → 50% B, 5) 1 min: 50% B.

[0380] To measure the change over time, the sample chamber was maintained at 37° C., and the sample was injected at each measurement time, which were 0, 20, 40, 60, 90, 120, 180, 240, 300, 360, 480, 600, and 720 minutes.

[0381] The lactone and carboxylate contents were calculated from their peak areas. Since the extinction coefficients of lactone and carboxylate are different, the lactone area was calculated by multiplying the carboxylate area by the following formula: Lactone EC 390 / Carboxylate EC 390 =0.918

[0382] The results of the lactone to carboxylate conversion experiment at pH 7.4 are shown in Figure 18. From the results of Figure 18, it was confirmed that the PBX-7016 compound is converted from lactone to carboxylate at a higher rate and faster than other reference compounds (exatecan, DXd, SN-38, FL118) at pH 7.4, which is the pH in blood. From this, it is predicted that the PBX-7016 compound will be present in the blood as an inactive carboxylate rather than as an active lactone, which shows cytotoxicity, relatively quickly and at a higher rate than the reference compounds, and will therefore be highly safe in normal cells.

[0383] Preparation Example 5: Synthesis of 25-4 and 25-6 from PBX-7014 and PBX-7016 and preparation of their ADCs (DAR7-8) (Trastuzumab-25-4 and Trastuzumab Zumab-25-6) Linker-payloads 25-4 (Chemical Formula 10) and 25-6 (Chemical Formula 11) containing two compounds (PBX-7014 and PBX-7016) and incorporating the enzymatically cleavable linker system GGFG were synthesized.

[0384] [ka]

[0385] [ka]

[0386] The molecular structure of 25-4 is GGFG-PBX-7014, and the molecular structure of 25-6 is GGFG-PBX-7016. That is, each uses the same GGFG linker as Enhertu (registered trademark).

[0387] Furthermore, the same GGFG linker and trastuzumab antibody as Enhertu® were used to produce ADCs with PBX-7014 and PBX-7016 as payloads.

[0388] The linker-payload compounds of formula 10 and formula 11 were conjugated with the Her2-targeting antibody trastuzumab to synthesize ADCs (Tra-25-4 and Tra-25-6, all DAR 8).

[0389] Trastuzumab-25-4 (Trastuzumab-7014) was produced as follows: The prepared trastuzumab was buffer-exchanged into a reaction buffer (150 mM NaCl, 50 mM histidine pH 6.0) using a PD-10 desalting column, and then 825 uM TCEP was treated with 27.5 uM of antibody at 25°C for 2 hours to create thiol sites necessary for the reaction from the disulfides of the antibody.

[0390] After that, excess TCEP was removed using a PD-10 desalting column, and the mixture was diluted with 61.9 μM 25-4 drug linker (Formula 10) and 13.8 μM 25-4 drug linker (Formula 11) in a reaction buffer containing 10% DMSO. The reduced trastuzumab was reacted at 25° C. for 1 hour to allow the primary conjugation reaction to proceed.

[0391] Trastuzumab-25-6 (trastuzumab-7016) was prepared by the same process, but using the same concentration of 25-6 drug linker (Formula 11) instead of 61.9 uM 25-4 drug linker (Formula 10).

[0392] Each ADC was then purified using SEC to confirm that it was purified as a monomer without aggregation (Figure 19). SDS-PAGE confirmed that the drugs were conjugated based on the band shift of the light and heavy chains (Figure 20).

[0393] Example 6. Anti-HER2 ADCs with PBX-payload: Binding affinity analysis Coating buffer was prepared by diluting the target antigen to 0.66μg / ml with ELISA plate coating buffer (R&D systems, #DY006). 100μL of the prepared coating buffer was used per well for coating. The plate cover was closed and the plate was allowed to proceed overnight (12-18 hours) while maintaining the temperature at 2-8℃. The plate used was Corning, #3590. 200μL or more of TBS was added. The plate was washed three times with Tween-20 buffer (thermo, #28360). The liquid was removed by pipetting and tapping on a paper towel to remove excess buffer. Blocking was performed with 200 μL of assay buffer for 1 hour at room temperature. The liquid was removed by pipetting and tapping on a paper towel to remove excess buffer. The control antibodies and ADCs to be measured were prepared at 1 μg / ml in assay buffer, and then the required amount was prepared at the following concentrations through 1 / 5 serial dilutions. For example, samples can be prepared by diluting the following concentrations with the replicate standard [240 ul assay buffer + 60 ul of sample at 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. Binding was performed with 100 μL of diluted control antibody and ADC in duplicate per coated well. The wells were incubated at room temperature for 1 hour. The wells were washed three times with 100 μl of TBS Tween-20 buffer (thermo, #28360). The liquid was removed with a pipette and then tapped lightly on a paper towel to remove excess buffer. The detection antibody was prepared by diluting it in the assay buffer according to the manufacturer's manual. For human antibodies, goat anti-human Fc-HRP (Novex, #A18817) was used as the detection antibody and diluted 1 / 2000. 100 μL of the diluted detection antibody was added to each well and incubated at room temperature for 1 hour. The wells were washed three times with 100 μl of TBS Tween-20 buffer (thermo, #28360). The liquid was removed with a pipette and then tapped lightly on a paper towel to remove excess buffer. The reaction was carried out at room temperature for 30 minutes using 100 μL of TMB substrate solution (Thermo, #N301). The reaction was stopped using 100 μL of stop solution (Thermo, N600). The reaction was stopped and absorbance was measured at 450 nm. After adding the stop solution, the reaction was completed within 30 minutes. When bubbles were present, 100-150 ul was transferred to a new plate and measured. Through this, trastuzumab, ADC conjugated with trastuzumab and PBX-7014, and ADC conjugated with trastuzumab and PBX-7016 were measured, and the results in FIG. 21 were obtained. As shown in FIG. 21, when the binding strength of ADC conjugated with trastuzumab and PBX-7014 or PBX-7016 to Her2 antigen was measured at various concentrations compared to the binding strength of trastuzumab to Her2 antigen, it was confirmed that there was no difference in binding strength.

[0394] Example 7. Anti-HER2 ADCs with PBX-payload: In vitro potency and target selectivity For the ADCs having Dxd, PBX-7014, and PBX-7016 as their payloads, in vitro cell viability assays were performed on the MDA-MB-453 (HER2++) cell line, FaDu (HER2+) cell line, and MDA-MB-468 (HER2-) cell line in the same manner as in Example 2-2, as described below.

[0395] In this case, the HER2 targeted therapeutic agent Herceptin (ingredient name: trastuzumab), Dxd drug, PBX-7014 (Production Example 2), PBX-7016 (Production Example 3), and its ADC, Tra-25-4, Tra-25-6 (Production Example 5), and Tra-Dxd (Enhertu (registered trademark)) were treated.

[0396] As a reference, Enhertz (Tra-Dxd, DAR 8) was treated with three cell lines based on the Her2 expression level, MDA-MB-453 (Her2++), FaDu (Her2+), and MDA-MB-468 (Her2-), and cell survival was observed through incubation for three days (Figure 22).

[0397] As shown in Figure 22, it was confirmed that the anti-Her2 ADC with a PBX-payload has excellent target selectivity.

[0398] Discussion: Evaluating the PBX-7014 and PBX-7016 as ADC payloads As shown in Figure 22, in the Her2++ MDA-MB-453, the ADC (Tra-25-6) with the PBX-7016 as the payload is about 5 times better than the Enhanced (Tra-Dxd) IC. 50 The negative cell line, MDA-MB-468, showed a similar IC value. 50 By confirming that the linker-payload systems of formulas 10 and 11 have the value, when developed into ADCs, they not only work to distinguish between positive and negative cell lines, but also have the same effect as the reference, Enhertz. It was confirmed that the compound showed excellent efficacy.

[0399] Discussion: Originality and excellence of PBX-7016 PBX-7016, a camptothecin-based compound newly designed and synthesized according to the present invention and represented by Chemical Formula 1, is a compound newly derived from FL118, which is an MD-CPT structure. PBX-7016 is a new topoisomerase I inhibitor that degrades DDX5, similar to the way FL118 acts not only as a topoisomerase I inhibitor but also as a DDX5 degrader (Figures 11 to 14).

[0400] In addition, when comparing cell survival simply, PBX-7016 had an IC of 1.5 to 2 times higher than Dxd. 50 Surprisingly, when used as an ADC payload, the ADC containing PBX-7016 had approximately 5-fold better cell survival than the ADC containing Dxd (Enhertz) (Figure 22).

[0401] 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 at 37°C with 5% CO2 and routinely subcultured twice a week by trypsin-EDTA treatment. Cells growing in exponential phase were harvested and counted for tumor inoculation.

[0402] 8-2.BALB / c nude mice SCID mice and BALB / c nude mice are both used as animal models to study the immune system and test treatments for a variety of diseases. Although SCID mice and BALB / c nude mice all have impaired immune systems, the underlying mechanisms of immune defects are different. SCID mice are mice that have been genetically engineered to lack functional T and B cells and therefore have severely impaired immune systems. They are used to study immune system development and function and to test potential treatments for diseases that affect the immune system, such as autoimmune disorders and certain types of cancer. BALB / c nude mice, on the other hand, lack a thymus gland and are unable to produce T cells. They are also born without hair, due to a defective hairless gene. These mice are used to study the role of T cells in the immune response and to test potential treatments for diseases that affect T cell function, such as certain types of cancer.

[0403] 8-3. Tumor inoculation and drug treatment JIMT-1 tumor cells (5x10) in 0.2 mL of DPBS with Matrigel 6 ) was inoculated subcutaneously into the right anterior flank of each BALB / c nude mouse. The average tumor volume was approximately 215 mm 3 Upon reaching this age, animals were randomized into groups and treatment for the drug efficacy study was initiated as described below. Treatment: IV, Q1W Vehicle group: Vehicle, 0 mg / kg, 5 μL / g, iv, twice (day 0, day 6 ) Isotype-Dxd (3) group: CTP63-Dxd, 3 mg / kg, 5 μL / g, iv, twice (days 0 and 6) Isotype-Dxd(10) group: CTP63-Dxd, 10 mg / kg, 5 μL / g, iv, twice (days 0 and 6) Isotype-PBX7016 (3) group; CTP63-PBX7016, 3 mg / kg, 5 μL / g, iv, twice (days 0 and 6) Isotype-PBX7016(10) group: CTP63-PBX7016, 10 mg / kg, 5 μL / g, iv, twice (days 0 and 6) Isotype-PBX7014 (3) group: CTP63-PBX7014, 3 mg / kg, 5 μL / g, iv, twice (days 0 and 6) Isotype-PBX7014(10) group: CTP63-PBX7014, 10 mg / kg, 5 μL / g, iv, twice (days 0 and 6) Trastuzumab-PBX7014 (3) group: CTP6-PBX7014, 3 mg / kg, 5 μL / g, iv, twice (days 0 and 6) Trastuzumab-PBX7014 (10) group: CTP6-PBX7014, 10 mg / kg, 5 μL / g, iv, twice (days 0 and 6) Trastuzumab-PBX7016 (3) group: CTP6-PBX7016, 3 mg / kg, 5 μL / g, iv, twice (days 0 and 6) Trastuzumab-PBX7016 (10) group: CTP6-PBX7016, 10 mg / kg, 5 μL / g, iv, twice (days 0 and 6) Enhertz (3) group: Enhertz, 3 mg / kg, 5 μL / g, iv, twice (day 0 Day 6) Enhertz (10) group: Enhertz, 10 mg / kg, 5 μL / g, iv, twice ( Day 0, Day 6)

[0404] 8-4. Tumor measurements and endpoints The primary endpoint was the time point at which tumor growth could be delayed or mice could be treated. The bidimensional size of the tumor was measured twice weekly using a caliper, and the volume was calculated using the formula (V=0.5×b 2 , where a and b are the long and short diameters of the tumor, respectively) in mm 3 It was displayed as.

[0405] Tumor growth inhibition (TGI) was calculated for each group. TGI(%)=[1-(T i -T0) / (V i -V0)] x 100 (T i is the mean tumor volume of the treatment group on a particular day, T0 is the mean tumor volume of the treatment group on the first day of treatment, V i is T i and the mean tumor volume of the vehicle control group on the same day, V0 is the mean tumor volume of the vehicle group on the first day of treatment).

[0406] Body weight loss ≥15% or tumor volume ≥3000mm 3 Any animals above this age were euthanized at a humane endpoint.

[0407] 8-5.Statistical analysis Results are expressed as mean and standard error (mean ± SEM). Data were analyzed using two-way RM ANOVA Dunnett's multiple comparison test using Graphpad Prism 6.0 software, and p < 0.05 was considered statistically significant.

[0408] Table 6 below shows the tumor volume (mm 3 ) over time (effect vs. time). [Table 7]

[0409] Figure 23, which is a graph of Table 6, shows the results of transplantation of JIMT-1 cell line in mice, i.e., Efficacy testing results for ADCs with various camptothecin-based drug payloads in an in vivo xenograft model are presented.

[0410] 8-6. Body weight of JIMT-1 tumor model The body weight of the JIMT-1 tumor model mice was monitored periodically (Figure 24).

[0411] Example 9: Anti-HER2 ADC with PBX-payload: Surrounding cell killing effect study The bystander effect is one of the factors that significantly affect the efficacy of ADC drugs. To confirm the bystander effect, FACS analysis was performed under 40 nM ADC treatment conditions.

[0412] In an antibody-drug conjugate (ADC) in which a payload-linker is attached to a trastuzumab antibody targeting a HER2 protein, the lead compound PBX-7016 of the present invention was used as a payload to verify the efficacy and usefulness of the ADC drug. In this example, the bystander effect was confirmed in vitro through a flow cytometric analysis experiment. ADC treatment was performed on a HER2-positive cell line, MDA-MB-453, which induced cell death, and the drug (payload) released during the cell death process induced toxicity in the surrounding HER2-negative cell line, resulting in cell death. This was defined as the bystander effect in this example.

[0413] In Preparation Example 5, in order to evaluate the in vitro surrounding cell killing effect of the prepared ADC, HER2-expressing negative cell lines and HER2-expressing positive cell lines were mixed and co-cultured in a 6-well cell culture plate at different ratios, and the presence or absence of a bystander effect due to ADC treatment was evaluated.

[0414] Specifically, GFP-MDA-MB-468 cells (3x10 5 cells) and HER2-positive cell line MDA-MB-453 cells (1x10 5 cells) were mixed at a ratio of 3:1 and co-cultured for 24 hours, after which trastuzumab antibody and all five ADC samples (trastuzumab-DXd, isotype IgG-DXd, trastuzumab-25-6, isotype IgG-25-6, Enhertz) were treated at a concentration of 40nM each, and the cells were cultured for 6 days. After that, GFP fluorescence measurement and cell count of GFP-MDA-MB-468 cells, a HER2-negative cell line, among the live cells was confirmed through flow cytometry analysis.

[0415] At the same time, MDA-MB-468 cells (3x10 5 cells) and HER2-positive cell line MDA-MB-453 cells (1x10 5 The HER2-positive cells were mixed with trastuzumab antibody and 5 types of ADC samples (trastuzumab-DXd, isotype IgG-DXd, trastuzumab-25-6, isotype IgG-25-6, Enhertz) at a ratio of 3:1 and co-cultured for 24 hours, then treated with trastuzumab antibody and all 5 types of ADC samples (trastuzumab-DXd, isotype IgG-DXd, trastuzumab-25-6, isotype IgG-25-6, Enhertz) at a concentration of 40nM each and cultured for 6 days. After that, the cells were stained using anti-HER2-FITC antibody, which is an antibody that can recognize HER2 protein and is labeled with FITC (Fluorescein isothiocyanate) fluorescent dye to show fluorescence, and the FITC fluorescence measurement and cell number of MDA-MB-453 cells, a HER2-positive cell line among living cells, were confirmed through flow cytometry analysis.

[0416] Flow cytometry analysis was performed by harvesting cells present in each well of a 6-well cell culture plate, diluting them with buffer (400 ul), and then scanning each sample using the FL1 laser. GFP or FITC fluorescence was measured at the same time (1 min each) and at the same flow rate.

[0417] As shown in Figures 25 and 26, flow cytometry analysis revealed that the PBX-7016-based ADC trastuzumab-25-6 not only killed HER2-positive cells, but also simultaneously killed HER2-negative cells, when compared with the DXd-based ADCs trastuzumab-Dxd and Enhertz, demonstrating a bystander effect equivalent to that of the existing DXd-based Enhertz. In contrast, it was confirmed that the negative control ADCs, isotype IgG-DXd and isotype IgG-25-6, did not significantly induce HER2-negative cell death compared to conditions where no ADC was treated.

[0418] In summary, the anti-HER2 ADC with PBX-7016 demonstrated a bystander effect similar to that of Enhertu.

Claims

1. (a) an activated camptothecin derivative represented by the following chemical formula 1, designed to bind to DDX5 protein and E3 ligase; or (b) a prodrug thereof designed to release the activated camptothecin derivative (a) from a target site in the body; or (c) a ligand-containing complex targeting DDX5 protein, in which the type I topoisomerase inhibitory ability of the activated camptothecin derivative (a) or the FL118 compound of chemical formula 2 has been inactivated via a linker bond. 【Chemistry 1】 X １ and X ３ are each independently carbon, oxygen, nitrogen, or sulfur; X １ and X ３ may be the same or different, X ２ is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X １ , (X ２ ) n and X ３ can form a 6- or 7-membered ring (n=values ​​of 1 to 2); Y １ , Y ２ and Y ３ may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus, or sulfur.

2. The camptothecin derivative (a) or its prodrug (b) according to claim 1, characterized in that the active camptothecin derivative (a) designed to bind to DDX5 protein and E3 ligase can act as a molecular glue degrader and a ligand that binds to E3 ligase.

3. The camptothecin derivative (a) or its prodrug (b) according to claim 1, characterized in that the active camptothecin derivative (a) designed to bind to DDX5 protein and E3 ligase kills target cells expressing DDX5 protein through a molecular glue degrader mechanism (MoA).

4. The active camptothecin derivative (a), designed to bind to DDX5 protein and E3 ligase, is characterized in that it has a mechanism of action (MoA) that degrades the oncoprotein DDX5, along with type I topoisomerase inhibitory activity. Item 2. A camptothecin derivative (a) or a prodrug thereof (b) according to Item 1.

5. The camptothecin derivative (a) or its prodrug (b) according to claim 4, characterized in that (1) in general formula 1 or chemical formula 2, the group C site optionally binds to type I topoisomerase and the group A site binds to DNA, stabilizing the covalent bond of the topoisomerase-DNA complex and preventing / preventing further ligation of the cleaved DNA fragments, or (2) in general formula 1 or chemical formula 2, the group A site binds to DDX5 and the group C site optionally binds to E3 ligase and induces DDX5 degradation. 【Chemistry 2】 【Chemistry 3】

6. A pharmaceutical composition for preventing or treating cancer or a composition for diagnosing cancer, comprising an active camptothecin derivative (a) described in any one of claims 1 to 5, a prodrug thereof (b), or a ligand-containing complex (c) that targets DDX5 protein.

7. After cancer diagnosis, it can be used as a first-line therapeutic agent or to (over)express DDX5 in cancer tissue. The pharmaceutical composition according to claim 6, characterized in that it is administered to an individual.

8. The pharmaceutical composition according to claim 6, which is administered to treat HER2-positive cancer, breast cancer, lung cancer, or colon cancer.

9. The pharmaceutical composition or cancer diagnostic composition according to claim 6, characterized in that the DDX5 protein is used as a biomarker for predicting drug sensitivity or tumor sensitivity.

10. The pharmaceutical composition according to claim 6, characterized in that DDX5 protein is a drug target for the active camptothecin derivative, thereby making it possible to avoid drug resistance, unresponsiveness to targeted therapy and / or resistance during treatment.

11. 7. The pharmaceutical composition according to claim 6, wherein the active camptothecin derivative blocks (turns off) the transcriptional induction of anti-apoptotic genes.

12. 7. The pharmaceutical composition according to claim 6, wherein the active camptothecin derivative degrades the transcription cofactor DDX5 protein, thereby maintaining or improving the sensitivity of cancer cells to chemotherapy or radiation therapy.

13. The pharmaceutical composition according to claim 6, characterized in that it transforms / transforms a solid tumor with high cell density and agglomeration into a diffuse tumor with low cell density, or transforms a cold tumor with low immune activity into a hot tumor with high immune activity.

14. The cancer diagnostic composition according to claim 6, which contains a ligand-containing complex (c) targeting DDX5 protein, and which is characterized in that it can be used to determine a patient group to which an active camptothecin derivative (a) or a prodrug thereof (b) is to be administered, or can predict drug response, recurrence rate and / or prognosis during chemotherapy and anticancer drug treatment.

15. A method for producing a ligand-containing complex targeting DDX5 protein, comprising: (a) an activated camptothecin derivative designed to bind to DDX5 protein and E3 ligase; (b) a prodrug thereof designed to release the activated camptothecin derivative (a) from a target site in vivo; or (c) the type I topoisomerase inhibitory ability of the activated camptothecin derivative (a) or the FL118 compound of formula 2 is inactivated via a linker linkage, The method for producing an active camptothecin derivative (a) comprises a step of designing the active camptothecin derivative (a) to contain a camptothecin-based skeleton represented by the following chemical formula 1 as a parent nucleus, selecting a compound designed in this manner, or synthesizing a compound designed in this manner. 【Chemistry 4】 X １ and X ３ are each independently carbon, oxygen, nitrogen, or sulfur; X １ and X ３ may be the same or different, X ２ is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X １ , (X ２ ) n and X ３ can form a 6- or 7-membered ring (n=values ​​of 1 to 2); Y １ , Y ２ and Y ３ may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus, or sulfur.

16. A method for producing an active camptothecin derivative, its prodrug or a ligand-containing complex targeting DDX5 protein as described in claim 15, characterized in that the active camptothecin derivative (a) designed to bind to DDX5 protein and E3 ligase has a mechanism of action (MoA) that degrades the oncoprotein DDX5 along with type I topoisomerase inhibitory ability.

17. The step of designing an active camptothecin derivative (a) containing a camptothecin-based skeleton represented by Chemical Formula 1 as a parent core or selecting a compound designed in this manner includes the steps of: (1) Selecting / selecting an active camptothecin derivative designed to have a mechanism of action (MoA) of inhibiting type I topoisomerase and / or a mechanism of action (MoA) of degrading oncoprotein DDX5 from a compound library containing a camptothecin-based skeleton represented by Chemical Formula 1 as a parent core, or (2) A method for producing an active camptothecin derivative, its prodrug or a ligand-containing complex targeting DDX5 protein as described in claim 15, characterized in that it is confirmed through in vitro and / or in vivo experiments whether a compound containing a camptothecin-based skeleton represented by chemical formula 1 as a parent nucleus inhibits type I topoisomerase and / or decomposes oncoprotein DDX5.

18. The active camptothecin derivative is X １ , (X ２ ) n, X ３ , Y １ , Y ２ and / or Y ３ The method for producing an active camptothecin derivative or a prodrug thereof according to claim 15, characterized in that the cell membrane permeability is adjusted as desired through deformation, thereby exerting a bystander effect in cancer tissue or controlling the degree of the effect.

19. The method for producing an active camptothecin derivative or a prodrug thereof according to claim 15, characterized in that the active camptothecin derivative (a) or a prodrug thereof (b) is intended for administration to a patient group in whose cells DDX5 acts as an oncoprotein.

20. The method for producing a camptothecin derivative, its prodrug or a ligand-containing complex targeting DDX5 protein according to claim 15, further comprising a step of quantitatively or qualitatively analyzing intracellular DDX5 protein in a tissue or liquid biopsy using the FL118 compound of Chemical Formula 2 or a ligand-containing complex targeting DDX5 protein (c) in which the type I topoisomerase inhibitory ability of the active camptothecin derivative (a) is inactivated via a linker linkage, thereby identifying a patient group to which the active camptothecin derivative (a) or its prodrug (b) is to be administered.

21. A method for producing an active camptothecin derivative that binds to DDX5, which acts as an oncoprotein in cells, and induces cell death through degradation of DDX5 protein, (b) a prodrug thereof designed to release the active camptothecin derivative (a) in vivo, or (c) the active camptothecin derivative (a) in which the type I topoisomerase inhibitory ability is inactivated via a linker linkage, the method comprising the steps of: a first step of selecting the active camptothecin derivative (a) from the group consisting of compounds of the following formulas: 3, 4, 5, 6, 7, 8, and 9, and isomers thereof; and Optionally, the method comprises a second step of selecting a prodrug (b) of the active camptothecin derivative (a) selected in the first step, the prodrug (b) being designed to release the active camptothecin derivative (a) in vivo. 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】

22. The method according to claim 21, characterized in that the active camptothecin derivative (a) or its prodrug (b) is for administration to a patient group in whose cells DDX5 acts as an oncoprotein.

23. The method of claim 21, further comprising a step of identifying a patient group to which the active camptothecin derivative (a) or its prodrug (b) is to be administered by quantitatively or qualitatively analyzing intracellular DDX5 protein in a tissue or liquid biopsy using the FL118 compound of Chemical Formula 2 or a ligand-containing complex (c) targeting DDX5 protein in which the type I topoisomerase inhibitory ability of the active camptothecin derivative (a) is inactivated via a linker linkage.

24. A compound represented by the following formula 3, 3-1, 4, 4-1, 5, 5-1, 6, 7, 8 or 9, or a pharma- ceutically acceptable salt thereof: 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】

25. A pharmaceutical composition for preventing or treating cancer, comprising a compound represented by the following formula 3, 3-1, 4, 4-1, 5, 5-1, 6, 7, 8 or 9, a pharma- ceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof: 【Chemical 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemical 27】 【Chemistry 28】 【Chemical 29】 【Chemistry 30】 【Chemistry 31】

26. A pharmaceutical composition for killing cells, comprising: (a) an activated camptothecin derivative represented by the following chemical formula 1, which is designed to bind to DDX5 protein and E3 ligase; or (b) a prodrug thereof designed to release the activated camptothecin derivative (a) from a target site in the body. 【Chemistry 32】 X １ and X ３ are each independently carbon, oxygen, nitrogen, or sulfur; X １ and X ３ may be the same or different, X ２ is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X １ , (X ２ ) n and X ３ can form a 6- or 7-membered ring (n=values ​​of 1 to 2); Y １ , Y ２ and Y ３ may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus, or sulfur.

27. The pharmaceutical composition for cell killing according to claim 26, characterized in that the active camptothecin derivative (a) designed to bind to DDX5 protein and E3 ligase kills target cells expressing DDX5 protein through a molecular glue degrader mechanism (MoA).

28. The pharmaceutical composition for cell killing described in claim 26, characterized in that the active camptothecin derivative (a) designed to bind to DDX5 protein and E3 ligase has a mechanism of action (MoA) of degrading the oncoprotein DDX5 along with type I topoisomerase inhibitory ability.

29. The active camptothecin derivative is X １ , (X ２ ) n, X ３ , Y １ , Y ２ and / or Y ３ The pharmaceutical composition for cell killing according to claim 26, characterized in that it is designed to exert a bystander effect in cancer tissue or to control the degree of the effect by adjusting cell membrane permeability as desired through deformation.

30. The pharmaceutical composition for cell killing according to claim 27, characterized in that the target cell is a cancer cell or a senescent cell.

31. An anti-cancer dosage form comprising: (a) an activated camptothecin derivative represented by the following chemical formula 1, which is designed to bind to DDX5 protein and E3 ligase; or (b) a prodrug thereof, which is designed to release the activated camptothecin derivative (a) from a target site in the body, administered in a dosage manner capable of stimulating antigen-presenting cells (APCs) through cell death of cancer cells and inducing an anti-tumor immune response. 【Chemical 33】 X １ and X ３ are each independently carbon, oxygen, nitrogen, or sulfur; X １ and X ３ may be the same or different, X ２ is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X １ , (X ２ ) n and X ３ can form a 6- or 7-membered ring (n=values ​​of 1 to 2); Y １ , Y ２ and Y ３ may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus, or sulfur.

32. The anti-cancer dosage form of claim 31, characterized in that the anti-cancer dosage form is designed to be targeted to tumor tissue.

33. A method for producing an anticancer drug based on an active camptothecin derivative (a), in which the bystander effect and / or ADME profile of the active camptothecin derivative (a) is controlled so that T cell exhaustion does not occur due to chronic antigen exposure, comprising: The active camptothecin derivative (a)-based anticancer drug is an anticancer dosage form containing: (a) an active camptothecin derivative designed to bind to DDX5 protein and E3 ligase via a molecular glue degrader; or (b) a prodrug thereof designed to release the active camptothecin derivative (a) from a target site in the body; A method for producing an anticancer agent, comprising the steps of: designing an active camptothecin derivative (a) or a prodrug thereof (b) to contain a camptothecin-based skeleton represented by the following chemical formula 1 as a parent nucleus, selecting a compound designed in this way, or synthesizing a compound designed in this way. 【Chemical 34】 X １ and X ３ are each independently carbon, oxygen, nitrogen, or sulfur; X １ and X ３ may be the same or different, X ２ is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X １ , (X ２ ) n and X ３ can form a 6- or 7-membered ring (n=values ​​of 1 to 2); Y １ , Y ２ and Y ３ may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus, or sulfur.

34. The active camptothecin derivative (a)-based anticancer drug is a topoisomerase I inhibitor. The method of claim 33, characterized in that the dosage form contains (a) an activated camptothecin derivative designed to have a mechanism of action (MoA) of degrading the oncoprotein DDX5 along with the ability to degrade the oncoprotein DDX5, or (b) a prodrug thereof designed to release the activated camptothecin derivative (a) in vivo.

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